CA3088187A1 - Compositions and methods to prevent and treat biofilms - Google Patents

Abstract

Compositions and methods to treat biofilms are disclosed based on the discovery of the role of the disaccharide trehalose in microbial biofilm development. In various embodiments to treat body-borne biofilms systemically and locally, the method includes administering trehalase, the enzyme which degrades trehalose, in combination with other saccharidases for an exposition time sufficient to adequately degrade the biofilm gel matrix at the site of the biofilm. The method also includes administering a combination of other enzymes such as proteolytic, fibrinolytic, and lipolytic enzymes to break down proteins and lipids present in the biofilm, and administering antimicrobials for the specific type(s) of infectious pathogen(s) underlying the biofilm. Additionally, methods are disclosed to address degradation of biofilms on medical device surfaces and biofilms present in industrial settings.

Description

CA Application CPST Ref: 40102/00001

2

3 Cross Reference

4 [0001] This application claims the benefit of U.S. Patent Application No.
16/686,437, filed November 18, 2019.

7 Field of the Invention 8 [0002] The present disclosure is generally related to compositions and methods to prevent and 9 treat biofilms.
11 Description of Related Art 12 [0003] Over the last century, bacterial biofilms have been described as a ubiquitous form of 13 microbial life in various ecosystems which can occur at solid-liquid, solid-air, liquid-liquid, and 14 liquid-air interfaces. The general theory of biofilm predominance was defined and published in 1978 (Costerton JW, Geesey GG, and Cheng GK, "How bacteria stick," Sci. Am., 1978; 238: 86-16 95.). The basic data for this theory initially came mostly from natural aquatic ecosystems 17 showing that more than 99.9% of the bacteria grow in biofilms on a variety of surfaces, causing 18 serious problems in industrial water systems as well as in various pipelines and vessels.
19 100041 Later this fundamental theory of bacterial biofilm was accepted in the medical and dental areas. New and advanced methods for the direct examination of various biofilms showed that 21 microorganisms that cause many medical device-related and other chronic infections in the 22 human body actually grow in biofilms in or on these devices, as well as on mucosal linings of 23 various organs and systems (oral cavity, respiratory tract, eyes, ears, GI tract, and urinary tract).
24 As stated in this theory, "bacteria have certain basic survival strategies that they employ wherever they are" (Donlan RM and Costerton JW, "Biofilms: Survival Mechanisms of 26 Clinically Relevant Microorganisms," Clinical Microbiology Reviews, Apr.
2002: 167-193.) 27 100051 The Nature and Structure of Biofilms CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 100061 Over decades, direct physical and chemical studies of various biofilms (mostly grown in 2 laboratory settings) show that they consist of single microbial cells and microcolonies, all 3 embedded in a highly hydrated exopolymer matrix comprising biopolymers of microbial origin, 4 such as polysaccharides (the major component), proteins, glycoproteins, nucleic acids, lipids, phospholipids, and humic substances; ramifying water channels bisect the whole structure, 6 carrying bulk fluid into the biofilm by convective flow, providing transport of nutrients and 7 waste products, and contributing to a pH gradient within the biofilm (Costerton JW and Irvin 8 RT, "The Bacteria Glycocalyx in Nature and Disease," Ann. Rev.
Microbiol., 1981; 35: 299-9 324.); (de Beer D, Stoodley P, and Lewandowski Z, "Liquid flow in heterogeneous biofilms,"
Biotechnol. Bioeng, 1994; 44: 636-641.); (Himmelsbach DS and Akin DE, "Near-Infrared 11 Fourier-Transform Raman Spectroscopy of Flax (Linum usitatissimum L.) Stems," J Agric Food 12 Chem, 1998; 46: 991-998.); (Maquelin K, Kirschner C, Choo-Smith LP, van den Braak N, Endtz 13 HIP, Naumann D, and Puppels GJ, "Identification of medically relevant microorganisms by 14 vibrational spectroscopy," J Microbiol Methods, 2002; 51: 255-271.);
(Neu TR and Marshall KC, "Bacterial Polymers: Physicochemical Aspects of Their Interactions at Interfaces," J
16 Biomater Appl, 1990; 5: 107-133.); (Neugebauer U, Schmid U, Baumann K, Ziebuhr W, 17 Kozitskaya S, Deckert V, Schmitt M, Popp J, "Toward a Detailed Understanding of Bacterial 18 Metabolism ¨ Spectroscopic Characterization of Staphylococcus Epidermidis," ChemPhysChem, 19 2007; 8: 124-137.); (Weldon MK, Zhelyaskov VR, Morris MD, "Surface-enhanced Raman spectroscopy of lipids on silver microprobes," Appl Spectrosc, 1998; 52: 265-269.). Depending 21 on the biofilm type and the microorganisms involved, microcolonies of microbial cells make up 22 approximately 10% - 15% of the biofilm by volume, and the biofilm matrix comprises 23 approximately 85% - 90%. Water, the major component of the biofilm matrix, can make up to 24 95% - 98% of the matrix volume, and the particulate fraction of the matrix can comprise the rest 2% - 5% correspondingly. Extracellular polysaccharides and proteins have been considered to be 26 the key components of the biofilm matrix and have been most extensively studied over decades 27 (Sutherland IW, "The biofilm matrix - an immobilized but dynamic microbial environment,"
28 Trends Microbiol, 2001; 9: 222-227.); (Stewart PS and Costerton JW, "Antibiotic resistance of 29 bacteria in biofilms," Lancet, 2001; 358: 135-138.); (Staudt C, Horn H, Hempel DC, Neu TR, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 "Volumetric measurements of bacteria and EPS-glycoconjugates in biofilms," Biotechnol 2 Bioeng, 2004; 88: 585-592.); (Zhang XQ, Bishop PL, and Kupferle MJ, "Measurement of 3 polysaccharides and proteins in biofilm extracellular polymers," Water Sci Technol, 1998; 37:
4 345-348.).
100071 Polysaccharides, postulated to be the key component of the biofilm matrix, provide 6 diverse structural variations of the glycocalux formed by saprophytic and pathogenic 7 microorganisms in a variety of environments (Barbara Vu, et al., "Review.
Bacterial extracellular 8 polysaccharides involved in biofilm formation," Molecules, 2009; 14: 2535-2554; doi:
9 3390/molecules 14072535.). The types of polysaccharides in microbial biofilms are of enormous range and depend on the genetic profile of microorganisms involved and the physicochemical 11 properties of local environment (Sutherland IW, "The biofilm matrix - an immobilized but 12 dynamic microbial environment," Trends Microbiol., 2001; 9: 222-227.).
Many polysaccharides 13 are constitutively produced by various bacteria as structural elements of the bacterial cell wall 14 and virulence factors; they can stay attached to the bacterial cell wall surface, forming a complex network surrounding the cell with electrostatic and hydrogen bonds involved, or they can be 16 released into media as exopolysaccharides (EPS) (Mayer C, Moritz R., Kirschner C., Borchar W, 17 Maibaum R, Wingender J, and Flemming HC, "The role of intermolecular interactions: studies 18 on model systems for bacterial biofilms," Int J Biol Macromol, 1999; 26:
3-16.).
19 Polysaccharides, as well as mono- and disaccharides, can be taken by bacteria from the environment and metabolized as a carbon source, and their metabolism is genetically regulated 21 via balanced production of enzymes for both synthesis and degradation pathways (Sutherland 22 IW, "Polysaccharases for microbial polysaccharides," Carbohydr Polym, 1999; 38: 319-328.).
23 Depending on their structure, EPS can bind various amount of water, and some of them (such as 24 cellulose, mutan or curdlan) can even exclude most water from their tertiary structure. Over the years, the gel-like viscosity of the biofilm matrix was attributed mainly to the physical and 26 chemical properties of the polysaccharides involved (Christensen BE, "The role of extracellular 27 polysaccharides in biofilms," J Biotechnol, 1989; 10: 181-202.);
(Stoodley P, et al., "Oscillation 28 characteristics of biofilm streamers in turbulent flowing water as related to drag and pressure 29 drop," Biotechnol Bioeng, 1998; 57: 536- 544.). Exopolysaccharides can be neutral CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 homopolymers (such as cellulose, dextrans, levans), but the majority are polyanionic (for 2 example, alginates, gellan, xanthan produced by Gram-negative bacteria) with attraction of 3 divalent cations (Ca, Mg) to increase binding force, and a few are polycationic, such as those 4 produced by some Gram-positive bacteria (Sutherland 1W, "Biotechnology of Exopolysaccharides," Cambridge: Cambridge University Press, 1990.); (Mack D, Fische W, 6 Krokotsc A, Leopold K, Hartmann R, Egge H, and Laufs R, " The intercellular adhesin involved 7 in biofilm accumulation of Staphylococcus epidermidis is a linear 13-1, 6-linked 8 glucosaminoglycan: purification and structural analysis," J Bacteriol, 1996; 178: 175-183.).
9 100081 Because only small amounts of the biofilm-derived EPS are normally available for direct studies, the researchers usually use data derived from planktonic cell cultures and extrapolate 11 them to biofilms. There is no conclusive evidence to support the idea of existence of the biofilm-12 specific polysaccharides, and to date, all studied polysaccharides present in various biofilms 13 resemble closely the corresponding polymers synthesized by planktonic cells. It has been 14 proposed that the increased amount of polysaccharides in biofilm (one or more, specific for a given bacteria in any given biofilm) can be part of a stress response in biofilm-grown 16 microorganisms, and bacteria form exopolysaccharides as a by-product to release reducing 17 equivalents accumulated in non-optimal growth conditions (Creti R, Koch S, Fabretti F, 18 Baldassarri L, and Huebneri J, "Enterococcal colonization of the gastro-intestinal tract: role of 19 biofilm and environmental oligosaccharides," BMC Microbiology, 2006; 6:
60 doi:
10.1186/1471-2180-6-60.); (Rinker KD, Kelly RM, "Effect of carbon and nitrogen sources on 21 growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon 22 Thermococcus litoralis and bacterium Thermotoga maritime," Biotechnol Bioeng, 2000; 69: 537-23 547.); (Sutherland IW, "Biofilm exopolysaccharides: a strong and sticky framework,"
24 Microbiology, 2001; 147: 3-9.).
[0009] Other extracellular products (specific substances or by-products of bacterial metabolism), 26 as well as detritus, can be either released into the biofilm from aging and lysed cells or trapped 27 within the biofilm matrix, and "cemented" there by mixture of exopolysaccharides (Christensen 28 BE, "The role of extracellular polysaccharides in biofilms," J.
Biotechnol., 1989; 10: 181-201.).
29 These extracellular products include small sugars (mono-, disaccharides), polyols, proteins, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 glycoproteins, enzymes, lipids, glycolipids, phospholipids, nucleic acids, and DNA (Boyd A and 2 Chakrabarty AM, "Role of alginate lyase in cell detachment of Pseudomonas aeruginosa," Appl 3 Environ Microbiol, 1994; 60: 2355-2359.); (Harz M, Rosch P, Peschke KD, Ronneberger 0, 4 Burkhardt H, and Popp J, "Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions," Analyst, 2005; 130:
6 1543-1550.); (Nottingher I, Verrier S, Haque S, Polak JM, Hench LL, "Spectroscopic study of 7 human lung epithelial cells (A549) in culture: living cells versus dead cells," Biopolymers, 2003;
8 72: 230-240.); (Sutherland IW, "A natural terrestrial biofilm," J Ind Microbiol, 1996; 17: 281-9 283.); (Webb JS et al, "Cell death in Pseudomonas aeruginosa biofilm development," J.
Bacteriol., 2003; 185: 4585-4592.); (Weldon MK, Zhelyaskov VR, Morris MD, "Surface-11 enhanced Raman spectroscopy of lipids on silver microprobes," Appl Spectrosc, 1998; 52: 265-12 269.); (Yarwood JM, et al., "Quorum sensing in Staphylococcus aureus biofilms," J. Bacteriol., 13 2004; 186: 1838-1850.). It has been suggested that extracellular DNA, released from the lysed 14 cells, plays an important role in supporting the biofilm structure and provides opportunities for microorganisms to exchange the genetic material for possible development of the biofilm-16 specific phenotypes (Costerton JW, Veeh R, Shirtliff M, Pasmore M, Post C, and Ehrich GD, 17 "The application of biofilm science to the study and control of chronic bacterial infections," J.
18 Clin. Invest., 2003; 112: 1466-1477.); (Gilbert P, Maira-Litran T, McBain AJ, Rickard AH, and 19 Whyte FW, "The physiology and collective recalcitrance of microbial biofilm communities,"
Adv. Microb. Physiol., 2002; 46: 202-256.); (Osterreicher-Ravid D, Ron EZ, &Rosenberg E, 21 "Horizontal transfer of an exopolymer complex from one bacterial species to another," Environ 22 Microbiol, 2000; 2: 366-372.); (Stoodley P, Sauer K, Davies DG, and Costerton JW, "Biofilms 23 as complex differentiated communities," Annu. Rev. Microbiol., 2002; 56:
187-209.);
24 (Whitchurch CB, et al., "Extracellular DNA required for bacterial biofilm formation," Science, 2002; 295: 1487.).
26 100101 It has been proposed that in the dynamic environment of biofilm, microorganisms use 27 special chemical signaling molecules to communicate (the process called quorum-sensing - QS), 28 and the presence of an adequate number of neighboring cells with coordinated chemical 29 signaling between them allow bacteria to properly respond to changes in environmental

5 CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 conditions, including insult from antimicrobials, and benefit from living in the biofilm 2 community. It was assumed that QS can regulate extracellular polysaccharide production, based 3 on the major alterations in the extracellular matrix of laboratory-grown Pseudomonas aeruginosa 4 biofilm when the mutant strain was unable to produce the N-(3-oxododecanoy1)-L-homoserine lactone signal specific for QS (Davies D, Parsek M, Pearson J, et al., "The involvement of cell-

6 to-cell signals in the development of a bacterial biofilm," Science, 1998; 280: 295-298.); (Singh

7 P, Schaeffer A, Parsek M, et al., "Quorum sensing signals indicate that cystic fibrosis lungs are

8 infected with bacterial biofilms," Nature, 2000; 407: 762-764.). But to date, the quorum-sensing-

9 regulated genes involved in Pseudomonas aeruginosa biofilm matrix production have not been identified, and the pel and/or psi genes (regulating production of other polysaccharides PEL and 11 PSL) have not been revealed as quorum-sensing-regulated genes as well (Branda SS, Vik A, 12 Friedman L, and Kolter R, "Biofilms: the matrix revisited," Trends in Microbiology, 2005;
13 13(1): 20-26.); (Whiteley M, et al., "Identification of genes controlled by quorum sensing in 14 Pseudomonas aeruginosa," Proc. Natl. Acad. Sci. U.S.A., 1999; 96: 13904-13909.). Also, the role of quorum sensing in resistance of biofilm to antimicrobials is not clear yet; for example, the 16 laboratory mutants defective in quorum sensing, are unaffected in their resistance to detergents 17 and antibiotics (Brooun A, et al., "A dose-response study of antibiotic resistance in Pseudomonas 18 aeruginosa biofilms," Antimicrob. Agents Chemother, 2000; 44: 640-646.).
19 100111 According to a classical model, any biofilm can be described as:
a non-homogenous multi-layer structure with dynamic environment; growing in a 3-dimensional mode, with 21 constant addition of the new layers and detachment of the parts of the biofilm; with spatial and 22 temporal heterogeneity within the biofilm and variations in bacterial growth rate; with different 23 metabolic and genetic activities of the microorganisms resulting in increased resistance to 24 antimicrobials (including antibiotics) and host defense mechanisms (Charaklis WG, Marshall KC , "Biofilm as a basis for interdisciplinary approach," pp.3-15, In:
Biofilms, 1990, John Wiley 26 and Sons, Charaklis WG. and Marshall KC. (ed.), New York, N.Y.); (Fux CA, et al., "Review.
27 Survival strategies of infectious biofilms", Trends in Microbiology, January 2005; Vol. 13, No 1:
28 34-40.). The heterogeneity within the biofilm has been confirmed for protein synthesis and 29 respiratory activity, but the DNA content remained relatively constant throughout biofilm CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 (Wentland EJ, et al., "Spatial variations in growth rate within Klebsiella pneumoniae colonies 2 and biofilm," Biotechnol. Prog., 1996; 12: 316-321.); (Xu KD, et al., "Biofilm resistance to 3 antimicrobial agents," Microbiology, 2000; 146: 547-549.). An oxygen tension gradient exists 4 within biofilm with the superficial areas being more metabolically active than the deeper areas where bacteria adapt to decreased oxygen availability (De Beer D, Stoodley P, Roe F, et al., 6 "Effects of biofilm structure on oxygen distribution and mass transport,"
Biotechnology 7 Bioengineering, 1994; 43: 1131-1138.). The outer layers of biofilm are more permeable to 8 antimicrobials due to slow build-up of polysaccharides and other constituents (proteins, lipids, 9 etc.), and the inner (deeper) layers are more dense, compressed, and less permeable. Bacteria in the outer layers of biofilm, exposed to the bulk medium, grow faster and can be less resistant to 11 antimicrobials. Conversely, the bacteria in the inner or deeper layers, located closer to the 12 attached surface, grow slower, adapting to decreased oxygen and nutrients availability, and in 13 time, can become more resistant to antimicrobials with possible consequent emergence of 14 biofilm-specific antibiotic-resistant phenotype (Brown MR, et al., "Resistance of bacterial biofilms to antibiotics: a growth-rate related effect?," J. Antimicrob.
Chemother., 1998; 22: 777-16 780.).
17 [0012] It has been proposed that "any given cell within the biofilm will experience a slightly 18 different environment compared with other cells within the same biofilm, and thus be growing at 19 a different rate" (Mah TC, and 0' Toole GA, "Review. Mechanisms of biofilm resistance to antimicrobial agents," Trends in Microbiology, Jan. 2001, 9(1): 34-39.). With continuous 21 bacterial growth, increased cell density triggers the general stress response in microbial cells, as 22 confirmed by increased production of osmoprotectant trehalose and degrading enzyme catalase, 23 with higher concentration of trehalose in proximity to the pathogenic cell colonies (Liu X, et al., 24 "Global adaptations resulting from high population densities in Escherichia coli cultures," J.
Bacteriol., 2000; 182: 4158- 4164.). These events result in physiological changes in biofilm, 26 including reduced flow of solutes (nutrients) into biofilm and diminished growth rate of bacterial 27 microcolonies for genotype survival (Brown M R, and Barker J, "Unexplored reservoirs of 28 pathogenic bacteria: protozoa and biofilms," Trends Microbiol., 1999; 7:
46-50.); (Mah TC., and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 .. 0' Toole GA, "Review: Mechanisms of biofilm resistance to antimicrobial agents", Trends in 2 .. Microbiology, Jan. 2001; 9(1): 34-39.).
3 .. [0013] About two decades ago, the existence of biofilm¨specific phenotypes of bacteria was an 4 .. emerging idea. Such biofilm-specific phenotypes, thought to be induced in a subpopulation of .. microorganisms upon attachment to a surface, were proposed to express specific biofilm-related 6 .. genes compared with their planktonic counterparts (Kuchma SL, and O'Toole G A, "Surface-7 .. induced and biofilm-induced changes in gene expression," Curr. Opin.
Biotechnol., 2000; 11:
8 .. 429-433.). Multiple research data, based mostly upon the genetic studies of the laboratory-9 .. constructed and laboratory-grown mutant strains, provided supportive evidence that the biofilm-.. grown cells differ from their planktonic counterparts in specific properties, including nutrients 11 .. utilization, growth rate, stress response, and increased resistance to antimicrobial agents and the 12 .. host defenses.
13 .. [0014] Biofilm Resistance to Antimicrobial Agents 14 .. 100151 The mechanism of resistance to antimicrobial agents (including antibiotics) in biofilm-.. related microorganisms is different from plasmid, transposons, and mutations that confer innate 16 .. resistance in individual bacterial cells (Stewart PS and Costerton JW, "Review. Antibiotic 17 .. resistance of bacteria in biofilms," Lancet, 2001; 358: 135-138.);
(Costerton JW, Stewart PS, and 18 .. Greenberg E, "Bacterial biofilms: a common cause of persistent infections," Science, 1999; 284:
19 .. 1318-1322.); (Costerton JW and Stewart PS, "Biofilms and device-related infections," In: Nataro .. JP, Blaser MJ, Cunningham-Rundles S., (eds.), Persistent bacterial infections. Washington, DC:
21 .. ASM Press, 2000; 432- 439.).
22 .. 100161 Multiple research studies provided basis for various mechanisms of biofilm resistance to 23 .. antimicrobials, including:
24 .. [0017] = physical and/or chemical diffusion barriers to penetration of antimicrobials and host .. defense cells into the exopolymer matrix of biofilm 26 .. 100181 = activation of a general stress response of the microorganisms 27 .. 100191 = slow growth of the microorganisms 28 .. [0020] = possible emergence of a biofilm-specific bacterial phenotype CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [0021] These mechanisms can be applied to any type of biofilm, varying with the bacteria 2 present and the type of antimicrobials being used (Geddes A, "Infection in the twenty-first 3 century: Predictions and postulates," J Antimicrob Chemother, 2000; 46:
873-878.); (Stewart PS, 4 "Theoretical aspects of antibiotic diffusion into microbial biofilms,"
Antimicrob. Agents Chemother., 1996; 40: 2517- 2522.); (Stewart PS, "Mechanisms of antibiotic resistance in 6 bacterial biofilms," Int J Med Microbiol, 2002; 292: 107-113.).
7 [0022] Most of the biofilm-resistance mechanisms are provided by the biofilm exopolymer 8 matrix as the initial physical and/or chemical barrier that can prevent, inhibit or delay penetration 9 of antimicrobials and host defense cells into the biofilm. The diffusion of antimicrobials through the biofilm can be inhibited by various means: for example, the common disinfectant chlorine is 11 consumed by chemical reaction within the matrix of a mixed Klebsiella pneumoniae and 12 Pseudomonas aeruginosa biofilm (de Beer D, et al., "Direct measurement of chlorine penetration 13 into biofilms during disinfection," Appl. Environ. Microbiol., 1994; 60:
4339- 4344.); antibiotic 14 ciprofloxacin binds to the biofilm components (Suci PA, et al., "Investigation of ciprofloxacin penetration into Pseudomonas aeruginosa biofilms," Antimicrob Agents Chemother, 1994; 38:
16 2125- 2133.); Pseudomonas aeruginosa biofilm prevents diffusion of piperacillin (Hoyle B, et al., 17 "Pseudomonas aeruginosa biofilm as a diffusion barrier to piperacillin,"
Antimicrob. Agents 18 Chemother., 1992: 36: 2054- 2056.); positively charged aminoglycosides bind to negatively 19 charged matrix polymers, such as I 1,4-glucosaminoglycan in Staphylococcus epidermidis biofilm and alginate in Pseudomonas aeruginosa biofilm (Lewis K, "Riddle of biofilm 21 resistance," Antimicrob Agents Chemother., 2001; 45: 999-1007.);
(Walters MC, et al., 22 "Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to 23 tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin," Antimicrob.
24 Agents Chemother., 2003; 47: 317- 323.); (Gordon CA, Hodges NA, Marriott C, "Antibiotic interaction and diffusion through alginate exopolysaccharide of Cystic fibrosis ¨ derived 26 Pseudomonas aeruginosa," J. Antimicrob. Chemother., 1988; 22: 667-674.); (Nichols WW, et 27 al., "Inhibition of tobramycin diffusion by binding to alginate,"
Antimicrob. Agents Chemother., 28 1988; 32: 518- 523.); the additional matrix component colanic acid, produced by mucoid 29 phenotype of E. coli, supports biofilm maturation and provides a thicker biofilm (Danese PN, et CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 al., "Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm 2 architecture," J. Bacteriol., 2000; 182: 3593- 3596.); penetration of antifungal agent nystatin into 3 the mycelium of Aspergillus fumigatus submerged in medium and covered by thin layer of 4 exopolymer matrix is higher than into the aerial-grown colony covered by thick layer of extracellular matrix (Beauvais A, et al., "An extracellular matrix glues together the aerial-grown 6 hyphae of Aspergillus fumigatus," Cellular Microbiology, 2007; 9 (6):
1588-1600.); secreted IgG
7 antibodies fail to penetrate biofilm because of matrix binding (de Beer D, et al., "Measurement 8 of local diffusion coefficients in biofilms by micro-injection and confocal microscopy,"
9 Biotechnol. Bioeng., 1997; 53: 151- 158.); alginate produced by mucoid phenotype of Pseudomonas aeruginosa protects bacteria from phagocytosis by host leukocytes and INF-y 11 activated macrophages (Bayer AS, et al., "Functional role of mucoid exopolysaccharide 12 (alginate) in antibiotic¨induced and polymorphonuclear leukocyte-mediated killing of 13 Pseudomonas aeruginosa," Infect. Immun., 1991; 59: 302- 308.); (Leid JG, Willson CJ, Shirtliff 14 ME, Hassett DJ, Parsek MR, and Jeffers AK, "The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing." J
16 Immunol, 2005; 175: 7512-7518.).
17 [0023] Antimicrobials diffusion can also be inhibited or delayed by specific active substances 18 produced by bacteria themselves: for example, enzyme catalase produced by Pseudomonas 19 aeruginosa spp. degrades hydrogen peroxide on diffusion into thick biofilm (Stewart PS, et al., "Effect of catalase on hydrogen peroxide penetration into Pseudomonas aeruginosa biofilms,"
21 Appl. Environ. Microbiol., 2000; 66: 836- 838.); ampicillin is unable to penetrate biofilm of 22 Klebsiella pneumoniae due to ampicillin-degrading enzyme Beta-lactamase (Anderi IN, et al., 23 "Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to 24 ampicillin and ciprofloxacin," Antimicrob. Agents Chemother., 2000; 44:
1818-1824.); (Bagge N, Hentzer M, Andersen JB, Ciofu 0, Givskov M, and Hoiby N, "Dynamics and spatial 26 distribution of beta-lactamase expression in Pseudomonas aeruginosa biofilms," Antimicrob 27 Agents Chemother, 2004; 48: 1168-1174.); extracellular slime derived from coagulase-negative 28 Staphylococci reduces the effect of glycopeptide antibiotics (Konig C, et al., "Factors 29 compromising antibiotic activity against biofilms of Staphylococcus epidermidis," Eur. J. Clin.
CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Microbiol. Infect. Dis., 2001; 20: 20-26.); (Souli M and Giamarellou H., "Effects of slime 2 produced by clinical isolates of coagulase-negative staphylococci on activities of various 3 antimicrobial agents," Antimicrob. Agents Chemother., 1998; 42: 939-941.); a PMN toxin, 4 rhamnolipid B, produced by Pseudomonas aeruginosa is known to kill neutrophils (Jensen PO, Bjarnsholt T, Phipps R, Rasmussen TB, Calum H, Christoffersen L, et al., "Rapid necrotic 6 killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of 7 rhamnolipid by Pseudomonas aeruginosa," Microbiology, 2007; 153: 1329-1338.).
8 [0024] Delayed penetration of antimicrobials into the biofilm can provide enough time for 9 bacteria to induce the expression of various genes regulating the stress response and mediating resistance to antimicrobials (Jefferson KK, Goldmann DA, and Pier GB, "Use of confocal 11 microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus 12 biofilms," Antimicrob Agents Chemother, 2005; 49: 2467-2473.); (Anwar H, Strap JL, and 13 Costerton JW, "Establishment of aging biofilms: a possible mechanism of bacterial resistance to 14 antimicrobial therapy," Antimicrob Agents Chemother, 1992; 36: 1347-1351.). The central regulator of a general stress response is the alternate sigma-factor RpoS
induced by high cell 16 density, and the presence of activated gene rpoS' mRNA was detected by RT-PCR in sputum 17 from Cystic Fibrosis patients with chronic Pseudomonas aeruginosa biofilm infections (Foley I, 18 et al., "General stress response master regulator rpoS is expressed in human infection: a possible 19 role in chronicity," J. Antimicrob. Chemother., 1999; 43: 164-165.).
Also, it has been shown that an additional sigma-factor Alg acted in concert with RpoS to control general stress response in 21 laboratory grown Pseudomonas aeruginosa during biofilm formation and maturation, and several 22 other genes were upregulated as well, including algC (controlling phosphomannomutase, 23 involved in exopolysaccharide alginate synthesis), algD, algU, and genes controlling 24 polyphosphokinase synthesis (Davis DG and Geesey GG, "Regulation of the alginate biosynsthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous 26 culture," Appl. Environ. Microbiol., 1995; 61: 860-867.). It has been demonstrated that as many 27 as 45 genes differed in expression between sessile cells and their planktonic counterparts during 28 the biofilm development in laboratory settings.
29 [0025] Biofilm-based Medical Conditions and Diseases CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 100261 Comprehensive review of the biofilm-based human infections as well as the biofilms on 2 medical devices was published by Rodney M. Donlan and J. William Costerton (Donlan RM and 3 Costerton JW, "Review. Biofilms: Survival mechanisms of clinically relevant microorganisms,"
4 Clinical Microbiology Reviews, Apr. 2002; 167-193.). Microbial biofilms are important factors in the pathogenesis of various human chronic infections, including native valve endocarditis 6 (NVE), line sepsis, chronic otitis media, chronic sinusitis and rhinosinusitis, chronic bronchitis, 7 cystic fibrosis pseudomonas pneumonia, chronic bacterial prostatitis, chronic urinary tract 8 infections (UTIs), periodontal disease, chronic wound infections, osteomyelitis (Costerton JW, 9 Stewart P, Greenberg E, "Bacterial biofilms: a common cause of persistent infections," Science, 1999; 284: 1318-1322.); (Hall-Stoodley Land Stoodley P, "Evolving concepts in biofilm 11 infections," Cellular Microbiology, 2009; 11(7): 1034-1043.). Microbial biofilms are detected 12 on various medical devices (prosthetic heart valves, central venous catheters, urinary catheters, 13 contact lenses, tympanostomy tubes, intrauterine devices), as well as on medical equipment 14 (endoscopes, dialysis systems, nebulizers, dental unit water lines), and on a variety of surfaces in hospitals and other medical settings (Costeron JW and Stewart PS, "Biofilms and device-related 16 infections," In: Nataro J.P., Blaser M.J., Cunningham-Rundles S., eds.
Persistent bacterial 17 infections. Washington, DC: ASM Press, 2000; 432-439.); (Bryers JD, "Medical Biofilms,"
18 Biotechnology and Bioengineering, 2008; 100 (1) May 1.). Due to their specific features, chronic 19 biofilm-based infections require different interventional approaches for effective treatment (Stewart PS and Costerton JW., "Review. Antibiotic resistance of bacteria in biofilms," Lancet, 21 2001; 358: 135-138.); (Donlan RM and Costerton JW, "Review. Biofilms:
Survival mechanisms 22 of clinically relevant microorganisms," Clinical Microbiology Reviews, Apr. 2002; 167-193.);
23 (Costerton JW, Stewart PS, and Greenberg EP, "Bacterial biofilms: a common cause of 24 persistent infections," Science, 1999; 284: 1318-1322.); (Costerton JW
and Stewart PS, "Biofilms and device-related infections," In: Nataro JP, Blaser MJ, Cunningham-Rundles S, eds.
26 Persistent bacterial infections. Washington, DC: ASM Press, 2000; 432-439.); (Wolcott RD, 27 M.D. and Ehrlich GD, Ph.D., "Biofilms and chronic infections," JAMA, 2008, Vol. 299, No 28 22.); (Costerton JW, Irvin RT, "The Bacteria Glycocalyx in Nature and Disease," Ann. Rev.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Microbiol., 1981; 35: 299-324.); (Costerton JW, et al., "The application of biofilm science to the 2 study and control of chronic bacterial infections," J. Clin. Invest., 2003; 112: 1466- 1477.).
3 [0027] Native Valve Endocarditis 4 [0028] The development of Native Valve Endocarditis (NVE) results from the interaction between the endothelium of the heart (generally, of the mitral, aortic, tricuspid, and pulmonic 6 valves) and microorganisms circulating in the bloodstream (Livornese LL
and Korzeniowski 7 OM, "Pathogenesis of infective endocarditis," pp. 19-35. In: Infective endocarditis, Kaye D.
8 (ed.), 2-nd ed., 1992; Raven Press, New York, N.Y.). Microorganisms usually do not adhere to 9 intact endothelium. There should be contributing factors that promote adherence, such as:
damaged endothelium (as in vasculitis), formation of initial thrombotic lesions of heart valves (as 11 in nonbacterial thrombotic endocarditis ¨ NBTE), accumulation of fibronectin secreted by 12 endothelial cells, platelets and fibroblasts in response to vascular injury, which can 13 simultaneously bind to fibrin, collagen, human cells, and bacteria, specific fibronectin receptors 14 in some bacteria (Streptococcus sanguis, Staphylococcus aureus), high-molecular weight dextrans produced by various Streptococci that promote adherence to the surface of the thrombus 16 in NBTE (Lowrance JH, Baddour LM, and Simpson WA, "The role of fibronectin binding on the 17 rate model of experimental endocarditis caused by Streptococcus sanguis," J. Clin. Investig. 86:
18 7-13.); (Roberts RB, "Streptococcal endocarditis: the viridins and beta hemolytic streptococci,"
19 pp.191-208. In: Infective endocarditis, Kaye D. (ed.), 2-nd ed., 1992;
Raven Press, New York, N.Y.). The most metabolically active colonies were detected on the surface of the thrombus, 21 forming initial biofilm there (Durack DT and Beeson PB, "Experimental bacterial endocarditis 22 II. Survival of bacteria in endocardial vegetations," Br. J. Pathol., 1972, 53: 50-53.). Clinical 23 research of 2345 cases of NVE demonstrated a variety of microorganisms involved: Streptococci 24 (including Streptococcus viridans, Streptococcus bovis), Enterococci, Pneumococci ¨ in 56% of cases; Staphylococci ¨ in 25% of cases (-19% - Coagulase positive and ¨6% -Coagulase 26 negative); Gram-negative bacteria ¨ in 11% of cases, and Fungi (Candida and Aspergillus spp.) 27 ¨ in 10% of cases; all these microorganisms gained access to the bloodstream primarily via the 28 oropharynx, gastrointestinal tract, and genitourinary tract (Tunkel AR
and Mandell GI,"

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Infecting microorganisms," pp. 85-97. In: Infective endocarditis, Kaye D.
(ed.), 2-nd ed., 1992;
2 Raven Press, New York, N.Y.).
3 [0029] Biofilm-based Chronic Infections in the Respiratory Tract 4 [0030] In the upper respiratory tract, bacterial biofilms have been demonstrated in chronic tonsillitis, chronic adenoiditis, chronic sinusitis and chronic rhinosinusitis (CRS), chronic otitis 6 media (OM), and cholesteatoma. In clinical specimens from patients with chronic and recurrent 7 tonsillitis, both attached and aggregated biofilm-associated bacteria were detected in mucosal 8 epithelium of tonsils removed for chronic tonsillitis (in 73% of cases) and in 75% of cases of 9 tonsils removed due to hypertrophy alone (Chole RA and Faddis BT, "Anatomical evidence of microbial biofilms in tonsillar tissues: a possible mechanism to explain chronicity," Arch 11 Otolaryngol Head Neck Surg, 2003; 129: 634-636.). Microbial biofilms associated with 12 epithelial lining with presence of a carbohydrate matrix in situ were demonstrated in clinical 13 specimens of human adenoids removed for chronic adenoiditis (Kania RE, Lamers GE, Vonk M
14 J, Dorpmans E, Struik J, Tran Ba Huy P, et al., "Characterization of mucosal biofilms on human adenoid tissues," Laryngoscope, 2008; 118: 128-134.); (Nistico L, Gieseke A, Stoodley P, Hall-16 Stoodley L, Kerschner JE, and Ehrlich GD, "Fluorescence 'in situ' hybridization for the 17 detection of biofilm in the middle ear and upper respiratory tract mucosa," Methods Mol Biol, 18 2009; 493: 191-213.).
19 100311 Chronic Rhinosinusitis [0032] In Chronic Rhinosinusitis (CRS), mucosal changes with different degrees of denudation 21 in epithelial cells result in a surface favorable for bacterial colonization and biofilm development 22 (Biedlingmaier J, Trifillis A, "Comparison of CT scan and electron microscopic findings on 23 endoscopically harvested middle turbinates," Otolaryngol Head Neck Surg, 1998; 118: 165-24 173.). Biofilm formation, mainly with Pseudomonas aeruginosa infection, was confirmed in patients who had surgery and continued to have symptoms despite medical treatment (Cryer J, 26 Schipor I, Perloff JR, Palmer JN, "Evidence of bacterial biofilms in human chronic sinusitis,"
27 ORE J Otolaryngol Relat Spec, 2004; 66: 155-158.). In patients with CRS
having surgery, 28 mucosal biopsies demonstrated different stages of the biofilm by scanning electron microscopy 29 (SEM) in five out of five patients, and all five patients showed aberrant findings of the mucosal CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 surface with various degrees of severity: from disarrayed cilia to complete absence of cilia and 2 goblet cells (Ramadan HH, Sanclement JA, Thomas JG, "Chronic rhinosinusitis and biofilms,"
3 Otolaryngol Head Neck Surg, 2005; 132: 414-417.). In most cases of CRS
and Pseudomonas 4 aeruginosa biofilms, clinical symptoms were refractory to culture-directed antibiotics, topical steroids, and nasal lavages, and only surgery (mechanical debridement) resulted in significant 6 improvement (Ferguson BJ, Stolz DB, "Demonstration of biofilm in human bacterial chronic 7 rhinosinusitis," Am J Rhinol, 2005; 19: 452-457.).
8 [0033] Chronic Otitis Media 9 100341 Chronic Otitis Media (OM) involves inflammation of the middle-ear mucoperiosteal lining and is caused by a variety of microorganisms, including: Streptococcus pneumoniae, 11 Haemophilus influenzae, Moraxella catarrhalis, group A beta-hemolytic streptococci, enteric 12 bacteria, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and 13 other organisms; mixed cultures can also be isolated (Feigin RD, Kline MW, Hyatt SR, and Ford 14 III KL, "Otitis media," pp. 174-189. In: Textbook of pediatric infectious diseases, Feigin RD and Cherry JD (ed.), 3-rd ed., vol. 1, 1992, W. B. Saunders Co., Philadelphia, Pa.); (Giebink GS, 16 Juhn SK, Weber ML, and Le CT, "The bacteriology and cytology of chronic otitis media with 17 effusion," Pediatric Infect. Dis., 1982; 1: 98-103.). Chronic OM as a biofilm-related infection 18 was demonstrated in clinical specimens and in animal models. Scanning electron microscopy 19 provided evidence of Haemophilus influenzae biofilm on the middle-ear mucosal surfaces of chinchillas that had been injected with a culture of this organism (Hayes JD, Veeh R, Wang X, 21 Costerton JW, Post JC, and Ehrlich GD, Abstr. 186, Am. Soc. Microbiol.
Biofilm, 2000;
22 Conf.2000.); (Hong W, Mason K, Jurcisek J, Novotny L, Bakaletz LO, and Swords WE, 23 "Phosphorylcholine decreases early inflammation and promotes the establishment of stable 24 biofilm communities of nontypeable Haemophilus influenzae strain 86-028NP in a chinchilla model of otitis media," Infect Immun, 2007b; 75: 958-965.). Biofilm aggregates of Streptococcus 26 pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were detected in biopsies of the 27 middle-ear mucosal lining in children with chronic or recurrent OM
undergoing TT placement 28 for treatment, but not in the middle-ear mucosal biopsies from patients undergoing surgery for 29 cochlear implantation (Hall-Stoodley L, Hu FZ, Gieseke A, Nistico L, Nguyen D, Hayes J, et al., CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 "Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis 2 media," JAMA, 2006; 296: 202-211.) 3 [0035] In chronic OM with effusion, the presence of highly viscous fluid in the middle ear 4 requires in many cases the implantation of tympanostomy tubes (TT) to alleviate pressure build-up and hearing loss. Tympanostomy tubes are subject to contamination, and biofilms build up on 6 their inner surfaces. The investigation of colonization and biofilm development by Pseudomonas 7 aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis on various tympanostomy 8 tubes, provided evidence that all three organisms developed biofilms on the Armstrong silicone 9 and the silver oxide-coated Armstrong-style silicone tubes; Pseudomonas aeruginosa also developed biofilms on the fluoroplastic tubes; only the ionized silicone tubes remained free of 11 contamination and biofilms (Biedlingmaier JF, Samaranayake R, and Whelan P, "Resistance to 12 biofilm formation on otologic implant materials," Otolaryngol Head Neck Surg, 1998; 118: 444-13 451.). Silver oxide-impregnated silastic tubes lowered the incidence of postoperative otorrhea 14 during the first postoperative week, possibly by preventing adherence and colonization of selected bacteria to the tube, but had no effect on the established infection in the middle ear 16 (Gourin CG and Hubbell RN, "Otorrhea after insertion of silver oxide-impregnated silastic 17 tympanostomy tubes," Arch. Otolaryngol Head Neck Surg, 1999; 125: 446-450.). Bacterial 18 biofilm was also detected on a human cochlear implant (Pawlowski KS, Wawro D, Roland PS, 19 "Bacterial biofilm formation on a human cochlear implant," Otol Neurotol, 2005; 26: 972-975.).
[0036] In the lower respiratory tract, microbial biofilms were associated with chronic bronchitis, 21 chronic obstructive pulmonary disease, and pneumonia, especially in patients with cystic 22 fibrosis. Scanning electron microscopy of clinical samples (sputum, bronchiolar lavage, lung and 23 bronchial lining biopsies) demonstrated microbial biofilms either attached to mucosal linings or 24 in the form of bacterial aggregates in mucus covering respiratory epithelium (Lam J, Chan R, Lam K, and Costerton JW, "Production of mucoid microcolonies by Pseudomonas aeruginosa 26 within infected lungs in cystic fibrosis," Infect Immun, 1980; 28: 546-556.); (Martinez-Solano L, 27 Macia MD, Fajardo A, Oliver A, and Martinez JL, "Chronic Pseudomonas aeruginosa infection 28 in chronic obstructive pulmonary disease," Clin Infect Dis, 2008; 47:
1526-1533.); (Starner TD, 29 Zhang N, Kim G, Apicella MA, and McCray PB Jr, "Haemophilus influenzae forms biofilms on CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 airway epithelia: implications in cystic fibrosis," Am J Respir Crit Care Med, 2006; 174: 213-2 220.); (Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al., 2002, "Effects 3 of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis 4 patients," J Clin Invest, 2002; 109: 317-325.); (Yang L, Haagensen JA, Jelsbak L, Johansen HK, Sternberg C, Hoiby N, and Molin S, "In situ growth rates and biofilm development of 6 Pseudomonas aeruginosa populations in chronic lung infections," J
Bacteriol, 2008; 190: 2767-7 2776.).
8 [0037] Cystic Fibrosis 9 [0038] Cystic fibrosis (CF), a chronic disease of the lower respiratory system, is the most common inherited disease: 70% of patients with CF are defective in the cystic fibrosis 11 transmembrane conductance regulator protein (CFTR), which functions as a chloride ion channel 12 protein, resulting in altered secretions in the secretory epithelia of the respiratory tract. In CF, 13 there is a net deficiency of water, which hinders the upward flow of the mucus layer thus altering 14 mucociliary clearance. Decreased secretion and increased absorption of electrolytes lead to dehydration and thickening of secretions covering the respiratory mucosa (Koch C and Hoiby N, 16 "Pathogenesis of cystic fibrosis," Lancet, 1993; 341: 1065-1069.). The hyperviscous mucus is 17 thought to increase the incidence of bacterial lung infections in CF
patients. Staphylococcus 18 aureus is usually the first pulmonary isolate from these patients, followed by Haemophilus 19 influenzae. Both of these infections can be treated effectively with antibiotics, but on persistence, they usually form biofilm and predispose the CF-affected lung to colonization with Pseudomonas 21 aeruginosa (colonization rate of 80%) and Burkholderia cepacia with lethal consequences 22 (Govan JR, and Deretic V, "Microbial pathogenesis in cystic fibrosis:
mucoid Pseudomonas 23 aeruginosa and Burkholderia cepacia," Microbiol. Rev., 1996; 60: 539-574.). As was 24 demonstrated in clinical studies, both organisms were nonmucoid during initial colonization, but on persistence in the lungs of patients with CF they ultimately undergo changes to mucoid 26 phenotype within a period of time from months to years (Koch C and Hoiby N, "Pathogenesis of 27 cystic fibrosis," Lancet, 1993; 341: 1065-1069.). The mucoid material, which was shown to be a 28 polysaccharide substance, later identified as alginate, was transiently produced by laboratory 29 strain of P. aeruginosa, following adherence to the surface (Hoyle BD, Williams LJ, and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Costerton JW, "Production of mucoid exopolysaccharide during development of Pseudomonas 2 aeruginosa biofilms," Infect. Immun., 1993; 61: 777-780.). It has been proposed that several in 3 vitro conditions, such as nutrient limitation, the addition of surfactants, and suboptimal levels of 4 antibiotics, may result in mucoidy due to increased production of alginate (May TB, Shinabarger D, Maharaj R, Kato J, Chu L, DeVault JD, Roychoudhury S, Zielinski NA, Berry A, Rothmel 6 RK, Misra TK, and Chakrabarty AM, "Alginate synthesis by Pseudomonas aeruginosa: a key 7 pathogenic factor in chronic pulmonary infections of cystic fibrosis patients," Clin. Microbiol.
8 Rev., 1991; 4: 191-206.). Early antimicrobial treatment with oral ciprofloxacin and inhaled 9 colistin has been shown to postpone chronic infection with Pseudomonas aeruginosa for several years (Koch C and Hoiby N, "Pathogenesis of cystic fibrosis," Lancet, 1993;
341: 1065-1069.).
11 100391 Periodontal Diseases 12 [0040] Periodontal diseases include infections of the supporting tissues of teeth, ranging from 13 mild and reversible inflammation of the gums (gingiva) to chronic destruction of periodontal 14 tissues (gingiva, periodontal ligament, and alveolar bone) and exfoliation of the teeth. The subgingival crevice (the channel between the tooth root and the gum) is the primary site of 16 periodontal infection and will deepen into a periodontal pocket with the progression of the 17 disease (Lamont RJ and Jenkinson HF, "Life below gum line: pathogenic mechanisms of 18 Porphyromonas gingivalis," Microbiol. Mol. Biol. Rev., 1998; 62: 1244-1263.). Microorganisms 19 isolated from patients with moderate periodontal disease include Fusobacterium nucleatum, Peptostreptococcus micros, Eubacterium timidum, Eubacterium brachy, Lactobacillus spp., 21 Actinomyces naeslundii, Pseudomonas anaerobius, Eubacterium sp. strain D8, Bacteroides 22 intermedius, Fusobacterium spp., Selenomonas sputigena, Eubacterium sp.
strain D6, 23 Bacteroides pneumosintes, and Haemophilus aphrophilus, and these bacteria are not found in 24 healthy patients (Moore WEC, Holdeman LV, Cato EP, Smilbert RM, Burmeister JA, and Ranney RR, "Bacteriology of moderate (chronic) periodontitis in mature adult humans," Infect.
26 Immun., 1993; 42: 510-515.). In adult patients with periodontitis, subgingival plaques harbor 27 spirochetes and cocci, and the predominant microorganisms of active lesions in subgingival 28 areas include Fusobacterium nucleatum, Wolinella recta, Bacteroides intermedius, Bacteroides 29 forsythus, and Bacteroides gingivalis (Porphyromonas gingivalis) (Omar AA, Newman HN, and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Osborn J, "Darkground microscopy of subgingival plaque from the top to the bottom of the 2 periodontal pocket," J. Clin. Periodontol., 1990; 17: 364-370.); (Dzink JI, Socransky SS, and 3 Haffajee AD, "The predominant cultivable microbiota of active and inactive lesions of 4 destructive periodontal diseases," J. Clin. Periodontol., 1988; 15: 316-323.).
100411 Proteinaceous conditioning films (called acquired pellicle), developed on the exposed 6 surfaces of enamel almost immediately after cleaning of the tooth surface, comprises albumin, 7 lysozyme, glycoproteins, phosphoproteins, lipids, and gingival crevice fluid. Within hours of 8 pellicle formation, single cells of primarily gram-positive cocci and rod-shaped bacteria from the 9 normal oral flora colonize these surfaces, binding directly to the pellicle through the production of extracellular glucans (Kolenbrander PE and London J, "Adhere today, here tomorrow: oral 11 bacterial adherence," J. Bacteriol., 1993; 175: 3247-3252.). After several days, actinomycetes 12 predominate followed by co-aggregation of various microorganisms, resulting in the 13 development of early biofilm with characteristic polysaccharide matrix and polymers of salivary 14 origin, with subsequent (within 2 to 3 weeks) formation of the dental plaque if left undisturbed (Marsh PD, "Dental plaque," pp. 282-300. In: Microbial biofilms. 1995; Lappin-Scott UM and 16 Costerton JW (ed.), Cambridge University Press, Cambridge, United Kingdom.). Plaque can be 17 mineralized with calcium and phosphate ions (called calculus or tartar) and develop more 18 extensively in protected areas (between the teeth, and between the tooth and gum). With the 19 increase of the plaque mass in these protected areas, the beneficial buffering and antimicrobial properties of saliva decrease, leading to dental caries or periodontal disease. Clinical research 21 data show that control of supragingival plaque by professional tooth cleaning and personal 22 hygienic efforts can prevent gingival inflammation and adult periodontitis (Corbet EF and 23 Davies Wilt, "The role of supragingival plaque in the control of progressive periodontal 24 disease," J. Clin. Periodontol., 1993; 20: 307-313.).
[0042] Chronic Bacterial Prostatitis 26 100431 The prostate gland may become infected by bacteria ascended from the urethra or by 27 reflux of infected urine into the prostatic ducts emptying into the posterior urethra (Domingue GJ
28 and Hellstrom WJG, "Prostatitis," Clin. Microbiol. Rev., 1998; 11: 604-613.). If bacteria were 29 not eradicated with antibiotic therapy at the early stage of infection, they continue to persist and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 can form sporadic microcolonies and biofilms that adhere to the epithelial cells of the prostatic 2 duct system, resulting in chronic bacterial prostatitis. The microorganisms involved in this 3 process include: E. coli (most common isolate), Klebsiella, Enterobacteria, Proteus, Serratia, 4 Pseudomonas aeruginosa, Enterococcus fecalis, Bacteroides spp., Gardnerella spp., Corynebacterium spp., and Coagulase-negative Staphylococci (CoNS) (Nickel JC, Costerton JW, 6 McLean RJC, and Olson M, "Bacterial biofilms: influence on the pathogenesis, diagnosis, and 7 treatment of the urinary tract infections," J. Antimicrob. Chemother., 1994; 33 (Suppl. A): 31-8 41.). The biopsies from patients with chronic bacterial prostatitis examined by either scanning 9 electron microscopy or transmission electron microscopy, demonstrated bacteria present in glycocalyx-encasted microcolonies, firmly adherent to the ductal and acinar mucosal layers 11 (Nickel JC and Costerton JW, "Bacterial localization in antibiotic-refractory chronic bacterial 12 prostatitis," Prostate, 1993; 23: 107-114.). Sporadic microcolonies of CoNS in the intraductal 13 space have been shown to be enveloped in a dehydrated slime matrix (Nickel JC and Costerton 14 JW, "Coagulase-negative staphylococcus in chronic prostatitis," J.
Urol., 1992; 147: 398-401.).
Treatment failures are common in chronic bacterial prostatitis due to the local environment and 16 biofilm formation, with changes in bacterial metabolism and possible development of resistance 17 to antimicrobials. In order to increase the effectiveness of the antimicrobial treatment, it has been 18 proposed to deliver higher antibiotic concentrations directly to the biofilm within the prostatic 19 ducts (Nickel JC, Costerton JW, Mclean RJC, and Olson M, "Bacterial biofilms: influence on the pathogenesis, diagnosis, and treatment of the urinary tract infections," J.
Antimicrob.
21 Chemother., 1994; 33 (Suppl. A): 31-41.).
22 100441 Biofilms on Medical Devices 23 100451 Over the last 20 years, biofilms on various medical devices, including prosthetic heart 24 valves, central venous catheters, urinary (Foley) catheters, contact lenses, intrauterine devices, and dental unit water lines, have been studied using viable bacterial culture techniques and 26 scanning electron microscopy, and for certain devices (contact lenses and urinary catheters) 27 additional evaluation of susceptibility of various materials to bacterial adhesion and biofilm 28 formation have also been implemented (Costerton JW, Stewart PS, and Greenberg EP, "Bacterial 29 biofilms: a common cause of persistent infections," Science, 1999; 284:
1318-1322.); (Donlan CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 RM and Costerton JW, "Review. Biofilms: Survival mechanisms of clinically relevant 2 microorganisms," Clinical Microbiology Reviews, Apr. 2002; 167-193.).
3 [0046] Prosthetic Heart Valves 4 [0047] Prosthetic valve endocarditis (PVE) is a microbial infection of the valve and surrounding tissues of the heart, ranging between 0.5% and 4%, and is similar for both types of valves 6 currently used ¨ mechanical valves and bioprostheses (Douglas JL and Cobbs CG, "Prosthetic 7 valve endocarditis," pp.375-396. In: Infective endocarditis, Kaye D.
(ed.), 2-nd ed., 1992; Raven 8 Press LTD., New York, N.Y.). Tissue damage resulting from surgical implantation of the 9 prosthetic valve, leads to accumulation of platelets and fibrin at the suture site and on the device, providing a favorable environment for bacterial colonization and biofilm development. PVE is 11 predominantly caused by microbial colonization of the sewing cuff fabric. The microorganisms 12 commonly invade the valve annulus, potentially promoting separation between the valve and the 13 tissue resulting in leakage. Infectious microorganisms involved in PVE
include Staphylococcus 14 epidermidis (at the early stages), followed by Streptococci, CoNS, Enterococci, Staphylococcus aureus, gram-negative Coccobacilli, fungi, and Streptococcus viridans spp.
(the most common 16 microorganism isolated during late PVE) (Hancock EW, "Artificial valve disease," pp.1539-17 1545. In: The heart arteries and veins; Schlant RC, Alexander RW, O'Rourke RA, Roberts R, 18 and Sonnenblick EH (ed.), 8-th ed., 1994; vol.2. McGraw-Hill, Inc., New York, N.Y.);
19 (Illingworth BL, Twenden K, Schroeder RF, and Cameron JD, "In vivo efficacy of silver-coated (silzone) infection-resistant polyester fabric against a biofilm producing bacteria, Staphylococcus 21 epidermidis, J. Heart Valve Dis., 1998; 7: 524-530.); (Karchmer AW and Gibbons GW, 22 "Infections of prosthetic heart valves and vascular grafts," pp.213-249.
In: Infections associated 23 with indwelling medical devices; Bisno AL and Waldovogel FA (ed.), 1994, 2-nd ed. American 24 Society for Microbiology, Washington, D.C.).
[0048] Central Venous Catheters 26 100491 For Central Venous Catheters (CVCs), the device-related infection rate is 3% to 5%.
27 Infectious biofilms are universally present on CVCs and can be associated with either the outside 28 surface of the catheter or the inner lumen. Colonization and biofilm formation may occur within 29 3 days of catheterization. Short-term catheters (in place for less than

10 days) usually have more CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 extensive biofilm formation on the external surfaces, and long-term catheters (up to 30 days) 2 have more extensive biofilm on the internal lumen. (Raad II, Costerton JW, Sabharwal, 3 Sacilowski UM, Anaissie W, and Bodey GP, "Ultrastructural analysis of indwelling vascular 4 catheters: a quantitative relationship between luminal colonization and duration of placement," J.
Infect. Dis., 1993; 168: 400-407.). Colonizing microorganisms originate either from the skin 6 insertion site, migrating along the external surface of the device, or from the hub, due to 7 manipulation by health care workers, migrating along the inner lumen (Elliott TSJ, Moss HA, 8 Tebbs SE, Wilson IC, Bonser RS, Graham TR, Burke LP, and Faroqui MH, "Novel approach to 9 investigate a source of microbial contamination of central venous catheters," Eur. J. Clin.
Microbiol. Infect. Dis., 1997; 16: 210-213.). Because the device is in direct contact with the

11 bloodstream, the surface becomes coated with platelets, plasma and tissue proteins such as

12 albumin, fibrinogen, fibronectin, and laminin, forming conditioning films to which the bacteria

13 are adherent: Staphylococcus aureus adheres to fibronectin, fibrinogen, and laminin, and

14 Staphylococcus epidermidis adheres only to fibronectin. Organisms colonizing CVCs include CoNS, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus 16 fecalis, and Candida albicans (Elliott TSJ, Moss HA, Tebbs SE, Wilson IC, Bonser RS, Graham 17 TR, Burke LP, and Faroqui MH, "Novel approach to investigate a source of microbial 18 contamination of central venous catheters," Eur. J. Clin. Microbiol.
Infect. Dis., 1997; 16: 210-19 213.).
[0050] Urinary Catheters 21 [0051] Urinary catheters are subject to bacterial contamination regardless of the types of the 22 catheter systems. In open systems, the catheter draining into an open collection container 23 becomes contaminated quickly, and patients commonly develop Urinary Tract Infection (UTI) 24 within 3 to 4 days. In closed systems, when the catheter empties in a securely fastened plastic collecting bag, the urine from the patient can remain sterile for 10 to 14 days in approximately 26 half the patients (Kaye D and Hessen T, "Infections associated with foreign bodies in the urinary 27 tract," pp.291-307. In: Infections associated with indwelling medical devices; Bisno AL and 28 Waldovogel FA (ed.), 1994; 2-nd ed., American Society for Microbiology, Washington, D. C.).
29 Regardless of the type of the system, with short-term catheterization (up to 7 days), 10% to 50%

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 of patients develop UTI, and with long-term catheterization (28 days and longer) essentially all 2 patients develop UTI (Stickler DJ, "Bacterial biofilms and the encrustation of urethral catheters,"
3 Biofouling, 1996; 94: 293-305.). The risk of catheter-associated UTI
increases by approximately 4 10% for each day the catheter is in place. Initially, catheters are colonized by a single microorganism, such as Staphylococcus epidermidis, Enterococcus fecalis, E.
coli, Proteus 6 mirabilis. Later, the number and diversity of bacteria increase, with mixed communities 7 containing Providencia stuartii, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella 8 pneumoniae, Morganella morganii, Acinetobacter calcoaceticus, and Enterobacter aerogenes 9 (McLean RJC, Nickel JC, and Olson ME, "Biofilm associated urinary tract infections," pp. 261-273. In: Microbial biofilms; 1995, Lappin-Scott HIM and Costerton JW (ed.), Cambridge 11 University Press, Cambridge, United Kingdom.).
12 [0052] Both in vivo and in vitro studies by scanning electron microscopy and transmission 13 electron microscopy provide evidence for biofilm formation on catheters.
The thickness of 14 biofilm on silicone and silicone-coated Foley catheters from patients undergoing long-term catheterization ranges from 200[tm to 500[tm, with the thickest biofilms formed by E. coli and 16 Klebsiella pneumoniae (up to 490[tm). The thinnest biofilms were formed by Morganella 17 morganii and diphtheroids (the average ¨ 10[tm), and these biofilms were also patchy 18 (Ganderton L, Chawla J, Winters C, Wimpenny J, and Stickler D, "Scanning electron 19 microscopy of bacterial biofilms on indwelling bladder catheters," Eur.
J. Clin. Microbiol. Infect.
Dis., 1992; 11: 789-796.).
21 [0053] Urinary catheter biofilms are unique, because certain microorganisms produce enzyme 22 urease which hydrolyzes the urea of the urine to form free ammonia, thus raising the local pH
23 and allowing precipitation of minerals hydroxyapatite (calcium phosphate) and struvite 24 (magnesium ammonium phosphate). These minerals become deposited in the catheter biofilms, forming a mineral encrustation which can completely block a urinary catheter within 3 to 5 days 26 (Tunney MM, Jones DS, and Gorman SP, "Biofilm and biofilm-related encrustations of urinary 27 tract devices," Methods Enzymol., 1999; 310: 558-566.). The primary urease-producing 28 organisms in urinary catheters are Proteus mirabilis, Morganella morganii, Pseudomonas 29 aeruginosa, Klebsiella pneumoniae, and Proteus vulgaris. Mineral encrustations were observed CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 only in catheters containing these bacteria Stickler D, Morris N, Moreno MC, and Sabbuba N, 2 "Studies on the formation of crystalline bacterial biofilms on urethral catheters," Eur. J. Clin.
3 Microbiol. Infect. Dis., 1998; 17: 649-652.); (Stickler D, Ganderton L, King J, Nettleton J, and 4 Winters C, "Proteus mirabilis biofilms and the encrustation of urethral catheters," Urol. Res., 1993; 21: 407-411.).
6 [0054] Contact Lenses 7 [0055] Bacteria adhere readily to both types of contact lenses: soft contact lenses (made of either 8 hydrogel or silicone) and hard contact lenses constructed of polymethylmethacrylate. Initial 9 adhesion of Pseudomonas aeruginosa to hydrogel contact lenses, resulted within 2 hours in biofilm formation with characteristic extracellular matrix polymers observed by transmission 11 electron microscopy and ruthenium red staining (Miller MJ and Ahearn G, "Adherence of 12 Pseudomonas aeruginosa to hydrophilic contact lenses and other substrata," J. Clin. Microbiol., 13 1987; 25: 1392-1397.). The degree of attachment depended on various factors, including the 14 nature of the substrate, pH, electrolyte concentration, ionic charge of the polymer, and bacterial strain tested.
16 [0056] Organisms that have been shown to adhere to contact lenses include: Pseudomonas 17 aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia spp., E. coli, Proteus 18 spp., and Candida spp. (Dart JKG, "Contact lens and prosthesis infections," pp. 1-30. In:
19 Duane's foundations of clinical ophthalmology; Tasman W and Jaeger EA
(ed.), 1996;
Lippincott-Raven, Philadelphia, PA.). An established biofilm was detected on the lens removed 21 from a patient with P. aeruginosa keratitis, as well as from the patients with clinical diagnosis of 22 microbial keratitis, in several cases containing multiple species of bacteria or bacteria and fungi 23 (Stapleton F and Dart J, "Pseudomonas keratitis associated with biofilm formation on a 24 disposable soft contact lens," Br. J. Ophthalmol., 1995; 79: 864-865.);
(McLaughlin-Borlace L, Stapleton F, Matheson M, and Dart JKG, "Bacterial biofilm on contact lenses and lens storage 26 cases in wearers with microbial keratitis," J. Appl. Microbiol., 1998;
84: 827-838.).
27 100571 The lens case has been implicated as the primary source of microorganisms for 28 contaminated lenses and lens disinfectant solutions, with contaminated storage cases in 80% of 29 asymptomatic lens users (McLaughlin-Borlace L, Stapleton F, Matheson M, and Dart JKG, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 "Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis," J.
2 Appl. Microbiol., 1998; 84: 827-838.). Also, the identical organisms were isolated from the lens 3 cases and the corneas of infected patients. Additionally, protozoan Acanthamoeba has been 4 shown to be a component of these biofilms (Dart JKG, "Contact lens and prosthesis infections,"
pp. 1-30. In: Duane's foundations of clinical ophthalmology; Tasman W and Jaeger EA (ed.), 6 1996; Lippincott-Raven, Philadelphia, PA.); (McLaughlin-Borlace L, Stapleton F, Matheson M, 7 and Dart JKG, "Bacterial biofilm on contact lenses and lens storage cases in wearers with 8 microbial keratitis," J. Appl. Microbiol., 1998; 84: 827-838.).
9 100581 Dental Unit Water Lines 100591 Dental procedures may expose both patients and dental professionals to opportunistic and 11 pathogenic organisms originating from various components of the dental unit. Small-bore 12 flexible plastic tubing supplies water (municipal or from separate reservoirs containing distilled, 13 filtered, or sterile water) to different hand pieces (air-water syringe, the ultrasonic scaler, the 14 high-speed hand piece), and elevated bacterial counts were detected in all these systems (Barbeau J, Tanguay R, Faucher E, Avezard C, Trudel L, Cote L, and Prevost AP, 16 "Multiparametric analysis of waterline contamination in dental units,"
Appl. Environ.
17 Microbiol., 1996; 62: 3954-3959.); (Furuhashi M and Miyamae T, "Prevention of bacterial 18 contamination of water in dental units," J. Hosp. Infect., 1985; 6: 81-88.); (Mayo JA, Oertling 19 KM, and Andrieu SC, "Bacterial biofilm: a source of contamination in dental air-water syringes," Clin. Prey. Dent., 1990; 12: 13-20.); (Williams HN, Kelley J, Folineo D, Williams 21 GC, Hawley CL, and Sibiski J, "Assessing microbial contamination in clean water dental units 22 and compliance with disinfection protocol," JADA, 1994; 125: 1205-1211.).
23 100601 Organisms generally isolated from dental water units include Pseudomonas spp., 24 Flavobacterium spp., Acinetobacter spp., Moraxella spp., Achromobacter spp., Methylobacterium spp., Rhodotorula spp., hyphomycetes (Cladosporium spp., Aspergillus spp., 26 and Penicillium spp.), Bacillus spp., Streptococcus spp., CoNS, Micrococcus spp., 27 Corynebacterium spp., and Legionella pneumophila (Tall BD, Williams HN, George KS, Gray 28 RT, and Walch M, "Bacterial succession within a biofilm in water supply lines of dental air-29 water syringes," Can. J. Microbiol., 1995; 41: 647-654.); (Whitehouse RLS, Peters E, Lizotte J, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 and Lilge C, "Influence of biofilms on microbial contamination in dental unit water," J. Dent., 2 1991; 19: 290-295.); (Mills SEP, Lauderdale W, and Mayhew RB, "Reduction of microbial 3 contamination in dental units with povidone-iodine 10%," JADA, 1986; 113:
280-284.); (Atlas 4 RM, Williams JF, and Huntington MK, "Legionella contamination of dental-unit waters," Appl.
Environ. Microbiol., 1995; 61: 1208-1213.); (Callacombe SJ and Fernandes LL, "Detecting 6 Legionella pneumophila in water systems: a comparison of various dental units," JADA, 1995;
7 126: 603-608.); (Pankhurst CL, Philpott-Howard JN, Hewitt JH, and Casewell MW, "The 8 efficacy of chlorination and filtration in the control and eradication of Legionella from dental 9 chair water systems," J. Hosp. Infect., 1990; 16: 9-18.). The variety of microorganisms observed, were embedded in an apparent polysaccharide matrix (Whitehouse RLS, Peters E, Lizotte J, and 11 Lilge C, "Influence of biofilms on microbial contamination in dental unit water," J. Dent., 1991;
12 19: 290-295.). Also, amebic trophozoites and cysts, and nematodes (in one biofilm sample) were 13 also observed (Santiago JI, Huntington MK, Johnston AM, Quinn RS, and Williams JF, 14 "Microbial contamination of dental unit waterlines: short- and long-term effects of flushing,"
Gen. Dent., 1994; 42: 528-535.). A positive correlation was found between biofilm and water 16 counts, and by 180 days of exposure, a thick, multiple layer of extracellular polymeric 17 substances covered the entire surface of the dental unit water line (Tall BD, Williams HN, 18 George KS, Gray RT, and Walch M, "Bacterial succession within a biofilm in water supply lines 19 of dental air-water syringes," Can. J. Microbiol., 1995; 41: 647-654.).
Biofilms containing extensive extracellular polymer matrix and both mixed skin flora and aquatic bacteria, were also 21 detected on the inner lumen of saliva ejectors (Barbeau J, ten Bocum L, Gauthier C, and Prevost 22 AP, "Cross contamination potential of saliva ejectors used in dentistry," J. Hosp. Infect., 1998;
23 40: 303-311.).
24 [0061] Methods of Treating Biofilms and Biofilm-Based Infections [0062] Many biofilm control strategies have been proposed, applied mostly to biofilm formed on 26 various medical devices, including long term antibiotics for patients using these devices, various 27 antimicrobials to cover the surfaces of devices, various polymer materials, ultrasound, and low-28 strength electrical fields along with disinfectants.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 100631 For biofilm-based infections in the human body, a few approaches aimed to either 2 eradicate or penetrate the extracellular polymeric substances have been offered: for example, a 3 mixture of enzymes was effective in eradicating laboratory-grown biofilms of several different 4 organisms (Johansen CP, Falholt P, and Gram L, "Enzymatic removal and disinfection of bacterial biofilm," Appl. Environ. Microbiol., 1997; 63: 3724- 3728.). Another more precise 6 approach was identifying the polysaccharides for a specific organism in the biofilm and treating 7 the biofilm with that enzyme: for example, the specific enzyme alginate lyase allowed more 8 effective diffusion of gentamycin and tobramycin through alginate, the biofilm polysaccharide of 9 mucoid Pseudomonas aeruginosa (Hatch RA, and Schiller NL, "Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas 11 aeruginosa," Antimicrob. Agents Chemother., 1998; 42: 974- 977.). In addition, for the 12 management of biofilm infections, various antibiotics have been examined extensively in vitro 13 and in vivo, including aminoglycosides, fluoroquinolones, macrolides, as well as the latest 14 protein synthesis inhibitors (Linezolid and Quinupristin) clinically available and appear promising for treatment of in vivo biofilm infections (In: Biofilms, Infection, and Antimicrobial 16 Therapy; Edited by Pace JL, Rupp ME, and Finch RG; Boca Raton, FL: CRC
Press, 2006.
17 Chapter 18, page 360.).
18 100641 A review of recent patent literature summarizes citations under six categories of current 19 treatment approaches: 1) antibiotics and small molecule inhibitors of new and established biofilms, 2) quorum sensing and signaling molecules inhibitors, 3) surface coating substances for 21 inhibition of biofilm formation, 4) antibodies and vaccines for infectious biofilm treatment, 5) 22 enzymes for degrading biofilms, and 6) bacteriophage treatment of infectious biofilms (Lynch 23 AS and Abbanat D, "New antibiotic agents and approaches to treat biofilm-associated 24 infections," Expert Opin. Ther. Patents, 2010; 20(10): 1373-1387.).
[0065] Additional approaches involve the use of various natural substances and combined 26 technologies. For example, naturally occurring impediments to biofilm adhesion have been 27 proposed such as, oral-ficin, a cysteine protease derived from the Ficus glabrata tree, which 28 prevents biofilm-forming bacteria from adhering to surfaces (Potera C, "A Potpourri of Probing 29 and Treating Biofilms of the Oral Cavity," Microbe Magazine, Oct.
2009.). The ability of honey CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 to prevent quorum sensing and thereby interfere with the formation or maintenance of biofilms 2 suggests it can be a candidate substance for the management of infected wounds ("The role of 3 biofilm in wounds," a thesis submitted to the University of Wales, Cardiff, UK, in candidature 4 for Ph.D. by Okhiria OA, May 2010, Chapter 5: Antimicrobial effect of honey on biofilm and quorum sensing: 190-234.).
6 100661 An example of the use of combined technologies is the treatment of biofilm infections on 7 implants using ultrasound in concert with antibiotics (Carmen JC, Roeder BL, Nelson JL, 8 Robison Ogilvie RL, Robison RA, Schaalje GB, and Pitt WG, "Treatment of Biofilm Infections 9 on Implants with Low-frequency Ultrasound and Antibiotics," Am J Infect Control. 2005, March; 33(2): 78-82.).
11 100671 Methods of Addressing Biofilm Contamination of Medical Equipment 12 [0068] Bacterial and fungal biofilms develop on the various types of medical equipment. This 13 includes medical diagnostic devices, such as: stethoscopes, colposcopes, nasopharyngoscopes, 14 angiography catheters, endoscopes, angioplasty balloon catheters; and various permanent, semi-permanent, and temporary indwelling devices, such as: contact lenses, intrauterine devices, 16 dental implants, urinary tract prostheses and catheters, peritoneal dialysis catheters, indwelling 17 catheters for hemodialysis and for chronic administration of chemotherapeutic agents (Hickman 18 catheters), cardiac implants (pacemakers, prosthetic heart valves, ventricular assisting devices -19 VAD), synthetic vascular grafts and stents, prostheses, internal fixation devices, percutaneous sutures, tracheal and ventilator tubing, dispensing devices such as nebulizers, and cleaning 21 devices such as sterilizers. Summarized herein are the current methods employed to diminish the 22 presence of microbial biofilms and associated pathogens on medical equipment.
23 100691 Implants 24 [0070] Biofilm infections associated with indwelling medical devices and implants are difficult to resolve using conventional antibiotics. Antibiotic treatment requires lengthy periods of 26 administration, with combined antibiotics at high dose, or the temporary surgical removal of the 27 device or associated tissue. Newer developments, aimed at interfering with the colonization 28 process comprise, for example, new biomaterials, the co-application of acoustic energy or low-29 voltage electric currents with antibiotics and the development of specific anti-biofilm agents CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 (Jass J, Surman S, and Walker JT, "Medical biofilms: detection, prevention, and control," Vol.
2 2., John Wiley, 2003: 261.).
3 [0071] Central Venous Catheters 4 [0072] Several studies have examined the effect of various types of antimicrobial treatment in controlling biofilm formation on venous catheters. The methods and materials used include 6 adding disinfectant to physiological flush of catheters for elimination of microbial colonization 7 (Freeman R, Gould FK. "Infection and intravascular catheters," [letter].
J. Antimicrob.
8 Chemother., 1985; 15: 258.), impregnation of catheters with polyantimicrobials (Darouiche RO
9 et al. , "A comparison of two antimicrobial-impregnated central venous catheters," N Engl J
Med, 1999; 340: 1-8.), coating of catheters with surfactants to bond antibiotics to catheter 11 surfaces (Kamal G.D., Pfaller M.A., Rempe L.E., Jebson P.J.R., "Reduced intravascular catheter 12 infection by antibiotic bonding. A prospective, randomized, controlled trial", JAMA, 1991; 265:
13 2364-2368.), and the use of an attachable subcutaneous cuff containing silver ions inserted after 14 local application of polyantibiotic (Flowers R.H., Schwenzer K.J., Kopel R.F., Fisch M.J., Tucker S.I., Farr B.M., "Efficacy of an attachable subcutaneous cuff for the prevention of 16 intravascular catheter-related infection", JAMA, 1989; 261: 878-883.).
17 [0073] Prosthetic Heart Valves 18 100741 The pathogenesis of infection associated with implanted heart valves is related to the 19 interface between the valve and surrounding tissue. Specifically, because implantation of a mechanical heart valve causes tissue damage at the site of its installation, microorganisms have 21 an increased tendency to colonize such locations (Donlan RM, "Biofilms and Device-Associated 22 Infections," Emerging Infectious Diseases Journal, Mar-Apr 2001; Vol. 7, No. 2: 277-281.).
23 Hence, biofilms resulting from such infections tend to favor development on the tissue 24 surrounding the implant or the sewing cuff fabric used to attach the device to the tissue. Silver coating of the sewing cuff has been found to reduce such infections (Illingworth BL, Tweden K, 26 Schroeder RF, Cameron JD, "In vivo efficacy of silver-coated (Silzone) infection-resistant 27 polyester fabric against a biofilm-producing bacteria, Staphylococcus epidermidis", J Heart 28 Valve Dis 1998; 7: 524. Abstract); (Carrel T, Nguyen T, Kipfer B, Althaus U, "Definitive cure CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 of recurrent prosthetic endocarditis using silver-coated St. Jude medical heart valves: a 2 preliminary case report," J Heart Valve Dis., 1998; 7: 531. Abstract.).
3 [0075] Urinary Catheters 4 [0076] Conventional approaches to the treatment of urinary catheter biofilms include: the use of antimicrobial ointments and lubricants, instillation or irrigation of the bladder with 6 antimicrobials, use of the collection bags containing antimicrobial agents, catheter impregnation 7 with antimicrobial agents, and the use of systemic antibiotics (Kaye D, Hessen MT, "Infections 8 associated with foreign bodies in the urinary tract," In: Bisno A.L., Waldovogel F.A., editors.
9 Infections associated with indwelling medical devices. 2nd ed.
Washington: American Society for Microbiology; 1994; pp. 291-307.). Such approaches have been found to have limited 11 efficacy, although silver impregnation of catheters has been found to delay onset of bacteriuria 12 (Donlan RM, "Biofilms and Device-Associated Infections," Emerging Infectious Diseases 13 Journal, Mar-Apr 2001; Vol. 7, No. 2: 277-281.). From various materials used for catheter 14 construction, silicone catheters obstruct less often than latex, Teflon, or silicone-coated latex in patients prone to catheter encrustation (Sedor J and Mulholland SG, "Hospital-acquired urinary 16 tract infections associated with indwelling catheter," Urol. Clin. N.
Am., 1999; 26: 821-828.).
17 [0077] A new product, the UroShieldTM System, produced by NanoVibronix uses low cost 18 disposable ultrasonic actuators which energize all surfaces of the catheter thereby interfering 19 with the attachment of bacteria, the initial step in biofilm formation (Nagy K, Koves B, Jackel M, Tenke P, "The effectiveness of acoustic energy induced by UroShield device in the 21 prevention of bacteriuria and the reduction of patient's complaints related to long-term 22 indwelling urinary catheters," Poster presentation at 26th Annual Congress of the European 23 Association of Urology (EAU); Vienna, March 2011: No. 483. Abstract.).
24 [0078] Dialysis systems [0079] The development of biofilms throughout hemodialysis systems has been substantiated. In 26 fact, some cases have been suspicious for the outbreak of infection within dialysis centers.
27 Furthermore, the endotoxins and other cytokines in these biofilms can cross the dialysis 28 membrane and trigger the inflammatory response in the patients (Vincent FC, Tibi AR, and 29 Darbord JC. "A bacterial biofilm in a hemodialysis system. Assessment of disinfection and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 crossing of endotoxins," ASAIO Trans., 1989; 35: 310-313.). In a study specific to the removal 2 of biofilms from dialysis tubing, the efficacy of 21 different decontamination procedures was 3 ascertained with the most effective treatment determined to be an acid pre-treatment, followed by 4 use of a concentrated bleach solution; treatments performed at high temperature did not improve the removal of biofilm (Marion-Ferey K, et al., "Biofilm removal from silicone tubing: an 6 assessment of the efficacy of dialysis machine decontamination procedures using an in vitro 7 model," Journal of Hospital Infection, 2003; 53(1): 64-71.).
8 [0080] Given the challenge of removing biofilms from the in-place water systems found in 9 clinical environments, a multi-step cleaning (removal of organic material), descaling (removal of inorganic material), and disinfection (removal of microorganisms) process is suggested. The 11 most common current protocols include the following: a) citric acid followed by bleach, b) 12 bleach alone, c) peracetic acid with acetic acid and hydrogen peroxide (PAA), d) citric acid 13 followed by autoclaving, e) citric acid at elevated temperature, f) glycolic acid at elevated 14 temperature, g) hot water, and h) citric acid followed by PAA. All of these disinfection protocols appear to be highly efficient with respect to microbial killing, but were inefficient in 16 reducing the amount of biofilm on affected surfaces.
17 [0081] No treatment thus far has shown complete biofilm removal (and consequently 18 endotoxins) from silicone surfaces. Descaling by itself is inadequate, even at high temperature.
19 Bleach appears to be a relatively good solitary agent for biofilm removal. Additionally, UV
irradiation has been shown to have limited impact on biofilms; and ozone has demonstrated a 21 higher removal efficacy, but limited biofilm killing. It has been postulated that destruction of 22 both the bacteria and associated endotoxins may be possible if super-oxidative concentrations 23 can be achieved ("The Role of Biofilms in Device-Related Infections,"
Ed. By Shirtliff M and 24 Leid JG; Springer-Verlag, Berlin, 2009.).
[0082] Endoscopes 26 100831 In a comparative study of the efficiency of numerous detergents to remove endoscope 27 biofilms, it was determined that "many commonly used enzymatic cleaners fail to reduce the 28 viable bacterial load or remove the bacterial EPS" (Vickery K, Pajkos A, and Cossart Y,"
29 Removal of biofilm from endoscopes: evaluation of detergent efficiency, "Am J Infect Control.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 2004, May; 32(3): 170-176.). Only one cleaner containing no enzymes (produced by Whiteley 2 Medical, Sydney, Australia) significantly reduced bacterial viability and residual bacterial 3 exopolysaccharide matrix.
4 [0084] Noteworthy is U.S. Patent 6,855,678, in which it is disclosed that through the use of scanning electron microscopy, it has been observed that biofilm consists of a number of layers 6 and most importantly, there exists a thin layer of biofilm which is adjacent and attaches tightly to 7 the surface of medical apparatus. The treatment formulation advocated herein includes in 8 combination surfactants, solvents, co-solvents, nitrogen containing biocide, and organic 9 chelating agents. This composition provides a simple non-corrosive, near neutral chemical detergent product that reliably cleans and disinfects endoscopes and other-medical apparatus.
11 The hypothesized method of action is that a) the solvent and co-solvent (example solvents 12 include low molecular weight polar water soluble solvents such as primary and secondary 13 alcohols, glycols, esters, ketones, aromatic alcohols, and cyclic nitrogen solvents containing 8 or 14 less carbon atoms, example co-solvents include low molecular weight amine, amide, and methyl and ethyl derivatives of amides) act to swell the biofilm, b) the organic chelating agent in 16 combination with the surfactant increases the ability of the nitrogen containing biocide to 17 penetrate the biofilm, and c) the organic chelating agent in combination with the nitrogen 18 containing biocide act to work synergistically to dislodge the biofilm and/or kill the 19 microorganisms therein.
[0085] Contact Lenses 21 [0086] Various cleaning solutions were tested against bacterial biofilms on contact lens storage 22 cases, including quaternary ammonium compounds, chlorhexidine gluconate, and hydrogen 23 peroxide 3%. Hydrogen peroxide 3% was most effective in inactivating 24 hr-old biofilms 24 formed by Pseudomonas aeruginosa, Staphylococcus epidermidis, and Streptococcus pyogenes.
Biofilm of Candida albicans was highly resistant to all of these treatments, and Serratia 26 marcescens could grow in chlorhexidine disinfectant solutions (Wilson LA., Sawant AD, and 27 Ahearn DG, "Comparative efficacies of soft contact lens disinfectant solutions against microbial 28 films in lens cases," Arch. Ophthalmol., 1991; 109: 1155-1157.); (Gandhi PA, Sawant AD, 29 Wilson LA, and Ahearn DG, "Adaptation and growth of Serratia marcescens in contact lens CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 disinfectant solutions containing chlorhexidine gluconate," Appl.
Environ. Microbiol. , 1993; 59:
2 183-188.). It has been found that sodium salicylate decreased initial bacterial adherence to lenses 3 and lens cases (Farber BF, His-Chia H, Donnenfield ED, Perry HD, Epstein A, and Wolff A, "A
4 novel antibiofilm technology for contact lens solutions," Ophthalmology, 1995; 102: 831-836.).
100871 Dental Unit Water Lines 6 100881 Dental unit water lines are ideal for colonization with aquatic bacteria and biofilm 7 formation due to their small diameter, very high surface-to-volume ratio, and relatively low flow 8 rates. Currently used flushing as treatment for reducing planktonic bacterial load that originates 9 from the tubing biofilm, does not provide sufficient results, and flushing alone is ineffective (Santiago JI, Huntington MK, Johnston AM, Quinn RS, and Williams JF, "Microbial 11 contamination of dental unit waterlines: short-and long-term effects of flushing," Gen. Dent., 12 1994; 42: 528-535.). Added povidone-iodine reduced contamination between 4 and 5 log fewer 13 bacteria per ml initially, but the levels returned to pretreatment within 22 days (Mills SE, 14 Lauderdale PW, and Mayhew RB, "Reduction of microbial contamination in dental units with povidone-iodine 10%," JADA, 1986; 113: 280-284.). Treatment with 0.5 to 1 ppm free chlorine 16 for 10 min. each day reduced normal bacterial counts by 2 logs from pretreatment levels, but the 17 counts increased again after chlorination was discontinued (Feigin RD
and Henriksen K, 18 "Methods of disinfection of the water system of dental units by water chlorination," J. Dent.
19 Res., 1988; 67: 1499-1504.). Chlorination with bleach (1:10 solution) of water systems already contaminated with bacterial biofilms was ineffective in removing them (Murdoch-Kinch CA, 21 Andrews NA, Atwan S, Jude R, Gleason MJ, and Molinari JA, "Comparison of dental water 22 quality management procedures," JADA, 1997; 128: 1235-1243.).
23 100891 Biofilms in Industrial Applications (Pipelines, Marine Biofouling, Food Sanitation, and 24 HVAC) [0090] Industrial systems suffer a number of deleterious effects due to the presence of biofilms.
26 For heating and cooling systems, as well as oil, water, and gas distributions systems, these 27 effects include flow restrictions in pipelines, flow contamination, and corrosion. For marine 28 systems such as ships, biofouling of hulls can lead to tremendous loss of ship fuel efficiency 29 owing to increased drag of the hull.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 100911 Current Approaches for Treating Biofilms in Water, Oil and Gas Distribution Systems 2 100921 In industrial systems for the distribution of water, oil, and gas, biofilms can form heavy 3 biomass that can reduce the effective diameter of a pipe or other conduit at a particular point or 4 increase friction along the flow path in the conduit. This increases resistance to flow through the conduit, reduces the flow volume, increases pump power consumption, decreasing the efficiency 6 of industrial operations. Further, this biomass can serve as a source of contamination to flowing 7 water or oil. Additionally, most biofilms are heterogeneous in composition and structure which 8 leads to the formation of cathodic and anodic sites within the underlying conduit metal thereby 9 contributing to corrosion processes.
100931 Currently, for pipeline treatment of biofilms, there is a trend to use strong oxidizing 11 biocides such as chlorine dioxide in cooling systems and ozone in water distribution systems 12 since low levels of chlorine have been found to be ineffective against biofilms. Also, a number of 13 non-oxidizing biocides are available, which are effective but their long-term effects on the 14 environment are still unclear. New techniques for biofilm control, such as ultrasound, electrical fields, hydrolysis of EPS and methods altering biofilm adhesion and cohesion are still in their 16 infancy at the laboratory level and are yet to be successfully demonstrated in large industrial 17 systems (Sriyutha Murthy P and Venkatesan R, "Industrial Biofilms and Their Control". In:
18 Marine and Industrial Biofouling; Editors: Fleming H, Murthy P, Venkatesan R, and Cooksey K;
19 Springer-Verlag, 2010.).
[0094] One of the major economic losses faced by the oil and gas companies is due to pipeline 21 corrosion. The internal corrosion of the pipelines is basically caused by sulfate reducing bacteria 22 (SRB). SRB are anaerobic and responsible for most instances of accelerated corrosion damage.
23 For biofilms created by SRB, some newer strategies include the use of:
a) calcium or sodium 24 nitrates which encourage more benign nitrate reducing bacteria to compete with SRB, b) molybdate as a metabolic inhibitor preventing sulfate reduction, c) anthraquinone which 26 prohibits sulfide production and its incorporation into the biofilm, and d) dispersants such as 27 filming amine technology which prevent biofilm adhesion. Also, since there is no continuous 28 water phase in oil pipelines (under typical flow conditions) by which to dose bactericides, the use CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 of water-oil emulsions have been suggested ("Petroleum Microbiology";
edited by 011ivier B and 2 Magot M, ASM Press, 2005.).
3 [0095] An example of the more recent biofilm altered adhesion concepts includes the disclosure 4 of International Patent Application PCT/US2006/028353 describing a non-toxic, peptide-based biofilm inhibitor that prevents Pseudomonas aeruginosa colonization of stainless steel (and likely 6 a wide variety of other metal surfaces) and non-metalic surfaces. The compositions and methods 7 describe a very high affinity peptide ligand that binds specifically to stainless steel and other 8 surfaces to prevent Pseudomonas biofilm formation. Another example of an inhibitor of biofilm 9 adhesion is the technology being developed by Australian firm BioSignal Ltd. involving the use of furanones from the red seaweed Delisea pulchra, which effectively avoids a broad spectrum of 11 bacterial infections without inciting any bacterial resistance to its defensive chemistry. Furanones 12 produced by this seaweed, bind readily to the same specific protein-covered bacterial receptor 13 sites that receive the bacterial signaling molecules (N-acyl homoserine lactone) which normally 14 induce surface colonization. BioSignal Ltd. is targeting the use of synthetic furanones to block bacterial communication and thereby prevent bacteria from forming groups and biofilms in 16 applications including pipelines, HVAC, and water lines treatment.
17 [0096] Methods of Decontamination of Food Processing, Storage, and Transport Systems in the 18 Food Iindustry 19 10097] In addition to the more conventional means of decontamination discussed above for other industrial applications, recently, the food industry has embarked upon the use of enzyme-based 21 schemes that have been carried over from the bio-processing of food stuffs. Specifically, efforts 22 have been undertaken to find ways to enzymatically degrade the EPS
itself and thereby 23 contribute to the removal of biofilms. Largely, these efforts have been directed at destruction of 24 the polysaccharide framework of the EPS. A premier example is found in the U. S. Patent Application 20110104141 to Novozyme which discloses the use of alpha-amylase as a primary 26 enzyme for the breakdown of biofilm polysaccharides with the potential inclusion of additional 27 enzymes such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, 28 chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-29 galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 haloperoxidase, invertase, laccase, lipase, mannosidase, oxidoreductases, pectinolytic enzyme, 2 peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, 3 transglutaminase, or xylanase. Products such as Biorem produced by Realco in coordination 4 with Novozyme to target applications in the food and beverage industry exploit a two step cleaning process that invokes use of this kind of multienzyme mixture followed by application of 6 a biocide.
7 [0098] In this industrial sector also, ultrasound has been found a useful tool; for sanitary control, 8 it was found that the combination of chelating agents with ultrasound has been useful for 9 removing selected biofilm-producing pathogens from metal surfaces (Oulahal N, Martial-Gros A, Bonneau M and Blum U, "Combined effect of chelating agents and ultrasound on biofilm 11 removal from stainless steel surfaces. Application to "Escherichia coli milk" and 12 "Staphylococcus aureus milk" biofilms", Biofilms, 2004; 1: 65-73, Cambridge University 13 Press.). The efficacy of such ensonification has been shown to exhibit dependency on the 14 frequency and duty cycle of the energy (Nishikawa T, et al., "A study of the efficacy of ultrasonic waves in removing biofilms," Gerontology, Sept 2010; Vol 27, Issue 3: 199-206.).
16 [0099] Current Methods for Treating Marine Biofouling 17 [00100] Biofouling occurs worldwide in various industries and one of the most common 18 biofouling sites is on the hulls of ships, where barnacles are often found. A significant problem 19 associated with biofilms on ships is the eventual corrosion of the hull, leading to the ship's deterioration. However, before corrosion occurs, organic growth can increase the roughness of 21 the hull, which will decrease the vessel's maneuverability and increase hydrodynamic drag.
22 Ultimately, biofouling can increase a ship's fuel consumption by as much as 30%. Parts of a 23 ship other than the hull are affected as well: heat exchangers, water-cooling pipes, propellers, 24 even the ballast water. Fishing and fish farming are also affected, with mesh cages and trawls harboring fouling organisms. In Australia, biofouling accounts for about 80%
of the pearling 26 industry's costs (Stanczak M, "Biofouling: It's Not Just Barnacles Anymore," CSA Discovery 27 Guide, 2004; http://www.csa.com/discoveryguides/biofoul/overview.php.).
28 [00101] The traditional method of control is to coat exposed surfaces with an anti-fouling 29 compounds. Most of these compounds rely on copper and tin salts that gradually leach from the CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 coating and contaminate the surrounding environment. One of the most widely used coatings to 2 date has been tributyl tin (TBT) which is highly toxic to marine organisms. Since it has been 3 found to have unwanted side-effects on non-target organisms, a world-wide ban on its use was 4 instituted in 2008. The race is on for an environmentally sound alternative (Scottish Association for Marine Science, http://www.sams.ac.uk/research/departments/microbial-molecular/mmb-6 proj ect-themes/algal-biofilms.).
7 [00102] Hence, in maritime applications such as shipping, there is an unmet need for viable, 8 cost-effective biofilm remediation.
9 [00103] Current Methods for Treating Biofims in Heating, Ventilation and Air-Conditioning (111/A C) and Refrigeration Systems 11 [00104] HVAC and refrigeration systems encounter problems associated with biofilms formed 12 on cooling coils, drain pans, and in duct work subjected to water condensation. Biofilm 13 formation on cooling coils diminishes heat exchange efficiency; its growth on other surfaces, 14 including drain pans and duct work, is a source of contamination in the air stream. Conventional methods of addressing biofilms in these applications include maintenance cleaning of coils, duct 16 work and drain pans, use of anticorrosion and antimicrobial coatings on system surfaces, and the 17 exposure of system surfaces to C-band ultraviolet light to break down biofilms and kill 18 pathogens.
19 1001051 Remediation of Biofilm Contamination in Household Applications [00106] The household products industry is vitally concerned with disinfection of household 21 surfaces, water and plumbing systems, and human hygienic needs.
Difficulties associated with 22 killing bacteria attached to these diverse surfaces are well known in this industrial sector and 23 considerable research currently is directed at developing products which kill or remove biofilms.
24 [00107] An innovation in this sector is probiotic-based cleaning. Some versions of these products lay down layers of benign bacteria that successfully compete with pathogenic bacteria 26 for resources on kitchen and bathroom surfaces. Other such products combine enzymes with 27 probiotic bacteria to digest biofilms and dead pathogens. A leading example of this class of 28 products is PIP produced by Chrisal Probiotics of the Netherlands.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00108] The conventional approaches to treatment of biofilm discussed for both medical and 2 industrial applications variously have been unproven, of limited effectiveness, time consuming, 3 costly in cases where large surface areas are involved or surfaces require repeated treatment, and 4 newer concepts have yet to demonstrate effectiveness and scalability to field applications.
Hence, there remains an urgent need for more effective and less costly methods to treat biofilms.
6 The present compositions and method offer the prospect of a new standalone approach to biofilm 7 treatment with higher efficacy and lower cost, with additional potential for augmenting certain 8 conventional treatments while reducing the costs of such treatments.

Summary 11 [00109] Trehalose (a universal general stress response metabolite and an osmoprotectant) can 12 play an important role in the formation and development of microbial biofilm and the specific 13 interactions of trehalose with water can be considered to be one of the most important 14 mechanisms of biofilm formation. The present compositions and methods have been conceived to target trehalose degradation as a key step in degrading biofilm.
16 [00110] In various embodiments of the compositions and methods, compounds that prevent, 17 degrade, and/or inhibit the formation of biofilms, compositions comprising these compounds, 18 devices exploiting these compounds, and methods of using the same are disclosed.
19 100111] Because trehalose serves to manipulate hydrogen bonds among water molecules and bacterial cells in the process of forming the biofilm gel, the degradation of trehalose ultimately 21 should result in degradation of the biofilm gel. A class of compounds that degrade trehalose 22 with high specificity, thereby degrading the biofilm matrix gel is disclosed. Specifically, the 23 naturally occurring enzyme trehalase will hydrolyze a molecule of trehalose into two molecules 24 of glucose. The small amount of enzyme trehalase produced in the human body must be augmented with the administration of much larger amounts to treat in vivo biofilm-based 26 infections. Various treatment formulations that incorporate trehalase enzymes and associated 27 delivery mechanisms are detailed for specific types of infections; these include systemic and 28 local treatment protocols. Additionally, trehalase-containing mixtures and associated processes 29 are disclosed to degrade biofilms present on medical instruments and to mitigate biofilm fouling CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 and biofilm-based biocorrosion for industrial applications. For degrading biofilms on medical 2 equipment, trehalase-containing mixtures can be used in concert with other processes, such as 3 ultrasound and ultrasound-assisted enzymatic activity to degrade biofilms. Biofilm prevention 4 approaches comprise the use of trehalase enzymes in surface coatings.
1001121 Following is a lexicon of terms and phrases that more particularly define the 6 compositions and methods and support the meaning of the claims:
7 [00113] Time-delayed release ¨ in the context of the present compositions and methods, time-8 delayed release refers specifically to trehalase (or other compounds) release that occurs at a 9 predetermined approximate time after the trehalase (and in some embodiments, other compounds) in pill, capsule, tablet or other form is ingested orally.
Typically, for the present 11 compositions and methods, the time delay means that the initial release of trehalase (or other 12 compounds) will occur in the small intestine, to avoid degradation by naturally occurring 13 proteolytic enzymes in the upper GI tract. Various pre-programmed temporal profiles for release 14 in the small intestine are within the scope of the compositions and methods, such as, for example, linearly increasing or decreasing rates of release with time, or a constant rate of release.
16 [00114] Sustained release ¨ in the context of the present compositions and methods, it refers 17 to the release of trehalase (or other compounds) for applications external to the body. This is a 18 continuous release of trehalase (or other compounds) that is not time-delayed, but is initiated at 19 first opportunity for the purpose of continuous, ongoing exposure of medical device and industrial surfaces to treatment enzymes.
21 [00115] Sufficient for efficacy ¨ pertains to treatment composition amounts and treatment 22 exposure durations adequate to breakdown the gel structure of biofilm for its dispersal and 23 further penetration by antimicrobial agents to treat the target infectious pathogens.
24 [00116] Trehalase ¨ refers to any enzyme selected from the category of trehalase isoenzymes.
There are two types of trehalase enzymes found in microorganisms: neutral trehalase (NT) 26 typically found in the cytosol and acid trehalase (AT) found in the vacuoles of the cytosol, either 27 of which type may find application in the present compositions and methods. Further, the 28 number of candidate enzymes is large; as many as 541 model variants (isoenzymes) of trehalase 29 can be found in the Protein Model Portal (http://www.proteinmodelportal.org), each exhibiting CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 varying potencies in the hydrolysis of trehalose into glucose. The present compositions and 2 methods anticipate a selection from among these isoenzymes that is optimized for the specific 3 biofilm application. For example, the ability to sufficiently purify a given isoenzyme for internal 4 bodily use may favor its selection for this purpose over another isoenzyme that exhibits higher enzymatic activity, but which would be relegated to industrial applications.
6 [00117] Digestive enzymes - are enzymes that break down polymeric macromolecules of 7 ingested food into their smaller building blocks, in order to facilitate their absorption by the 8 body. In the present icompositions and methods, treatment formulations comprising trehalase (or 9 other compounds) are disclosed which should: a) avoid degradation by the digestive enzymes naturally occurring in the upper GI tract and b) be combined in time-delayed release form with 11 digestive enzyme supplements to avoid degradation by proteolytic enzymes in such supplements.
12 [00118] Medical devices - comprise devices that are installed either temporarily or 13 permanently in the body and medical instruments that may or may not contact the body, but at 14 least contact tissue or bodily fluids. Examples of temporarily installed medical devices include catheters, endoscopes, and surgical devices. Permanent devices examples include devices such as 16 orthopedic implants, stents, and surgical mesh. Examples of devices used external to the body 17 include stethoscopes, dialysis machines, and blood and urinary analysis instruments. Each of the 18 aforementioned devices exhibit surfaces that are vulnerable to biofilm formation and therefore 19 can benefit from treatment by specific embodiments of the presently disclosed compositions and methods.
21 [00119] Antimicrobials - are substances that kill or inhibit the growth of microorganisms such 22 as bacteria, fungi, or protozoans. Antimicrobials either kill microbes (microbiocidal) or prevent 23 the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-24 living objects or outside the body.
[00120] Other saccharidases (enzymes hydrolyzing saccharides) - include various di-, oligo-, 26 and polysaccharidases.
27 1001211 Living organisms ¨ pertains to the spectrum of living entities from microbes to 28 animals and humans.
CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00122] GI tract ¨ refers to the gastrointestinal tract; the upper GI tract comprising the mouth, 2 esophagus, stomach, and duodenum, and the lower GI tract comprising the small and large 3 intestines.
4 [00123] Administering via the GI tract - relates to three main alternative treatment delivery methods: first is oral administration in which the treatment compounds are administered via the 6 mouth; for the patients that may not be able to receive treatment by mouth, the second method 7 available is by the naso-gastric tube; and a third method includes delivery by colonic irrigation.
8 [00124] Administering via systemic use ¨ relates to administration of treatment compounds by 9 percutaneous injection, intramuscular injection, intra-venous injection, and venous catheter administration.
11 1001251 Other aspects, advantages, and features of the present disclosure will become 12 apparent after review of the entire application, including the following sections: Brief 13 Description of the Drawings, Detailed Description, and the Claims.

Brief Description of the Drawings 16 [00126] FIG. 1A is diagram of the chemical structure of the dissacharide trehalase.
17 [00127] FIG. 1B is a pictorial diagram of the backbone structure of trehalase.
18 1001281 FIG. 2A is a ribbon model pictorial diagram of an enzyme of trehalase derived from 19 Sacharomyces cerevi siae.
[00129] FIG. 2B is a ribbon model pictorial diagram of an enzyme of trehalase derived from 21 Penicillium marneffei.
22 1001301 FIG. 2C is a ribbon model pictorial diagram of an enzyme of trehalase derived from 23 Homo sapiens.
24 [00131] FIG. 2D is a ribbon model pictorial diagram of an enzyme of trehalase derived from Candida albicans.
26 1001321 FIG. 3 is a summary chart showing the biofilm produced by P.
Aeruginosa PA01 in 27 accordance with a non-limiting example.
28 [00133] FIG. 4 is a summary chart of the biofilm produced by S. Aureus ATCC25923 in 29 accordance with a non-limiting example.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00134] FIG. 5 is another summary chart of the biofilm produced by S.
Aureus ATCC25923 in 2 accordance with a non-limiting example.
3 [00135] FIG. 6 is another summary chart of the biofilm produced by P.
Aeruginosa PA01 in 4 accordance with a non-limiting example.
1001361 FIG. 7 is a summary chart for the results of the MIC determination with selected S.
6 Aureus strains in accordance with a non-limiting example.
7 [00137] FIG. 8 is a summary table for the results of the screening of clinical isolates in 8 accordance with a non-limiting example.
9 1001381 FIGS. 9A through 9C are charts for the results of the Trehalase testing on clinical isolates where the biomass has crystal violet staining in accordance with a non-limiting example.
11 1001391 FIGS. 10A through 10C are charts for the results of the Trehalase testing on clinical 12 isolates for cell viability with resazurin staining in accordance with a non-limiting example.
13 [00140] FIGS. 11A through 11C are charts for the results showing the effectiveness of 14 Trehalase added during biofilm growth on a catheter segment up to 24 hours in accordance with a non-limiting example.
16 [00141] FIGS. 12A through 12C are charts for the results of the Trehalase added after 24 17 hours biofilm growth on a catheter segment in accordance with a non-limiting example.
18 1001421 FIG. 13 is a chart listing the planktonic cell counts for an initial pH of 5.5 and 19 different treatments of Trehalase and THPS alone or in combination.
[00143] FIG. 14 is a chart similar to that shown in FIG. 13, but listing the planktonic cell 21 counts for an initial pH 7Ø
22 1001441 FIG. 15 is a chart showing the sissle cell count versus different treatments for 23 Trehalase and THPS either alone or in combination at an initial culture medium of pH 5.5.
24 [00145] FIG. 16 is another chart similar to that shown in FIG. 15 showing the sissle cell count versus the different treatments, but for an initial culture medium pH of 7Ø
26 [00146] FIG. 17 is a chart showing the weight loss of carbon steel coupons after a 7-day 27 biofilm prevention test using the different treatments.
28 [00147] FIG. 18 is a chart showing the pit depth of carbon steel coupons after a 7-day biofilm 29 prevention test using the different treatments.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00148] FIGS. 19A trough 19K are confocal laser scanning microscope images of sulfate 2 reducing bacteria biofilms on carbon steel coupons after a 7-day biofilm prevention test using the 3 different treatments.
4 [00149] FIGS. 20A through 20K are scanning electron microscope images of sulfate reducing bacteria biofilms on carbon steel coupons after a 7-day biofilm prevention test using the different 6 treatments.
7 [00150] FIGS. 21A through 21K are scanning electron microscope images of corrosion pits on 8 carbon steel surfaces after a 7-day biofilm prevention test using the different treatments.

Detailed Description 11 [00151] Since any bacterial biofilm can be defined as a living dynamic structure with spatial 12 and temporal heterogeneity for both components of microbial biofilm (the exopolymer gel matrix 13 and the microcolonies of infectious microorganisms embedded in this gel matrix), the treatment 14 approaches for biofilm-based chronic infections should be aimed simultaneously at both components of microbial biofilm: prevention of formation, inhibition of growth and degradation 16 of biofilm gel matrix, and killing the infectious pathogens embedded in the biofilm gel matrix. In 17 this context, trehalase should be included into existing approaches for prevention and treatment 18 of microbial biofilms, being used in combination with existing natural and synthetic 19 amtimicrobials and anti-biofilm substances and methods of their use to increase their effectiveness. A review of recent patent literature summarizes citations under six categories of 21 current treatment approaches: 1) antibiotics and small molecule inhibitors of new and established 22 biofilms and biofilm-forming infectious pathogens, 2) quorum sensing and signaling molecules 23 inhibitors, 3) surface coating substances for inhibition of biofilm formation on medical devices 24 and equipment, 4) antibodies and vaccines for infectious biofilm treatment, 5) enzymes for degrading biofilms, and 6) bacteriophage treatment of infectious biofilms (Lynch AS and 26 Abbanat D, "New antibiotic agents and approaches to treat biofilm-associated infections," Expert 27 Opin. Ther. Patents, 2010; 20(10): 1373-1387.).
28 [00152] All microorganisms in their natural environments encounter a multitude of stresses, 29 including osmotic stress, oxidative stress, membrane and cell envelope stress, ribosomal stress, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 nutrient and oxygen limitations, temperature stress, and many other stresses as characteristics of 2 various specific environmental conditions. Infectious pathogens encounter the same stresses in 3 their environment ¨ the internal milieu of their host (including human body). The infectious 4 process of biofilm-based infections in the human body should be considered as a constant battle between infectious microorganism (for its adaptation, survival and proliferation in the milieu of a 6 human body) and a host who uses all its natural defense mechanisms to kill the pathogen and 7 eliminate it from the body, restore normal homeostasis, and repair damaged body tissues. The 8 exposure to various stresses, impact bacterial susceptibility to a variety of antimicrobials through 9 their initiation of stress responses that recruit various resistance determinants or promote physiological changes that compromise antimicrobial activity (Keith Poole, "Bacterial stress 11 responses as determinants of antimicrobial resistance", J Antimicrob Chemother 2012; 67: 2069 12 ¨ 2089.). One of the most important survival mechanisms for any bacteria in any environment is 13 the general stress response (ubiquitous in nature) triggered by various environmental stresses and 14 their interactivities via genetic regulation. In general stress response, the increased production of trehalose (as a general stress response metabolite and an osmoprotectant) plays an important role 16 in adaptation and survival of infectious pathogen in the host body, and plays an important role in 17 antimicrobial resistance as well.
18 1001531 Trehalose is a disaccharide that is ubiquitous in the biosphere and present in almost 19 all forms of life except mammals. In various bacteria and fungi, it is one of the most important storage carbohydrates, serving as a source of energy and as a carbon source for synthesis of 21 cellular components; it can play a transport role and control certain metabolic pathways; it 22 functions as a protectant for cell membranes and cell proteins against the adverse effects of 23 various stresses, including the osmotic stress, heat, cold, desiccation, dehydration, deprivation of 24 nutrients, oxidation, and anoxia (Elbein AD, "The metabolism of a, a-trehalose," Adv.
Carbohyd. Chem. Biochem., 1974; 30: 227-256.); (Crowe J, Crowe L, and Chapman D, 26 "Preservation of membranes in anhydrobiotic organisms. The role of trehalose," Science, 1984;
27 223: 209-217.); (Takayama K and Armstrong EL, "Isolation, characterization and function of 6-28 mycoly1-6'acetyltrehalose in the H37Rv strain of Mycobacterium tuberculosis," Biochemistry, 29 1976; 15: 441-446.); (Christopher Askew, Adnane Sellam, Ellias Epp, Herve Hogues, Alaka CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Mullick, Andre Nantel, Malcolm Whiteway, "Transcriptional regulation of carbohydrate 2 metabolism in the human pathogen Candida albicans", PLoS Pathogens 2009-10-01.); (Joke 3 Serneels, Helene Tournu, Patrick Van Dijck, "Tight control of trehalose content is required for 4 efficient heat-induced cell elongation in Candida albicans", Journal of Biological Chemistry, 2012-10-26.).
6 1001541 Trehalose may be partially responsible for the virulence and antimicrobial resistance 7 properties in various opportunistic and pathogenic microorganisms, including those known to 8 cause chronic infections with biofilm formation in the human body, including: Pseudomonas 9 spp., Bacillus spp., Staphylococci spp., Streptococci spp, Haemophilus influenza, Klebsiella pneumoniae, Proteus spp., Mycobacteria spp., Corynebacteria spp., Enterococci spp., 11 enteropathogenic E. coli, various human pathogenic yeasts and fungi (Candida spp., 12 Cryptococcus neoformansõ Aspergillus spp.). As demonstrated in some strains of Candida 13 albicans, interference with the production of trehalose strongly reduces their virulence.
14 Specifically, C. albicans mutants with deleted gene TSP2, which encodes trehalose-6-phosphate phosphatase, one of two enzymes involved in trehalose synthesis, exhibited diminished virulence 16 in in vivo mouse model of systemic infection and, being grown within in vitro biofilm systems, 17 displayed significantly less biofilm formation than selected non-mutant strains (Coeney T, Nailis 18 H, Tournu H, Van Dick P, and Nelis H, "Biofilm Formation and Stress Response in Candida 19 Albicans TSP2 Mutant," ASM Conference on Candida and Candidiasis, Edition 8, Denver, CO;
March 12-17, 2006.). The in vivo studies in the pathobiology of Cryptococcus neoformans, 21 identified the presence of a functioning trehalose pathway during experimental infection in the 22 mouse and rabbit models and suggested its importance for C. neoformans survival in the host;
23 using created null-mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-24 phosphate phosphatase (TP52), and neutral trehalase (NTH1) genes, it was demonstrated that both TPS1 and TP,S2 are required for high-temperature (37 C) growth and glycolysis, but the 26 block at TP,S2 results in the apparent toxic accumulation of T6P, which makes the enzyme 27 trehalose-6-phosphate phosphatase a fungicidal target (Elizabeth Wills Petzold, Uwe 28 Himmelreich, Eleftherios Mylonakis, Thomas Rude, Dena Toffaletti, Gary M. Cox, Jackie L.
29 Miller, and John R. Perfect, "Characterization and Regulation of the Trehalose Synthesis CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans", Infection and 2 Immunity, Oct. 2006, p. 5877-5887.).
3 [00155] All microorganisms can synthesize trehalose intracellularly and/or take it from the 4 environment using various synthesis and degradation pathways for trehalose metabolism. The specific use of these pathways by various microorganisms depends on their genetic program for 6 trehalose utilization and availability of substrates for trehalose biosynthesis in their environment.
7 [00156] Many microorganisms, including human pathogenic bacteria and fungi, synthesize 8 trehalose intracellularly mostly via pathways that utilize various nucleoside diphosphate glucose 9 derivatives as glucosyl donors (ADP-D-glucose, CDP-D-glucose, GDP-D-glucose, TDP-D-glucose and UDP-D-glucose) and a-D-glucose-6-phosphate in a two-step reaction:
in the first 11 step ¨ formation of intermediate metabolite trehalose 6¨phosphate (T6P) by the action of enzyme 12 trehalose-6-phosphate synthase (TPS), and in the second step - formation of final product 13 trehalose (a, a ¨ trehalose), by the action of enzyme trehalose-6-phosphate phosphatase (TPP) 14 (Styrvold OB and Strom AR, "Synthesis, accumulation, and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the 16 periplasmic trehalase," J. Bacteriol, 1991; 173(3): 1187-1192. PMID:
1825082). It should be 17 mentioned that some mycobacteria, such as Mycobacterium smegmatis and Mycobacterium 18 tuberculosis, possessing unusual trehalose-6-phosphate synthases, are capable of utilizing all five 19 nucleoside diphosphate glucose derivatives as glucosyl donors (Lapp D, Patterson BW, Elbein AD, "Properties of a trehalose phosphate synthetase from Mycobacterium smegmatis. Activation 21 of the enzyme by polynucleotides and other polyanions," J. Biol. Chem., 1971; 246 (14): 4567-22 4579.).
23 1001571 Also, many microrganisms, can synthesize trehalose directly from disaccharide 24 maltose (degradation product of glycogen and starch) independently of the presence of phosphate compounds trehalose-6-phosphate and glucose-6-phosphate. This pathway involves the 26 intramolecular rearrangement of maltose (glucosyl-alphal,4-glucopyranoside) to convert the 1,4-27 linkage into the 1,1-linkage of trehalose by the action of enzyme trehalose synthase (TS), 28 forming free trehalose (a, a ¨ trehalose) as the initial product. It is postulated that in 29 Corynebacterium glutamicum this pathway may work in the opposite direction, compensating CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 for the absence of a trehalase enzyme, by converting excess trehalose back into maltose, for 2 reuse as a carbon source (De Smet KA, Weston A, Brown IN, Young DB, Robertson BD, "Three 3 pathways for trehalose biosynthesis in mycobacteria," Microbiology, 2000;
146 (Pt 1): 199-208.
4 PMID: 10658666); (Wolf A, Cramer R, Morbach S, "Three pathways for trehalose metabolism in Corynebacterium glutamicum ATCC 13032 and their significance in response to osmotic 6 stress," Mol Microbiol, 2003; 49(4): 1119-1134. PMID: 12890033.).
7 [00158] In an additional pathway, trehalose can be formed from polysaccharides (such as 8 glycogen or starch) in multi-step process by the action of several enzymes: in the first step - the 9 enzyme isoamylase hydrolyzes the a-1,6-glucosidic linkage in glycogen or the a-1,4-glucosidic linkages in other polysaccharides (such as starch from plants), to produce a maltodextrin 11 (oligosaccharide); in the next step ¨ the enzyme maltoolgosyl-trehalose synthase (MTS) converts 12 maltodextrin to maltooligosyl-trehalose by forming an a, a-1,1-glucosidic linkage via 13 intermolecular transglucosylation; and in the third step ¨ the enzyme maltooligosyl-trehalose 14 trehalohydrolase (MTTH) hydrolyzes the product, forming free trehalose (a, a ¨ trehalose) and a maltodextrin which becomes shorter by two glucosyl residues. In Corynebacterium glutamicum, 16 which possess three different pathways for trehalose biosynthesis, this is the main route for 17 trehalose biosynthesis (Maruta K, Mitsuzumi H, Nakada T, Kubota M, Chaen H, Fukuda S, 18 Sugumoto T, Kurimoto M, "Cloning and sequencing of a cluster of genes encoding novel 19 enzymes of trehalose biosynthesis from thermophilic archaebacterium Sulfolobus acidocaldarius," Biochim Biophys Acta, 1996; 129 (3): 177-181. PMID:
8980629.).
21 [00159] For degradation of trehalose, various microorganisms, including human pathogenic 22 bacteria and fungi, can utilize several alternative pathways. Unmodified trehalose (a, a ¨
23 trehalose) may be degraded by a hydrolyzing enzyme trehalase (a, a ¨
trehalohydrolase), yielding 24 two 3-D-glucose molecules, or it may be split by the action of the enzyme trehalose phosphorylase (TP), yielding 3-D-glucose-6-phosphate as the end product.
Trehalose 26 phosphorylase (TP), can also catalyze the reversible synthesis and degradation of trehalose 27 from/to al3-D-glucose-1-phosphate and 3-D-glucose, or a-D-glucose-1-phosphate and a-D-28 glucose. Phosphorylated form, trehalose-6-phosphate, can be either hydrolyzed by trehalose-6-29 phosphate hydrolase, yielding13-D-glucose and13-D-glucose-6-phosphate, or degraded by CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalose-6-phosphate phosphorylase, yielding 0-D-glucose-1-phosphate and 0-D-glucose-6-2 phosphate. All end products of the degradation pathways can be metabolized via glycolysis. All 3 end products of trehalose degradation pathways can be metabolized via glycolysis. (Helfert C, 4 Gotsche S, Dahl MK, "Cleavage of trehalose-phosphate in Bacillus subtilis is catalyzed by a phospho-alpha-(1-1)-glucosidase encoded by the TreA gene," Mol Microbiol, 1995; 16(1): 111-6 120. PMID: 7651129.); (Levander F, Andersson U, Radstrom P, "Physiological role of beta-7 phosphoglucomutase in Lactococcus lactis," Appl Environ Microbiol, 2001;
67(10): 4546-4553.
8 PMID: 11571154.).
9 1001601 For survival in the live environment of a human body, the pathogenic microorganisms must continuously adapt to temporal and spatial fluctuations in osmolarity of body fluids. In 11 osmoadaptation, bacteria constitutively use the universal mechanism of uptake and release of 12 osmotically active compounds (osmolytes). Bacteria adapt to the conditions of increased external 13 osmolarity by importing charged ions from the environment, and importing or synthesizing 14 compatible solutes. Upon a shift to a low-osmolarity media, the excretion of osmolytes is required to restore normal turgor and prevent the cells from bursting. The pathways for import 16 and efflux of compatible solutes include PTS system, ABC transporters, mechanosensitive 17 channels, and porins (Berrier CM, Besnard M, Ajouz B, Coulombe A, and Ghazi A, "Multiple 18 mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied 19 pressure," J. Membr. Biol., 1996; 151: 175-187.); (Bremer R and Kraemer R, "Coping with osmotic challenges: osmoregulation through accumulation and release of compatible solutes in 21 bacteria," pp. 79-97. In G. Storz and R.Hengge-Aronis (ed.), Bacterial stress responses. 2000;
22 ASM Press, Washington, D.C.); (Chang G, Spencer R, Lee AT, Barclay MT, and Rees DC, 23 "Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive 24 ion channel," Science, 1998; 282: 2220-2225.); (Morbach S and Kraemer R, "Body shaping under water stress: osmosensing and osmoregulation of solute transport in bacteria,"
26 ChemBioChem, 2002; 3: 384-397.).
27 1001611 Compatible solutes are small, zwitterionic, highly soluble organic molecules, which 28 include diverse substances, such as amino acids (proline,glutamate), amino acid derivatives 29 (glycine betaine, ectoine), and sugars (trehalose and sucrose), that are thought to stabilize CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 proteins and lead to the hydration of the cell (Steator RD and Hill C, "Bacterial osmoadaptation:
2 the role of osmolytes in bacterial stress and virulence," FEMS Mocrobiol.
Rev., 2002; 26:
3 49-71.). Various bacteria may prefer different osmolytes taken from the environment, but all of 4 them constitutively utilize trehalose (taken from the environment or synthesized intracellularly) as a universal osmoprotectant. For example, E. coli and Vibrio Cholerae in human GI tract prefer 6 glycine betaine, but its synthesis relies on an external supply of proline, betaines, or choline 7 which may not be readily available in the environment or significantly reduced in the deeper 8 layers of microbial biofilm. When these compounds are not available, a microbial cell can 9 achieve a moderate level of osmotic tolerance by accumulation of glutamate and trehalose (Styrvold OB, Strom AR, "Synthesis, accumulation, and excretion of trehalose in osmotically 11 stressed Escherichia coli K-12 strains: influence of amber suppressors and function of 12 periplasmic trehalase," J Bacteriol, 1991; 173 (3): 1187-1192. PMID:
1825082.); (Kapfhammer 13 D, Karatan E, Pflughoeft KJ, and Watnik PI, "Role for Glycine Betaine Transport in Vibrio 14 cholera Osmoadaptation and Biofilm Formation within Microbial Communities," Applied and Environmental Microbiology, July 2005: 3840-3847.).
16 [00162] As demonstrated in laboratory-grown bacteria, the first adaptive response to osmotic 17 stress comprises both the increased uptake rate and the amount of cytosolic potassium, followed 18 by the accumulation of glutamate and synthesis of trehalose (Dinnbier U, Limpinsel E, Schmid 19 R, and Bakker EP, "Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium 21 chloride concentrations," Arch. Microbiol., 1988; 150: 348-357.);
(McLaggan D, Naprstek J, 22 Buurman ET, and Epstein W, "Interdependence of K and glutamate accumulation during 23 osmotic adaptation of Escherichia coli," J. Biol. Chem., 1994; 269: 1911-1917.); (StromAR and 24 Kaasen I, "Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression," Mol. Microbiol., 1993; 8: 205-210.). The time-dependent (10 to 60 minutes) 26 alterations in the proteome of E. coli (grown under aerobic conditions) in response to osmotic 27 stress, demonstrated upregulated genes for synthesis of both trehalose and cytosolic trehalase 28 (trehalose-degrading enzyme with regulatory properties) in the middle phase (10 to 30 minutes) 29 and in the long phase (30 to 60 minutes) of bacterial adaptation to hyperosmotic stress, with the CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalase synthesis genes (TreF) upregulated in the early phase of adaptation (0 to 10 minutes) 2 (Weber A, Kogl SA, and Jung K, "Time-Dependent Proteome Alterations under Osmotic Stress 3 during Aerobic and Anaerobic Growth in Escherichia coli," Journal of Bacteriology, Oct. 2006:
4 7165-7175. doi: 10.1128/JB.00508-06.).
[00163] Trehalose is a stable dissacharide with glycosidic bond [0-a-D-Glucopyranosyl-(1-6 1)- a-D-glucopyranoside] formed from a condensation between the hydroxyl groups of the 7 anomeric carbons of two molecules of glucose, preventing them from interacting with other 8 molecules and thereby rendering trehalose among the most chemically inert sugars (Birch GG, 9 .. "Trehaloses," Adv. Carbohydr. Chem. Biochem., 1963; 18: 201-225.);
(Elbein AD, "The .. metabolism of alpha, alpha-trehalose," Adv. Carbohydr. Chem. Biochem., 1974; 30: 227-256.).
11 The flexible glycosidic bond, together with the absence of internal hydrogen bonds, yields a 12 supple molecule, but this glycosidic bond does not break easily: the lkcal/mol linkage is highly 13 resilient, enabling the trehalose molecule to withstand a wide range of temperature and pH
14 conditions (Pava CL and Panek AD, "Biotechnological applications of the disaccharide trehalose," Biotechnol. Annu. Rev., 1996; 2: 293-314.). Because of the unusual glycosidic bond 16 between the anomeric carbons (1-1), there are no more accessible carbons for further 17 polymerization, so that trehalose exists only as a disaccharide, being rather distributed as 18 disaccharide molecules in the gel-like matrix of biofilm, influencing its density via interaction 19 between trehalose and water molecules. Intermolecular hydrogen bonds (H
bonds), the strongest of intermolecular forces, are central to trehalose interaction with water.
Specifically, such bonds 21 modify the structure of water surrounding trehalose molecules and account for the self-22 aggregation phenomena of trehalose molecules observed in molecular dynamic simulations and 23 .. supported by experimental studies.
24 [00164] The chemical structure of trehalose is depicted in Figure la indicating an alpha-linked disaccharide formed by an a,a-1,1-glucosidic bond between two a-glucose units.
The backbone 26 .. structure of this enzyme is shown in Figure lb depicting the two planes established by the 27 glucose units.
28 .. [00165] Trehalose has a unique ability to capture water through extensive solvation. Water 29 molecules are arranged in a solvation complex around trehalose molecules, with water CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 associating with trehalose functional groups through H bond formation; at infinite dilution, the 2 solvation number approaches 15 (the highest among all disaccharides).
Trehalose is able to 3 restructure water even at minimum aqueous concentrations, supporting the gelation phenomena 4 in these conditions. With respect to water restructuring behavior, trehalose enhances the hydrogen bonding between water molecules by approximately 2%. This is sufficient to 6 destructure the pure water tetrahedral network in conformity with a restructuring imposed by 7 trehalose clusters. Stronger, more linear, and better optimized H bonds are formed between water 8 molecules, while weaker bonds are relegated to trehalose-water interactions (Sapir L and Harries 9 D, "Linking Trehalose Self-Association with Binary Aqueous Solution Equation of State," J.
Phys. Chem. B, 2011; 115: 624-634.).
11 1001661 Trehalose self-associates in aqueous solutions in a concentration dependent manner to 12 form clusters of increasing size, until finally forming percolating, infinitely connected, clustering 13 networks (at concentrations of 1.75 M and higher), affecting the dynamic properties of the 14 solution. The lack of intramolecular hydrogen bonds in trehalose, compared with other disaccharides (sucrose, maltose, isomaltose), accounts for its higher tendency to aggregate, 16 thereby already affecting the dynamic properties of water at lower trehalose concentrations 17 (Lebret A, Bordat P, Affouard F, Descamps M, Migliardo FJ, Phys. Chem.
B, 2005; 109:
18 11046.); (Lebret A, Affouard F, Bordat P, Hedoux A, Guinet Y, and Descamps M, Chem. Phys., 19 2008; 345:267.); (Peric-Hassler L, Hansen HS, Baron R, and Hunenberger PH, "Conformational properties of glucose-based disaccharides investigated using molecular dynamics simulations 21 with local elevation umbrella sampling," Carbohydr. Res., 2010; 345:
1781.) 22 1001671 In a ternary mixture of protein (lysozyme), sugar, and water, at a moderate 23 concentration of 0.5 M, trehalose can cluster around the protein, thereby trapping a thin layer of 24 water molecules with modified solvation properties, playing the role of a "dynamic reducer" for solvent water molecules in the hydration shell around the protein. A
remarkable conformational 26 rigidity of the trehalose molecule due to anisotropic hydration (very little hydration adjacent to 27 the glycosidic oxygen of trehalose), provides stable interactions with hydrogen-bonded water 28 molecules; trehalose makes an average of 2.8 long-lived hydrogen bonds per each step of 29 molecular dynamic simulation compared with the average of 2.1 for the other sugars (Lins RD, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Pereira CS, and Hunenberger PH, "Protein-Trehalose Interactions in Aqueous Solution,"
2 Proteins, 2004; 55: 177.); (Choi Y, Cho KW, Jeong K, and Jung S, "Molecular dynamic 3 simulations of trehalose as a 'dynamic reducer' for solvent water molecules in the hydration 4 shell," Carbohydr Res., June 12, 2006; 341(8): 1020-1028.).
1001681 In a simulated ternary mixture of lipid membranes, composed of DPPC
6 (dipalmitoylphosphatidylcholine), with aqueous solution of trehalose, the trehalose molecules 7 cluster near membrane interfaces, forming hydrogen bonds both between trehalose molecules 8 and with the lipid headgroups (Pereira CS, Hunenberger PH, "The effect of trehalose on a 9 phospholipid membrane under mechanical stress," Biophys. J., 2008; 95:
3525.); (Sum AK, Faller R, and de Pablo JJ, "Molecular simulation study of phospholipid bilayers and insights of 11 the interactions with disaccharides," Biophys. J., 2003; 85: 2830.).
Trehalose may compete with 12 water binding to both carbonyls and phosphates in cell membranes, forming the OH bridges that 13 are stronger than the H-bonds of water with those groups, and the displacement of water is 14 compensated with the insertion of sugar. Trehalose, a dimer of glucose with the ability to form at least 10 hydrogen bonds, inserts in a lipid interface nearly normal to the lipid bilayer plane and 16 can decrease water activity in the cell membrane up to 70% at a concentration of trehalose as low 17 as 0.1 mM. The insertion of trehalose, replacing water simultaneously at the carbonyls and the 18 phosphates, does not cause the surface defects in the cell membrane with respect to hydrated 19 lipids (Pereira CS and Hunenberger PH, "The effect of trehalose on a phospholipid membrane under mechanical stress," Biophys. J., 2008; 95:3525.); (Sum AK, Faller R, and de Pablo JJ, 21 "Molecular simulation study of phospholipid bilayers and insights of the interactions with 22 disaccharides," Biophys. J., 2003; 85: 2830.); (Villareal M, Diaz SB, Disalvo EA, Montich G, 23 Langmuir, 2004; 20: 7844.). We hypothesize that disaccharide trehalose, being inserted into the 24 phospholipid bilayer(s) of bacterial cell membrane(s) for protection of their integrity, can affect the fluidity of the cell membranes (via its specific interactions with water, carbonyls and 26 phosphates), and cause conformational changes in trans-membrane proteins (including ion 27 channels, efflux pumps and porins), resulting in changes of their functional properties.
28 [00169] As a result of water displacement, trehalose may affect the cell surface potential and 29 hence microbial cell aggregation and attachment to surfaces. There can be at least two CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 mechanisms for these phenomena. First, the magnitude of cell surface potential can be 2 modulated by trehalose displacement of water in its attachment to cell membrane phospholipids 3 and carbonyl compounds. Second, this same displacement of water (in a non-uniform manner) 4 can lead to heterogeneity of surface potential, also imparting the adhesion properties of microorganism. (Poortinga AT, Bos R, Norde W, and Busscher HJ, "Electric double layer 6 interactions in bacterial adhesion to surfaces," Surface Science Reports, 2002; 47: 1-31.);
7 (Disalvo EA, Lairion F, Martini F, Almaleck H, Diaz S, and Gordillo G, "Water in Biological 8 Membranes at Interfaces: Does it Play a Functional Role?," An. Asoc.
Quim. Argent., 2004;
9 V.92 n. 4-6 Buenos Aires ago./dic.).
[00170] Trehalose and biofilm formation 11 [00171] Based on the unique properties of trehalose as a universal general stress response 12 metabolite and an osmoprotectant, and the specific features of its interactions with water (which 13 comprises up to 95% of biofilm matrix), trehalose can be one of the most important components 14 of microbial biofilm, and its specific interactions with water can be considered to be one of the most important mechanisms of biofilm formation.
16 [00172] Since the formation of microbial biofilm can be seen as a continuous process of 17 adaptation of a microorganism to its environment, trehalose and its interactions with water can 18 play an important role in all stages of microbial biofilm development (the early stage of initial 19 biofilm formation, maturation of the biofilm, and .dispersal of the biofilm).
[00173] From the first moment, when a microorganism enters the human body in a planktonic 21 form, it is subjected to various stresses (first of all, osmotic stress) and undergoes the general 22 stress response with the production of trehalose, which in its initial interactions with water 23 begins the process of microbial biofilm formation. In this initial stage, trehalose facilitates 24 adhesion of planktonic bacteria to surfaces by various means: as a result of its interaction with water and the lipid headgroups at the cell membrane interfaces, it decreases the microbial cell 26 surface potential and enhances the bacterial cell aggregation, initial adsorption and attachment to 27 the surfaces, both biotic and abiotic. Also, trehalose favors the bacterial cell aggregation and 28 attachment to various surfaces by forming a hydration layer with modified solvation properties 29 around the bacterial cell and reducing the dynamic properties of water in this layer (and up to the CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 3-rd and 4-th hydration layers), thus slowing down the bacterial cell movement. In addition, 2 trehalose self-associates in aqueous solution in a concentration dependent manner to form 3 clustering networks, affecting the dynamic properties of the solution.
Through extensive 4 solvation, trehalose has a potent ability to restructure water in the solution and enhance the hydrogen bonding between water molecules, thus contributing to the gelation phenomena and the 6 biofilm formation.
7 [00174] During the next stage of the biofilm development (the formation of bacterial colonies 8 and the maturation of biofilm), the bacteria will continuously produce trehalose as a general 9 stress response metabolite and an osmoprotectant in response to constantly varying environmental conditions, such as increased cell density, nutrients limitations, and waste 11 products accumulation in the biofilm. Then, the continuous trehalose ¨
water interactions, with 12 attraction of new water molecules and further restructuring of water, will result in formation of 13 new layers of the biofilm and gradually increased biofilm volume. During this stage, bacteria 14 will release into the biofilm matrix various extracellular substances, including specific proteins (adhesins, matrix interacting factors), compatible solutes, signaling molecules, metabolic end- or 16 by-products, such as polysaccharides, lipids, phospholipids, and the detritus from aging and 17 lysed cells, which will contribute to the formation of the tertiary structure of the biofilm, 18 stabilization of the biofilm architecture, thickening of the biofilm matrix, and increased density 19 of the biofilm.
[00175] During the late stage (the dispersal stage of microbial biofilm), as the biofilm ages, 21 the amount of trehalose in the superficial layers of the biofilm will gradually decrease due to 22 higher accumulation of trehalose in the deeper layers adjacent to the microbial cells, so that the 23 trehalose restructuring effect on water, the strengthening effect on the hydrogen bonds between 24 water molecules, and the aggregation forces between the microbial cells will gradually diminish and favor the sloughing off of the superficial layers of the biofilm, containing the pathogenic 26 microorganisms that already have been exposed to the host defense factors and various 27 antimicrobial substances (if used). These microorganisms, disseminating from the sloughing off 28 superficial layers of the "parenting biofilm", will be covered by the thin layer of their 29 "parenting" matrix gel, or just covered by superficial water layer(s) formed by their own CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalose for protection from osmotic stress during their move through the body tissues and 2 fluids. These disseminating microorganisms may express higher resistance to the host defense 3 factors and antimicrobials (due to their previous exposure to higher concentrations of 4 antimicrobials in the superficial layers of "parenting biofilm") and potentially increased virulence, so that the newly formed disseminated biofilm sites, containing these pathogenic 6 microorganisms, will contribute to further increase in antimicrobial resistance and virulence in 7 the initial microorganisms forming the "initial biofilms".
8 [00176] At any stage of the biofilm development, the microorganisms embedded in the 9 biofilm matrix will respond to any environmental assault on the biofilm, including the use of various harmful substances (i.e. antimicrobials, disinfectants, and various other anti-biofilm 11 compounds) by additional production of trehalose as a general stress response metabolite and an 12 osmoprotectant, that will result in further increase in the biofilm gel matrix volume and density, 13 thus preventing the penetration of harmful substances into the biofilm and protecting the 14 microorganisms from killing.
1001771 Trehalose was detected along with other sugars, di-, oligo-, and polysaccharides in 16 the laboratory-grown microbial biofilms, in the reseach studies mostly aimed at either evaluating 17 the effect of various nutrients on microbial biofilm formation, or analyzing the content of the 18 biofilm exopolymer matrix.
19 1001781 For example, trehalose was detected in a small amount (3%), along with glycerol (5%), mannitol (18%), and glucose (74%), in the monosaccharide-polyol fraction of the aerial-21 grown hyphae of the Aspergillus fumigatus biofilm; all hexoses and polyols were found 22 intracellularly in the same proportion as extracellularly (Beauvais A, Schmidt C, Guadagnini S, 23 Roux P, Perret E, Henry C, Paris S, Mallet A, Prevost M, and Latge JP, "An extracellular matrix 24 glues together the aerial-grown hyphae of Aspergillus fumigates,"
Cellular Microbiology, 2007;
9(6): 1588-1600.). In another example, biofilm development on stainless steel by Listeria 26 monocytogenes (the most common biofilm-producing pathogen in the food industry), was 27 enhanced by the presence of mannose or trehalose as nutrients in the growth media, with 28 trehalose being superior to mannose in constant biofilm production during 12 days of incubation 29 at 21 degrees C (Kim KY and Frank JF, "Effect of nutrients on biofilm formation by Listeria CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 monocytogenes on stainless steel," Journal of food protection, 1995;
58(1): 24-28.). In another 2 study, the formation of a structurally and metabolically distinctive biofilm by Streptococcus 3 mutans (the most common pathogen in dental biofilms), was enhanced by the combination of 4 sucrose and starch, compared with sucrose alone, in the presence of surface-adsorbed salivary a-amylase and bacterial glucosyltransferases, with upregulation of genes associated with maltose 6 uptake/transport and fermentation/ glycolysis (Klein MI, DeBaz L, Agidi S, Lee H, Xie G, Lin 7 AH, Hamaker BR, Lemos JA, and Koo H, "Dynamics of Streptococcus mutans Transcriptome in 8 Response to Starch and Sucrose during Biofilm Development," PLoS ONE, 2010; 5(10): 1-13.).
9 In the next study, the yeasts from hydrocarbon-polluted alpine habitats (Cryptococcus terreus -strain PB4, and Rhodotorula creatinivora - strains PB7 and PB12) synthesized and accumulated 11 glycogen (both acid- and alkali-soluble) and trehalose during growth in culture media, containing 12 either glucose or phenol as a sole carbon and energy source, with higher biofilm formation by 13 both strains of Rhodotorula creatinivora (Krallish I, Gonta S, Savenkova L, Bergauer P, and 14 Margesin R, "Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora," Extremophiles, 2006; 10(5): 441-449.).
16 [00179] In contrast to the previous results, the laboratory-grown wild type Enterococcus 17 faecalis formed strong biofilm in the presence of maltose or glucose in the growth media, and 18 formed very little amount of biofilm in medium containing trehalose (Creti R, Koch S, Fabretti 19 F, Baldassarri L, and Johannes H, "Enterococcal colonization of the gastro-intestinal tract: role of biofilm and environmental oligosaccharides," BMC Microbiology, 2006; 6: 660-668.).
21 [00180] Since trehalose is the most abundant disaccharide in yeasts and fungi, the biofilm 22 matrix of any biofilm-based yeast or fungal infections, and/or multispecies biofilms which 23 include yeasts and/or fungi, can be more resistant to penetration by antimicrobials.
24 [00181] Enzyme trehalase (a,a-trehalase; a,a-trehalose-1-C-glucohydrolase, EC 3.2.1.28) [00182] Enzyme trehalase (a, a-trehalase), highly specific for the non-reducing disaccharide 26 trehalose, directly degrades trehalose into two molecules of glucose on hydrolysis, being this the 27 only trehalose degradation pathway reported up to today. Trehalase phylogeny unveiled three 28 major branches comprising those from bacteria, plant and animals, and those from fungal origin.
29 Crystallographic study of bacterial trehalase indicated that this enzyme's structures are highly CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 conserved in spite of the marked differences found at the sequence level, suggesting a bacterial 2 origin for the trehalases in contrast to an eukaryotic origin, as previously proposed (Barraza A, 3 Sanchez F. "Trehalases: A neglected carbon metabolism regulator?", Plant Signal Behva 2013;
4 8: e24778.). Trehalose degradation by trehalase appears to be important, perhaps essential, in the life functions of various lower organisms, including yeasts, fungi, bacteria, insects, and 6 invertebrates (Nwaka S and Holzer H, "Molecular biology of trehalose and trehalases in the 7 yeast, Saccharomyces cerevisiae," Prog. Nucleic Acid Res. Mol. Biol., 1998; 58: 197-237.).
8 Enzyme trehalase also has been reported to be present in many macroorganisms (including 9 mammals - animals and humans) and vascular plants, but the functions and properties of this enzyme were not fully elucidated (Elbein AD, "The metabolism of a,a-trehalose," Adv.
11 Carbohyd. Chem. Biochem, 1974; 30: 227-256.); (Elbein AD, Pan YT, Pastuszak I, and Carroll 12 D, "New insights on trehalose: a multifunctional molecule,"
Glycobiology, 2003; Vol. 13, No 4:
13 17R-27R.).
14 1001831 As many as 541 model variants of trehalase can be found in the Protein Model Portal (http://www.proteinmodelportal.org/). A few of these models corresponding to different enzyme 16 variants (isoenzymes) are shown in Figures 2a through 2d.
17 [00184] In lower forms of life (yeasts, fungi, bacteria), there are two main types of trehalase 18 enzyme: neutral trehalase (NTH) and acid trehalase (ATH), which are encoded by two different 19 genes. Most of the trehalase activity in these microorganisms, comes from the neutral trehalase, located in the cytosol, with the pH optimum of about 7, highly specific for trehalose as the 21 substrate, and inactive on cellobiose, maltose, lactose, sucrose, raffinose, and mellibiose; this 22 enzyme has also a specific regulatory function (App H and Holzer H, "Purification and 23 characterization of neutral trehalase from the yeast ABYS1 mutant," J.
Biol. Chem., 1989; 264:
24 17583-17588.). The acid or vacuolar trehalase has a pH optimum of 4.5 and is also very specific for trehalose as the substrate, showing no activity with cellobiose, maltose, lactose, sucrose, and 26 mellibiose; this enzyme acts in the periplasmic space where it binds exogenous trehalose to 27 internalize it for further cleavage it in the vacuoles to produce free glucose (Mittenbuhler K and 28 Holzer H, "Purification and characterization of acid trehalases from the yeast SUC2 mutant," J.
29 Biol. Chem., 1988; 263: 8537-8543.); (Stambuk BU, de Arujo PS, Panek AD, and Serrano R, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 "Kinetics and energetics of trehalose transport in Saccharomyces cerevisiae," Eur. J. Biochem., 2 1996; 237: 876-881.).
3 [00185] The activities of both trehalases (NTH and ATH) are low in yeast cells growing 4 exponentially, but high during stationary phase growth after glucose has been depleted (Winkler K, Kienle I, Burgert M, Wagner JC, and Holzer H, "Metabolic regulation of the trehalose content 6 of vegetative yeast," FEBS Lett., 1991; 291: 262-272.). ATH1 deletion mutant of the yeast S.
7 cerevisiae cannot grow in the medium with trehalose as the carbon source, but a Candida utils 8 mutant strain is able to utilize extracellular trehalose as carbon source despite of the lack of ATH
9 activity. Various bacteria, such as E. coli, have trehalases that also may supply exogenous trehalose and glucose via the phospho-transferrase system (PTS) (Horlacher R, Uhland K, Klein 11 W, Erhmann M, and Boos W, "Characterization of a cytoplasmic trehalase of Escherichia coli,"
12 J. Bacteriol., 1996; 178: 625-627.).
13 [00186] In the plant kingdom, enzyme trehalase is ubiquitous, being involved in carbon 14 metabolism in both lower and higher plants. Although sugar trehalose is rare in higher (vascular) plants, it has been demonstrated that trehalase in these plants could take a part in the degradation 16 of trehalose derived from the plant-associated bacteria in symbiotic interactions; it has been also 17 suggested that trehalase in higher plants could play additional role in the defense against 18 parasites and other pathogenic organisms (in plant ¨ pathogen interactions) (Muller J, Wiemken 19 A, and Aeschbacher R, "Trehalose metabolism in sugar sensing and plant development," Plant Sci., 1999; 147: 37-47.); (Muller J, Aeschbacher RA, Wingler A, Boller T, and Wiemken A, 21 "Trehalose and trehalase in Arabidopsis," Plant Physiol., 2001; 125:
1086-1093.).
22 1001871 Though disaccharide trehalose is not produced in mammals, the enzyme trehalase 23 exists in mammals (including humans) in the kidney brush border membranes and in the 24 intestinal villi membranes; the role of trehalase in kidney is still not clear, but in the intestine its function is to hydrolyze ingested trehalose (Dahlqvist A, "Assay of intestinal disaccharidases,"
26 Anal. Biochem., 1968; 22: 99-107.); (RufJ, Wacker H, James P, Maffia M, Seiler P, Galand G, 27 Kiekebusch A, Semenza G, and Mantei N, "Rabbit small intestine trehalase. Purification, cDNA
28 cloning, expression and verification of GPI-anchoring," J. Biol. Chem, 1990; 265:
29 15034-15040.); (Yonemaya Y and Lever JE, "Apical trehalase expression associated with cell CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 patterning after inducer treatment of LLC-PK monolayers," J. Cell.
Physiol., 1987; 131:
2 330-341.). Only one type of trehalse (a,a-trehalase, highly specific enzyme for direct 3 degradation of trehalose into two molecules of glucose) is present in humans. Trehalase 4 produced by the glands of Lieberkuhni in the small intestine is a constituent of the intestinal juice along with other specific saccharidases, such as maltase, sucrase-isomaltase complex, and Beta-6 glycosidase-lactase (Mayes PA, "Carbohydrates of physiologic significance," In: Harper's 7 Biochemistry, 25th ed, 2000, pp. 149-159, Appleton & Lange, Stamford, CT.); (Rodwell VW
8 and Kennelly PJ, "Enzymes: General Properties; Enzymes: Kinetics," In:
Harper's Biochemistry, 9 25th ed, 2000, pp. 74-102, Appleton & Lange, Stamford, CT.). As with all other disaccharidases, trehalase remains attached to the brush border of the enterocyte in the intestinal lumen while the 11 catalytic domain is free to react with the substrate. There is little free trehalase activity in the 12 intestinal lumen; most activity is associated with small "knobs" on the brush border of the 13 intestinal epithelial cells. A small fraction (approximately 0.5%) may be absorbed by passive 14 diffusion, as shown for other disaccharides, in patients with trehalase deficiency (van Elburg RM, Uil JJ, Kokke FTM, Mulder AM, van dr Broek WGM, Mulder CJJ, and Heymans HSA, 16 "Repeatability of the sugar-absorption test, using lactulose and mannitol, for measuring intestinal 17 permeability for sugars," J. Pediatr. Gastroenterol. Nutr., 1995; 20:
184-188.). Trehalase activity 18 have been found also in the renal cortex, plasma, urine, liver and bile, although function of the 19 enzyme in these locations is not clear yet; it is likely that trehalase in the urine and bile can be incidental to its presence in the kidney and liver (Eze LC, "Plasma trehalase activity and diabetes 21 mellitus," Biochem Gen., 1989; 27: 487-495.). A complete cDNA clone encoding human 22 trehalase (a protein of 583 amino acids with calculated molecular weight of 66 595 kDa), a 23 glycoprotein of brush-border membranes, has been isolated from a human kidney library; the 24 deduced amino acid sequence of the human trehalase enzyme showed similarity to sequences of the enzyme trehalase from rabbit (81%), silk worm, Tenebrio molitor (43%), Escherihia coil 26 (33%); human trehalase also resembled yeast acidic trehalase - ATH (25%
identity) and neutral 27 trehalase ¨ NTH (26,5% identity); by homology with mammalian trehalase from other species, 28 human enzyme is an ectoenzyme whose hydrophobic region at the carboxyl terminus is linked to 29 the plasma membrane by GPI anchor (Reiko Ishihara, Shigeru Taketani, Misa Sasai-Takedatsu, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Minoru Kino, Rikio Tokunaga, Yohnosuke Lobayashi, "Molecular cloning, sequencing and 2 expression of cDNA encoding human trehalase", Cene 202 (1997) 69-74.).
The research data 3 showed that mammalian trehalase is encoded by a single gene and is probably expressed as one 4 .. form in various tissues (Ruf, J., et. al., 1990, "Rabbit small intestinal trehalase: purification, cDNA cloning, expression, and verification of glucosylphosphatidylinositol anchoring", J. Biol.
6 Chem. 265, 15034-15039; Reiko Ishihara, et. al., "Molecular cloning, sequencing and expression 7 of cDNA encoding human trehalase", Gene 202 (1997) 69-74.
8 [00188] Biochemical properties of the human enzyme a,a--trehalase include:
9 [00189] = high specificity for the substrate (disaccharide trehalose ¨ a, a-trehalose) [00190] = method of activation ¨ direct contact with the substrate (trehalose) 11 [00191] = optimal conditions for activity ¨ in the range of pH
between 5.0 and 7.0 (similar 12 to the other disaccharidases) 13 [00192] = end product of action ¨ 2 molecules of D-glucose 14 1001931 = heat sensitivity ¨ as a glycosylated protein is probably similar to the other disaccharidases 16 [00194] = catalytic efficiency ¨ high due to the high specificity for the substrate trehalose 17 [00195] = coenzymes or metal ions for activity ¨ not needed 18 1001961 = co-variants of enzyme ¨ unknown 19 1001971 The main function of human a, a-trehalase is to hydrolyze ingested disaccharide trehalose into glucose. Trehalase deficiency is a known metabolic condition in humans, when the 21 body is not able to degrade disaccharide trehalose into two molecules of glucose and digest it.
22 People with enzyme trehalase deficiency experience vomiting, abdominal discomfort and 23 diarrhea after eating mushrooms (rich in trehalose) of any other food containing trehalose.
24 Trehalase enzyme deficiency in most cases appear to be inherited in an autosomal recessive manner (Kleinman RE, Goulet 0, Mieli-Vergani G, Sherman PM, In: Walker's Pediatric 26 Gastrointestinal Disease: Physiology, Diagnosis, Management, 5-th edition, 2008); (Semenza, 27 G., Auricchio, S., and Mantei, N. In: The Metabolic & Molecular Bases of Inherited Disease;
28 8-th ed., 2001; Chapter 75: Small Intestinal Disaccharidoses. McGraw-Hill, New York.). Isolated 29 intestinal trehalase deficiency is found in approximately 8% of Greenlanders; it is not infrequent CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 among Finns, but is believed to be rare elsewhere. The low (2%) incidence of isolated trehalase 2 enzyme deficiency was described in the populations from the USA, UK, and mainland Europe 3 (Bergoz R, Valloton MC, and Loizeau E, "Trehalase deficiency," Ann. Nutr.
Metab., 1982; 26:
4 191-195.). In the UK, from 400 patients investigated for suspected malabsorption, 369 (92%) had normal intestinal histology on biopsy, with the normal range of trehalase at 4, 79-37, 12 U/g 6 protein; 31(8%) patients with villous atrophy had a diagnosis of coeliac disease and significantly 7 reduced activity of disaccharidases, including trehalase, with recovered function of all enzymes 8 (except lactase) after treatment with a gluten-free diet; the authors concluded that there is no 9 basis for routine determination of trehalase activity in the population of the UK (Murray IA, Coupland K, Smith JA, Ansell ID, Long RG, "Intestinal trehalase in a UK
population:
11 Establishing a normal range and the effect of disease," Br. J. Nutr., 2000; 83(3): 241-245.). In 12 Belgium, in intestinal biopsy samples from 200 patients with abdominal symptoms and diarrhea, 13 total aaa-trehalase deficiency (0-12 U/g mucosa) was detected in 18 (9%) cases, partial 14 deficiency (3-12 U/g mucosa) ¨ in 39 (19.5%) cases, and only 4 patients (2%) presented selective aaa-trehalase deficiency with otherwise normal other disaccharidases; these data 16 suggested that aaa-trehalase deficiency can be more common than it is believed (Buts JP, 17 Stilmant C, Bernasconi P, Neirinck C, De Keyser N, "Characterization of alpha, alpha-trehalase 18 released in the intestinal lumen by the probiotic Saccharomyces boulardii," Scandinavian Journal 19 of Gastroenterology, 2008; 43 (12): 1489-1496.).
[00198] The importance of enzyme trehalase was demonstrated in certain pathologic 21 conditions, including birth defects and genetic abnormalities: low or absent intestinal trehalase 22 was detected in the sample of amniotic fluid from a fetus with anal imperforation, whereas a 23 higher than normal level of renal trehalase activity was found in amniotic fluid from a fetus with 24 polycystic kidney disease (Elsliger MA, Dallaire L, Potier M, "Fetal intestinal and renal origins of trehalase activity in human amniotic fluid," Clin Chim Acta, July, 16, 1993; 216(1-2): 91-26 102.). Also, low intestinal trehalase enzyme level was detected in amniotic fluid on 27 amniocentesis in 14 pregnant women at 1 in 4 risk for a child with cystic fibrosis, screened at the 28 18-th week of gestation; and in two terminated at the 19-th week cases, histochemical lesions 29 characteristic of cystic fibrosis were seen in exocrine glands, including the pancreas and CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 intestinal mucosa of both fetuses, and the total protein content in the meconium of these fetuses 2 was also significantly higher than in the controls (Szabo M, Teichmann F, Szeifert GT, Toth M, 3 Toth Z, Torok 0, Papp Z, "Prenatal diagnosis of cystic fibrosis by trehalase enzyme assay in 4 amniotic fluid," Article first published online: 23 APR 2008; DOT:
10.1111/j. 1300-0004.
1985.tb01211.x.). The trehalase enzyme assay in amniotic fluid was recommended as a genetic 6 test for prenatal diagnosis of cystic fibrosis. The latest genetic studies in 2942 full-heritage Pima 7 Indians and 3897 "mixed" heritage Native Americans with Diabetes type 2 (T2D), found strong 8 correlation with trehalase enzyme activity in plasma of people with T2D;
four single nucleotide 9 polymorphisms (SNPs) were detected in their trehalase gene (TREH) that were associated with T2D (Yunhua L Muller, Robert L Hanson, William C Knowler, Jamie Fleming, Jayita Goswani, 11 Ke Huang, Michael Traurig, Jeff Sutherland, Chris Wiedrich, Kim Wiedrich, Darin Mahkee, 12 Vicky Ossowski, Sayuko Kobes, Clifton Bogardus, Leslie J Baier, "Identification of genetic 13 variation that determines human trehalase activity and its association with type 2 diabetes", 14 Humangenetik 2013-06-01.). The Chinese research studies on genetic risk factors for glioma (one of the most aggressive human tumors), indicated that more than 100 single nucleotide 16 polymorphism (SNPs) are associated with the risk of glioma, including the SNPs in trehalase 17 (TREH) gene, and provided evidence for three glioma susceptibility genes ¨ TREH, IL4, and 18 CCDC26 (Shangu Li, Tianbo Jin, Jiayi Zhang, Huiling Lou, Bo Yang, Yang Li, Chao Chen, 19 Yongsheng Zhang, Shanqu Li, Tianbo Jin, Jiayi Zhang, Huiling Lou, Bo Yang, Yang Li, Chao Chen, Yongsheng Zhang, "Polymorphisms of TREH, IL4 and CCDC26 genes associated with 21 risk of glioma", Cancer Epidemiology 2012-06-01.). The correlation between intestinal histology 22 and trehalase activities during intestinal injury has been shown in clinical studies in patients with 23 intestinal ischemia-reperfusion injury (Stefan Toth, Timea Pekarova, Jan Varga, Vladimira 24 Tomeckova, Stephan Toth, Lucia Lakyova, Jarmila Vesela, "Trehalase as a possible marker of intestinal ischemia-reperfusion injury", Acta Biochimica Polonica 2013-01-01.).
26 1001991 Since ingestion of large quantities of foods containing trehalose is not common 27 worldwide, the real frequency of trehalase deficiency in various populations around the world is 28 mostly unknown. However, it should be noted, that over the last two decades, in addition to 29 natural sources of trehalose in the food (mostly, mushrooms, algae, baker's yeasts), it has been CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 approved in some countries, including the USA, as an additive in the preparation of dried, 2 frozen, and processed food, and as a moisture retainer in various products (including ice cream, 3 and baked goods), with no requirements for labeling of this constituent in prepared food or other 4 products (Abbott PJ and Chen J, WHO Food Additives Series 46: Trehalose.
International Programme on Chemical Safety. Accessed Feb 4, 2010, available at:
6 http ://www.inchem.org/documents/j ecfa/j ecmono/v46j e05.htm.).
7 [00200] The amount of enzyme trehalase normally produced for digestion and utilization of 8 exogenous trehalose is appropriate for healthy people, but is far less than what is needed for 9 people with biofilm-based chronic infections, especially for individuals with trehalase enzyme deficiency. Therefore, the use of enzyme trehalase, along with other enzyme formulations and 11 antimicrobials (including antibiotics), can greatly enhance the effectiveness of various treatment 12 protocols for biofilm-based chronic infections.
13 [00201] Therefore, at least one basis for the presently disclosed compositions and methods is 14 the addition of enzyme trehalase, highly specific to the hydrolysis of the trehalose constituent of microbial biofilms, to treatment protocols for biofilm-based chronic infections in order to 16 increase the effectiveness of existing treatment modalities.
17 [00202] To avoid possible immunogenicity and/or toxicity of any other sources of trehalase 18 (animal-derived or microbial-derived enzymes) while used in humans, the best source of 19 trehalase for use in humans can be a human genetic recombinant enzyme (human trehalase gene script expressed in rice genetically modified for human enzyme production);
this manufacturing 21 method will be analogous to production of synthetic human enzyme Lysosyme ("Lysobac") 22 produced by Sigma-Aldrich, USA. Also, plant-derived trehalase (sugar cane trehalase - a,a-23 trehalase, that is located in the cytosol of the plant cells), can be used in humans; in anecdotal 24 cases of using natural row sugar cane sticks for chewing as a sweet treat (substitute for chewing gum and candy) and using a raw (non-processed) fresh pressed sugar cane juice for food (as 26 sugar substitute), it was demonstrated that the adult and children population in remote regions of 27 Central America had healthy teeth, absence of dental caries and periodontal diseases, and healthy 28 condition of the epithelial lining of the oral cavity (personal observations, not published research 29 data). So that, the enzyme trehalase in the raw sugar cane juice (sugar cane trehalase - a,a-CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalase,) seems to be safe for use in humans, and can be used in combination with 2 antimicrobials for prevention and treatment of microbial biofilm in the oral cavity. Also, the 3 process of manufacturing of sugar cane trehalase can be cost-efficient regarding its possible wide 4 use in medical and health care fields. Trehalse can be manufactured in various forms (powder, liquid, gel, tablets, and capsules) that can be tailored to specific applications in humans and 6 delivered to any specific location in the body where biofilm is the issue (mostly mucosal linings 7 of the oral cavity, GI tract, respiratory tract, and urinary tract, and skin lesions and wounds);
8 trehalase can be used in combination with other saccharidase enzymes (dextranase and lyase), 9 included in existing formulations of digestive enzymes, included in various existing formulations of vitamins, used in combination with various natural substances (anti-inflammatory and 11 immune-modulating compounds and anti-oxidants), and can be used in combination with various 12 antibiotics and natural antimicrobials (antimicrobial peptides of human-or plant-origin) to treat 13 biofilm-based infections in a human body and to control biofilms on medical devices, medical 14 equipment, and various medical implants. For industrial applications (water, gas, and oil pipelines, HV/AC systems), trehalase can be obtained from natural sources (plants, algae, fungi) 16 using known manufacturing methods (for example, the United States Patent Number 5,593,869 17 Jan.14, 1997 "Method of manufactirung sugars by trehalase" includes the description of 18 manufacturing novel enzyme trehalase from green algae of Lobosphaera, Chlorellaceae, 19 Chlorofyceae, Chlorophyta families); in another example ¨ method of extraction of trehalase from Sugar Cane is described in PH Dissertation of Susan Bosch, 2005, "Trehalose and Carbon 21 Partitioning in Sugar Cane, Chapter 5, pp. 123-146, University of Stellenbosch, South Africa).
22 However, to date, no available medical/health scientific information shows evidence of enzyme 23 Trehalase as a component of any prescription drugs, natural antimicrobials preparation and their 24 combinations, OTC products, nutritional supplements, vitamins combinations, digestive enzymes formulations and/or enzymatic formulations used for local treatment of microbial biofilms on 26 skin and mucosal lining surfaces of humans, or as a component of systemic enzymes 27 formulations, or as a component of compositions and methods for biofilm treatment on medical 28 devices, implants, and equipment, or being a component of various disinfecting combinations for 29 prevention and treatment of microbial biofilm on various surfaces in hospitals (including surgical CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 units), medical offices and other medical/health settings, and public places. Also, there is no 2 available information about the enzyme trehalase being a component of any existing anti-biofilm 3 formulations and/or any methods for microbial biofilm prevention and treatment in industrial 4 fluid conduits (water, gas, oil pipelines), and various industrial and household equipment.
1002031 Embodiments for the treatment of biofilm-based infections 6 1002041 To increase effectiveness of existing treatment modalities and protocols for biofilm-7 based chronic infections in the human body, combination of trehalase with antimicrobials can be 8 used alone or in combination with other enzymes and substances (antioxidants, anti-9 inflammatory and immune-modulating), being included in various formulations and methods of their use, for direct application to the sites of infectious biofilm (directly accessible mucosal 11 linings of the respiratory tract, GI-tract, genito-urinary tract, eyes, skin, open wounds, etc.) 12 and/or for systemic use to treat biofilm-based infections in directly inaccessible (or hardly 13 accessible) sites of infection and in the bloodstream. Simultaneously, trehalase can be used in 14 combination with natural substances (natural antimicrobials, human antimicrobial peptides, anti-oxidants, anti-inflammatory and immune-modulating substances) to support and/or enhance 16 body's own abilities to prevent microbial biofilm formation or degrade formed biofilm, to kill 17 invading pathogens and eliminate them from the body, to restore normal homeostasis and 18 immune system function, and to repair any tissue damages resulted from biofilm-based 19 infections.
[00205] In direct application to the sites of microbial biofilm in a human body, trehalase 21 enzyme could be used as a component of formulations of hydrolytic enzymes (including 22 dextranase and alginate-lyase) and antimicrobilals, applied in a single step to the site of 23 infectious biofilm; or trehalase can be included in anti-biofilm combinations of enzymes and 24 antimicrobials used in a multi-step procedure, starting as the first (pretreatment) step with application of combination of hydrolytic enzymes (trehalase, dextranase, alginate-lyase) with an 26 exposition time sufficient to initiate degradation of the biofilm matrix, followed by the second 27 step with application of combination of proteolytic, fibrinolytic, and lipolytic enzymes over a 28 corresponding appropriate exposition time to further degrade the biofilm matrix, followed in a 29 third step by application of antimicrobials (or their combinations) specific for the infection(s) CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 involved, or polymicrobial antibiotics, in combination with trehalase for prolonged use. In this 3-2 step method, the initial degrading effect on biofilm matrix (provided by trehalase in combination 3 with other hydrolytic enzymes), will ease and potentiate the action of proteolytic, fibrinolytic, 4 and lipolytic enzymes to further degrade the biofilm matrix and provide access to microbial cells for antimicrobal substances combined with trehalase to gradually kill infectious pathogens and 6 prevent the Biofilm Induction Response (BM) to sub-MIC doses of antimicrobials at the 7 initiation of treatment course. The BM is a well- known phenomenon that is of concern in 8 clinical practice, when killing concentration of antibiotics gradually increases at the site of 9 microbial biofilm on initiation of treatment, allowing bacteria to initiate BIR at the very initial time of treatment (within minutes to a few hours). In addition, at the end of treatment course, 11 some dormant microorganisms (so called persister cells) who survived antimicrobial treatment, 12 will come back to active growing state from dormancy and will form biofilm as BM, being 13 exposed to the decreasing dose of antimicrobials (down to sub-MIC dose) (Jeffrey B. Kaplan, 14 "Antibiotic-Induced Biofilm Formation. Review; Int J Artif Organs 2011;
34(9): 737-751.).
Therefore, the presence of trehalase in combination with antimicrobials during full treatment 16 course, will increase the effectiveness of used antimicrobials and possibly reduce antimicrobial 17 resistance in biofilm-forming infectious pathogens.
18 1002061 As a systemic enzyme, trehalase in combination with other saccharidases should be 19 used as a time-delayed release substance(s), or being included in multi-enzyme formulations with time-delayed release of constituent(s) to avoid early degradation of trehalase and other 21 hydrolytic enzymes (dextranase and alginate-lyase) by proteolytic enzymes in the upper GI tract 22 (stomach and duodenum) and/or by proteolytic enzymes in administered formulations, and 23 finally being released in the small intestine for further absorption. In this way, trehalase can be 24 supplied for direct absorption and distribution via the bloodstream to hardly accessible "niches"
of biofilm-based infections, for example, on the inner lining of the blood vessels, in bones, joints, 26 on medical devices and implants).
27 1002071 The length of the time for delayed release can be established for trehalase in 28 combination with other hydrolytic enzymes (dextranase and alginate-lyase), so that the release of 29 these enzymes combination will occur in the small intestine, being protected from proteolytic CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 enzymes in the upper GI tract (stomach and duodenum). Also, differential time delays can be 2 established for combination of trehalase with other hydrolytic enzymes (dextranase and alginate-3 lyase) and any co-administered proteolytic enzymes (having different pH
for their proteolytic 4 activities) or a combination of these proteolytic enzymes, to avoid deleterious action of proteolytic compounds on trehalase and other hydrolytic enzymes (dextranase and alginate-6 lyase). In conventional digestive or systemic enzyme formulations currently on the market, the 7 contained hydrolytic enzymes (di-, oligo-, and polysaccharidases) typically are not protected 8 from deleterious action of various proteolytic enzymes included in existing formulations.
9 [00208] Upper Respiratory Tract [00209] The major biofilm-forming species of pathogens affecting the upper respiratory tract 11 include Haemophilus influenzae, Klebsiella pneumoniae, Pneumococcus, Streptococcus spp., 12 Staphylococcus spp., Pseudomonas aeruginosa, Candida spp., and Aspergillus spp. For local 13 .. treatment of biofilm-based infections in the upper respiratory tract (chronic sinusitis, 14 rhinosinusitis, tonsillitis, pharyngitis, and otitis media), trehalase enzyme can be used in combination with other enzymes, antimicrobials specific to the present pathogens or 16 polymicrobial antibiotics, or natural antimicrobials, and anti-inflammatory and immune-17 modulating substances in direct application to the sites of infectious biofilm on mucosal lining, 18 in liquid form as a saline-based solution for instillations, irrigations, and sprays, as well as in 19 gel, ointment, and powder forms.
[00210] In direct application to the sites of infectious biofilm on mucosal lining of the upper 21 respiratory tract, trehalase enzyme could be used as a component of formulations of hydrolytic 22 enzymes (di-, oligo-, and polysaccharidases, including dextranase and alginate-lyase as most 23 frequently used hydrolytic enzymes) and antimicrobilals, applied in a single step to the site of 24 infectious biofilm; or trehalase can be included in anti-biofilm combinations of various enzymes, antimicrobials, anti-inflammatory and immune-modulating substances, and used in a multi-step 26 procedure, starting as the first (pretreatment) step with application of combination of hydrolytic 27 enzymes (trehalase, dextranase, alginate-lyase) with an exposition time sufficient to initiate 28 degradation of the biofilm matrix, followed by the second step with application of combination 29 of proteolytic, fibrinolytic, and lipolytic enzymes over a corresponding appropriate exposition CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 time to further degrade the biofilm matrix, followed in a third step by application of 2 antimicrobials (or their combinations) specific for the infection(s) involved, or polymicrobial 3 antibiotics, in combination with trehalase, anti-inflammatory and immune-modulating substances 4 for prolonged use. Local treatment can be reinforced by systemic use (introduced via GI tract for absorption and distribution via blood stream to the sites of infectious biofilm) of multi-enzyme 6 formulations (including trehalase in a time-delayed release form), anti-inflammatory and 7 immune-modulating substances, along with systemic use of antibiotics preferably with 8 polymicrobial activity.
9 1002111 For local treatment of Pseudomonas aeruginosa infectious biofilm on mucosal lining of the upper respiratory tract, the enzyme alginate-lyase (highly specific for degradation of the 11 polysaccharide alginate ¨ an important constituent of Pseudomonas aeruginosa biofilm), should 12 be also added to combination of trehalase with antibiotics, anti-inflammatory and immune-13 modulating substances for prolonged use as the third step of local application.For Streptococcus 14 spp. infections, the enzyme dextranase (highly specific for degradation of the dextrans -oligosaccharides produced by Streptococcus spp., which facilitate microbial adhesion to the 16 mucosal surfaces and biofilm formation, should also be added to combination of trehalase with 17 antibiotics, anti-inflammatory and immune-modulating substances for prolonged use as the third 18 step of treatment in local application to treat microbial biofilm in the upper respiratory tract.
19 1002121 Otitis Media [00213] For otitis media with or without effusion, treatment should include a systemic 21 enzymes formulation (including trehalase in combination with other saccharidases in time-22 delayed release form) along with systemic antibiotics. The systemic treatment can be reinforced 23 with local treatment applied to the lining of the nasal cavity to address possible spread of 24 infection to the middle ear from the nasal and sinus cavities; this local treatment should include combination of trehalase with anti-inflammatory and immune-modulating substances, and 26 polymicrobial antibiotics or natural antimicrobial substances with wide range of anti-infectious 27 activity (for example, colloidal silver spray). For specific treatment of Pseudomonas aeruginosa 28 and/or Streptococcal biofilms, the enzymes alginate-lyase and dextranase sjould be added to CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 combination of trehalase with antimicrobials. Delivery of the local treatment to the inner ear can 2 be done by a nasal instillation with a pathway through the Eustachian tube into the middle ear.
3 [00214] For otitis media with effusion and installed tympanic tubes, the abovementioned 4 systemic and local treatments should be reinforced by an additional step:
the installed tympanic tubes can be covered inside with trehalase in combination with polymicrobial antibiotics or 6 natural antimicrobials with wide range of antimicrobial activities.
7 [00215] Lower respiratory Tract 8 [00216] Treatment of biofilm-based infections in the lower respiratory tract, should include: a) 9 the use of systemic enzymes (with trehalase and other saccharidases in time-delayed release form) along with systemic antibiotics; b) local treatment using bronchi-alveolar or whole lung 11 lavage in a multi-step procedure, including as the first step - the use of trehalase in combination 12 with other saccharidases (alginate-lyase, dextranase) in a saline-based solution, followed by 13 proteolytic, fibrinolytic, and lipolytic enzymes in a saline-based solution as the second step, and 14 antibiotics in combination with trehalase in a saline-based solution as the third step; c) nasal instillation of trehalase in combination with anti-inflammatory and immune-modulating 16 substances, and polymicrobial antibiotics or natural antimicrobial substances with wide range of 17 antimicrobial activities.
18 1002171 Additional contributing factors to chronic biofilm-based infectious conditions are:
19 genetic trehalase enzyme deficiency (a rare genetic disease listed by NII-I Genetic and Rare Diseases Information Center), genetic trehalase enzyme deficiency in individuals with cystic 21 fibrosis, and artificial trehalase deficiency due to widespread use of trehalose in the food industry 22 as an approved additive in the preparation of dried and frozen foods, and as a moisture 23 conservant, in various foods, such as an ice cream and baked goods.
24 [00218] Taking into account genetic trehalase deficiency in cystic fibrosis patients, uncontrolled consumption of trehalose in food is a favorable factor for thick biofilm formation 26 on the mucosal lining of the upper and lower respiratory tracts in such individuals. Pseudomonas 27 aeruginosa, in symbiosis with other bacteria and fungi, exploits this environment, creating thick 28 polymicrobial biofilm which is almost impossible to eradicate with long-term antibiotic therapy 29 alone (although such therapy can support the patient). Including the enzyme trehalase in CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 combinations with antibiotics, anti-oxidants, anti-inflammatory and immune-modulating 2 substances in various protocols for local and systemic treatments of biofilm-based chronic 3 infections in patients with cystic fibrosis, will significantly increase positive outcomes of such 4 treatment protocols and improve patients' quality of life. To address this thick polymicrobial biofilm at any stages of its development, treatment should include: a) the use of systemic 6 enzymes (with trehalase and other saccharidases in time-delayed release form), along with 7 systemic antibiotics; b) local treatment using bronchi-alveolar or whole lung lavage in a multi-8 step procedure, including as the first step - the use of trehalase in combination with other 9 saccharidases (alginate-lyase, dextranase) in a saline-based solution, followed by proteolytic, fibrinolytic, and lipolytic enzymes in a saline-based solution as the second step, and antibiotics 11 in combination with trehalase in a saline-based solution as the third step; c) using special enzyme 12 "Pulmozyme" to thin the mucus in the airways of CF patients. This treatment can be reinforced 13 by using nasal instillation of trehalase in combination with anti-inflammatory and immune-14 modulating substances, and polymicrobial antibiotics or natural antimicrobials with wide range of antimicrobial activities.
16 [00219] Native Valve Endocarditis (NVE), Infectious Endocarditis, and Line Sepsis 17 [00220] A preferred treatment protocol for NVE, Infectious Endocarditis, and Line Sepsis as 18 blood stream infections, should include use of systemic enzyme formulations, with included 19 trehalase and other saccharidases (preferably, specific to present pathogens) in time-delayed release form; and trehalase in combination with antibiotics directed to specific infectious agents, 21 or polymicrobial antibiotics. The typical organisms involved in these biofilm-mediated infections 22 include Streptococci spp, Enterococci spp., Pneumococcus, Staphylococci spp.(both coagulase 23 positive and negative), gut bacteria, and fungi (most often, Candida albicans and Aspergillus 24 spp.). Because all these pathogens gain access to the bloodstream primarily via the oropharynx, GI-tract, and genito-urinary tract, systemic treatment of NVE, Infectious Endocarditis, and Line 26 Sepsis should be reinforced by local treatment of those infections at the sites of origin, including 27 the previously described multi-step procedure (with application of trehalase, other enzymes, and 28 antimicrobials) if the sites of origin represent biofilm-based infections.
29 [00221] Chronic Bacterial Prostatitis (CBP) and Urinary Tract Infections (UTI) CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1002221 Use of systemic enzymes with included trehalase and other saccharidases in time-2 delayed release form, and antimicrobials in combination with trehalase will address the presence 3 of biofilm-based chronic infections in both CBP and UTI. For local treatment of UTI via bladder 4 instillation, a method after the fashion of a single-step procedure or the multi-step procedure disclosed above for treating biofilm on mucosal linings, should be employed:
trehalase in 6 combination with other hydrolytic enzymes (dextranase, alginate-lyase), and trehalase in 7 combination with polymicrobial antibiotics and/or natural antimicrobials, can be applied in a 8 single step to the site of infectious biofilm; or trehalase can be included in anti-biofilm 9 combinations of enzymes and antimicrobials used in a multi-step procedure, starting as the first (pretreatment) step with application of combination of trehalase with other hydrolytic enzymes 11 (dextranase, alginate-lyase) with an exposition time sufficient to initiate degradation of the 12 biofilm matrix, followed by the next step - application of combination of proteolytic, 13 fibrinolytic, and lipolytic enzymes over a corresponding appropriate exposition time to further 14 degrade the biofilm matrix, and in the next step - - application of trehalase in combination with antibiotics (or their combinations) specific for the present infection(s) involved, or polymicrobial 16 antibiotics, and/or natural antimicrobials for prolonged use. For local treatment of CBP, again, 17 trehalase can be used in combination with other enzymes and antimicrobials in a single-step 18 application or used in multi-step procedure, but with higher concentrations of polymicrobial 19 antibiotics and/or natural antimicrobials delivered directly to the biofilm location within the prostatic ducts by instillation means (via a medical device such as a catheter).
21 [00223] GI Tract Infections 22 1002241 GI tract infections are characterized by polymicrobial biofilm communities along 23 with chronic parasitic and helmintic infections (nematodes are known to produce and accumulate 24 trehalose). For treating microbial biofilms in the upper GI tract, trehalase can be included in combinations of digestive enzymes for fast release, along with use of trehalase in combination 26 with antibiotics specific for present infection(s) and/or natural antimicrobials. For treatment of 27 biofilm-based infections in the lower GI tract, the formulations of digestive enzymes should 28 include trehalase with other saccharidases in time-delayed release form to avoid early 29 degradation by proteolytic enzymes in the upper GI tract or by proteolytic enzymes in the same CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 formulations. Digestive enzymes formulations that include trehalase in a time-delayed release 2 form, shouls be used along with trehalase combined with antibiotics (specific for pathogenic 3 microorganism involved or polymicrobial antibiotics, anti-parasitic, anti-helmintic and anti-4 protozoa drugs) and/or natural antimicrobials active against the pathogens involved. Also, optionally, trehalase can be included in combinations of digestive enzymes and specific 6 antibiotics and/or natural antimicrobials for colonic irrigation treatment method in single-step or 7 multi-step treatment procedures (as described for biofilm treatment on mucosal linings in the 8 previous paragraphs).
9 1002251 Dental and Periodontal Diseases 1002261 The two groups of bacteria responsible for initiating dental caries, including 11 Streptococcus mutans and Lactobacillus (known to possess multiple pathways for biosynthesis of 12 trehalose), have direct access to high concentrations of orally ingested simple sugars and other 13 saccharides, as well as those produced by the action of salivary amylase on ingested 14 carbohydrates (polysaccharides), that favors the increased synthesis of trehalose and formation of microbial biofilm. Enzyme trehalase in combination with antimicrobials (ex.
Chlorhexidine 16 Gluconate), and/or with other saccharidases (ex. Dextranase) can be used for prevention of 17 dental caries by inhibiting the formation of bacterial biofilms on the teeth and surrounding tissue 18 surfaces.
19 1002271 Periodontal disease is a classic biofilm-mediated condition that is refractory to treatment by antimicrobials alone. Applied treatments, which include combination of trehalase 21 with antimicrobials (ex. Chlorhexidine Cluconate) and added other saccharidases (ex.
22 Dextranase), can be both preventive and curative. Combination of trehalase with antimicrobials 23 (alone or with other saccharidases) can be used in oral application for treatment of periodontal 24 diseases and/or during a professional dental cleaning procedure. Also, the multi-step local treatment, including the application of trehalase in combination with antimicrobials (alone or 26 with other saccharidases), followed by the application of proteolytic, fibrinolytic, and lipolytic 27 enzymes, and finally by the application of trehalase in combination with antimicrobials, as 28 disclosed above for treating infectious biofilm on mucosal linings, can be used as a curative 29 method for periodontal biofilm-based infections. Since the bacterial biofilm is the essence of the CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 dental plaque, the use of trehalase in combination with antimicrobials (alone or with other 2 saccharidases) in the mouthwash or gel form can diminish the formation of the dental plaque, 3 and in prolonged use of trehalase in combination with antimicrobials can gradually degrade and 4 eliminate the existing bacterial biofilms.
1002281 For dental surgery, the use of combination of trehalase with antimicrobials in 6 prepared formulations can serve as prophylaxis against biofilm-based infections. Trehalase in 7 combination with antimicrobials (ex. Chlorhexidine Gluconate) can be used in pre- and post-8 operative dental surgery procedure. Additionally, it can be combined with the other materials 9 commonly used to treat teeth in endodontics, such as dental cements.
1002291 A prophylactic application of trehalase in combination with antimicrobials (including 11 natural antimicrobials, such as medicinal plant-derived essential oils or their active compounds) 12 in dental hygiene includes its use in mouthwashes, toothpastes, dental floss, and chewing gum.
13 Trehalase in combination with antimicrobials can be included into conventional non-alchohol-14 containing mouthwashes (to avoid alcohol-induced denaturation of the enzyme); such compositions also typically include menthol, thymol, methyl salicylate, and eucalyptol. Inclusion 16 of trehalase in combination with antimicrobials in toothpaste is straightforward, without 17 chemical interaction with components of conventional toothpaste; typical toothpaste 18 formulations comprise: abrasive 10-40%, humectant 20-70%, water 5-30%, binder 1-2%, 19 detergent 1-3%, flavor 1-2%, preservative 0.05-0.5% and therapeutic agent 0.1-0.5%.
Impregnation of dental floss fibers with trehalase in combination with antimicrobials is 21 analogous to inclusion of flavorings used in dental floss materials such as silk, polyamide, or 22 Teflon. Finally, trehalase in combination with antimicrobials (alone or with other saccharidases) 23 can be included in chewing gum compositions to prevent the formation of dental plaques and 24 bacterial biofilms, as well as to treat oral biofilm-based infections in treatment protocols with antimicrobials.
26 1002301 Mitigation of Ingestion of Excess Trehalose by Susceptible Individuals 27 1002311 Owing to its unique chemical structure and properties, trehalose remains stable under 28 low pH conditions, even at elevated temperatures, and has the ability to protect proteins in a wide 29 range of temperature changes, including deep freezing, that makes it an attractive substance for CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 use as a stabilizer and conservant for various products in medical and food industries. Over the 2 last two decades, the agro-food industry has introduced the use of trehalose in many foodstuffs as 3 a food stabilizer, sweetener, and a moisture retainer, since the high stability of trehalose enables 4 the original product characteristics to be retained even after heat processing, freezing, and prolonged storage. Usually, the product labeling does not indicate the presence or amount of this 6 food additive. Patients exhibiting biofilm-based infections, especially those with genetic 7 trehalase enzyme deficiency, can be at increased risk upon consumption of the dietary trehalose, 8 as the excess of this sugar can be used by the gut bacteria for local GI
tract biofilm formation.
9 For mitigation of these negative events, enzyme trehalase can be added in a time-delayed release form to existing formulations of digestive and systemic enzymes, to avoid negative 11 consequences upon consumption of excess amount of dietary trehalose.
12 [00232] Embodiments for the Treatment of Biofilm-Based Infections on Medical Devices and 13 Medical Equipment 14 1002331 The methods for treatment of biofilm-based infections on medical devices and medical equipment exposed to bodily fluids and tissues comprise two categories: preventive and 16 curative. The preventive methods of the present compositions and methods rely on altering the 17 device and equipment surfaces by using trehalase in combination with antimicrobials, whereas 18 curative methods exploit temporary exposure of such surfaces to treatment compositions of 19 trehalase with antimicrobials and other compounds in various treatment protocols.
[00234] Preventive Methods 21 [00235] To prevent microbial biofilm growth on medical devices and equipment surfaces, 22 trehalase in combination with antimicrobials can be used in coatings (both delayed-release and 23 non-delayed release) and for immobilization on the surfaces of such devices and equipment.
24 Simple (non-delayed release) coatings, comprising trehalase with antimicrobials, can be applied to metal and polymer surfaces, and fabric materials to provide a brief initial exposure of biofilm 26 and biofilm-forming pathogens to treatment combinations. Delayed-release coatings can release 27 trehalase and antimicrobials into the surrounding environment over time to gradually degrade 28 biofilm and kill the embedded pathogens, ultimately depleting the initial amount of coating 29 contained enzyme and antimicrobials. In contrast to these coatings, when combinations of CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalase with antimicrobials are immobilized on a surface of medical devices and equipment, 2 the enzyme can act as a permanent, reusable catalyst, providing the potential for ongoing 3 degradation of biofilm and providing antimicrobials with continuous direct access to biofilm-4 embedded pathogens.
1002361 Treatment coatings, containing trehalase in combination with antimicrobials, can be 6 applied to non-porous surfaces (metal and polymer surfaces of medical devices and equipment) 7 and porous surfaces, such as those of fabric and fabric-based surgical sewing material, surgical 8 mesh used for hernia repair, surgical wounds, burns and skin lesions dressing materials. A
9 foremost example is a method to prevent biofilm formation and growth on prosthetic heart valves by impregnating the fabric sewing cuff with trehalase in combination with antimicrobials before 11 attachment of the cuff to the heart valve assembly; additionally, the heart valve assembly can be 12 covered with an immobilized coating comprising trehalase in combination with antimicrobials.
13 Delayed-release coatings that discharge trehalase in combination with antimicrobials over time 14 offer the prospect of prophylactic action against the formation of microbial biofilms on the biofilm-vulnerable surfaces of medical devices and on temporary and permanent bodily implants.
16 The delayed-release coatings can include combination of trehalase with antimicrobials embedded 17 in surface porosity either pre-existing or specially-created at the surface, surface-attached 18 microencapsulated trehalase with antimicrobials, and dissolvable coatings overlaying the 19 combination of trehalase with antimicrobials on the surface. The methods of adhesion to the device or implant surfaces, and the mechanisms of time release of agents of interest are well 21 known in the prior art and can be modified to exploit the use of trehalase in combination with 22 antimicrobials in the present compositions and methods.
23 1002371 Trehalase in combination with antimicrobials can be immobilized (as discussed below 24 in greater detail with respect to curative methods) on the biofilm-vulnerable surfaces of medical devices and equipment. The methods of enzyme immobilization on polymer and metal surfaces 26 is described in detail by Drevon GF ("Enzyme Immobilization into Polymers and Coatings,"
27 PhD Dissertation, University of Pittsburgh, 2002). Immobilization of trehalase with 28 antimicrobials on the surface of medical devices and equipment can be combined with other 29 materials of antimicrobial nature, such as medical silver and medical copper, and/or with biofilm CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 attachment preventives like BacticentTM KB. Combination of trehalase with antimicrobials can 2 be immobilized on a compound that serves as a support structure, and this support structure 3 compound can be bound to device surfaces. This method insures that trehalase enzymatic activity 4 is preserved by avoiding possible direct interaction of trehalase with the device surfaces. From among the numerous candidate support structure compounds, a choice can be optimized with 6 respect to maintaining the enzymatic activity of trehalase while achieving high binding affinity 7 to the device surfaces.
8 [00238] Combination of trehalase with antimicrobials, as treatment coatings both delayed-9 release and non-delayed release, as well as immobilized trehalase in combination with antimicrobials, can be used on the interior and exterior surfaces of central venous catheters and 11 urinary catheters, and on the biofilm-vulnerable surfaces of endoscopes and implants of various 12 types, including orthopedic implants.
13 [00239] The surfaces of implantable and bodily-inserted devices are targets of both the 14 immune response and bacterial colonization, a so-called "race for the surface" (Gristina A, "Biomedical-centered infection: microbial adhesion versus tissue integration,"
Clinical 16 Orthopedics and Related Research, 2004, No. 427, pp. 4-12.). In the case of the immune 17 response acting first, the macromolecule adhesion and general inflammatory action can lead 18 ultimately to the enclosure of the device surface by a nonvascular fibrous capsule which further 19 can support bacterial colonization and biofilm formation. If bacterial colonization occurs before overt immune response, biofilm can form immediately adjacent to the device surface. Since both 21 the accumulation of host cells at the device surface and bacterial colonization of the surface have 22 initial macromolecule adhesion in common, defeat of such adhesion in vivo is synergistic with 23 use of trehalase in combination with antimicrobials to impede biofilm formation.
24 [00240] For this purpose, trehalase in combination with antimicrobials can be combined with new coatings that offer the promise of deterring macromolecule adhesion to synthetic surfaces.
26 Among examples are Semprus SustainTM technology, a polymeric approach to harnessing water 27 molecules at device surfaces to impede macromolecule attachment, Optichem antifouling 28 coating with microporosity excluding macromolecule contact with the protected device surface, 29 and zwitterionic coatings (Brault ND, Gao C, Xue H, Piliarik M, Homola J, Jiang S, Yu Q, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 "Ultra-low fouling and functionalizable zwitterionic coatings grafted onto SiO2 via a biomimetic 2 adhesive group for sensing and detection in complex media," Biosens Bioelectron., 2010 Jun 15, 3 25(10): 2276-2282.) that suggest the prospect of defeating protein adhesion through the 4 exploitation of periodic reversal of polarity in the surface coating.
Delayed-release coatings which include trehalase in combination with antimicrobials, can be used in concert with 6 macromolecule-repellant coatings in various modes. For example, the combination of trehalase 7 with antimicrobials time release sites can be established with adequate density within the 8 confines of a macromolecule-repellant coating. Alternatively, disparate coatings can be 9 interleaved in various geometries both parallel and perpendicular to the device surface.
1002411 Curative Methods 11 1002421 Methods of the present compositions and methods that address degradation and 12 removal of biofilms and associated pathogens from surfaces involve various soak (immersion) 13 and rinse protocols. Solutions of trehalase in combination with antimicrobials and other 14 compounds, such as other enzymes, chelating agents, and stabilizers are anticipated. In a preferred embodiment of a soak solution, the present inventive use of trehalase enzyme to 16 degrade the biofilm gel matrix can be viewed as an important addition to enzyme mixtures found 17 in such products as the aforementioned Biorem. Immersive exposure to trehalase-based soak 18 solutions can be followed by exposure to various biocidal treatments, as are well known in the 19 prior art, for elimination of biofilm-forming pathogens. Rinse and soak solutions containing trehalase should be maintained at the temperature of maximum enzyme activity.
Also, soak and 21 immersion durations should be made sufficient for effectiveness.
22 1002431 A preferred method of solution-based treatment comprises the following multi-step 23 procedure:
24 [00244] 1 - creating a first treatment solution taken from the group comprising trehalase with antimicrobials and saccharidases in aqueous or saline solution, 26 1002451 2 - creating a second treatment solution taken from the group comprising: a) 27 proteolytic enzymes in aqueous or saline solution, b) fibrinolytic enzymes in aqueous or saline 28 solution, and c) lipolytic enzymes in aqueous or saline solution, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00246] 3 - creating a third treatment solution taken from the group comprising: a) biocides in 2 aqueous or saline solution, b) antibiotics, specific to the infectious agents present in aqueous or 3 saline solution, or c) polymicrobial antibiotics in aqueous or saline solution, d) natural 4 antimicrobials of wide range of action in aqueous or saline solution, [00247] 4 - flushing or rinsing the surface under treatment with these solutions (or immersing 6 such surface in these solutions) in the sequence given.
7 [00248] The exposure time for the treated surface should be sufficient for effectiveness, and 8 such solution treatments should take place in a manner that avoids exposure of trehalase with 9 antimicrobials and other saccharidases to proteolytic enzymes.
1002491 This multi-step procedure can be applied to treatment of central venous and urinary 11 catheters, endoscopes, contact lenses and lens cases, dialysis system components, dental unit 12 water lines, and other medical devices that can be subjected to immersion, rinse, or fluid 13 injection. In the case of dialysis systems, various surfaces that contact biological fluids must be 14 disinfected: some surfaces can be immersed in treatment solutions with the option of ultrasound-assisted cleaning, other surfaces are not immersible and simply must be soaked and flushed with 16 treatment solutions. Also, for dialysis system components and dental unit water line treatment, 17 the aforementioned third solution additionally can contain chelating agents and enzyme 18 stabilizers.
19 [00250] An alternative avenue of delivery of trehalase in combination with antimicrobials involves immobilization of such combination by attachment to a support structure compound of 21 some kind. In contrast to immobilization on device surfaces, as discussed above, the 22 combination of trehalase with antimicrobials can be immobilized on a support structure 23 compound that is in liquid suspension for use as a treatment liquid.
Such immobilization of 24 trehalase in combination with antimicrobials can permit its extended presence and repeated use in catalysis. Additionally, it can increase the enzyme's catalytic efficiency and thermal stability 26 based on the specifics of its attachment to the support structure. There are five general categories 27 of such immobilization: a) adsorption, b) covalent binding, c) entrapment, d) encapsulation, and 28 e) cross-linking (Walker JM, Rapley R, and Bickerstaff GF, "Immobilization of Biocatalysts" in 29 Molecular Biology and Biotechnology, 4th edition, edited by J. M. Walker and R. Rapley, RSC

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Publishing, 2007). All such mechanisms are within the scope of the present compositions and 2 methods. In the delivery of trehalase in combination with antimicrobials to biofilm, some 3 immediate implementations of immobilization are envisioned herein. For example, trehalase in 4 combination with antimicrobials can be covalently bound to microspheres, as discussed below, or encapsulated in liposomes after the fashion of U. S. Patent 7,824,557 (which discloses the use 6 of antimicrobial-containing liposomes to treat industrial water delivery systems). These delivery 7 mechanisms can be incorporated by uptake into the biofilm matrix to provide sustained exposure 8 to trehalase in combination with antimicrobials.
9 1002511 The feasibility of immobilization of trehalase in combination with antimicrobials is underscored by examples of trehalase immobilization for various non-treatment purposes that 11 can be found in the recent research literature. For analytical purposes, Bachinski et al.
12 demonstrated the immobilization of trehalase on aminopropyl glass particles by covalent 13 coupling that allowed the enzyme to retain its catalytic activity (N.
Bachinski, A. S. Martins, V.
14 M. Paschoalin, A. D. Panek, and C. L. Paiva, "Trehalase immobilization on aminopropyl glass for analytical use," Biotechnol Bioeng., 1997 Apr 5, 54(1): 33-39.). For reactor reuse, trehalase 16 has been immobilized on chitin as well (A. S. Martinsa, D. N. Peixotoa, L. M.C. Paivaa, A. D.
17 Paneka and C. L.A. Paivab, "A simple method for obtaining reusable reactors containing 18 immobilized trehalase: Characterization of a crude trehalase preparation immobilized on chitin 19 particles," Enzyme and Microbial Technology, February 2006, Volume 38, Issues 3-4, Pages 486-492.). The present compositions and methods include immobilization of enzyme trehalase 21 in combination with antimicrobials on support structures that have particular affinity for 22 biofilms. U. S. Patent Application No. 20060121019 discloses the covalent and non-covalent 23 attachment of biofilm degrading enzymes to "anchor" molecules that have an affinity for the 24 biofilm; moieties cited as having a known affinity for biofilms included Concanavalin A, Wheat Germ Agglutinin, Other Lectins, Heparin Binding Domains, enzyme Elastase, Amylose Binding 26 Protein, Ricinus communis agglutinin I, Dilichos biflorus agglutinin, and Ulex europaeus 27 agglutinin I.
28 [00252] A preferred method of using immobilized trehalase in combination with 29 antimicrobials in liquid treatment comprises the same solution-based multi-step procedure CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 outlined above, but using immobilized trehalase in aqueous or saline suspension. Likewise, the 2 method is similarly applicable to treatment of the same categories of medical devices disclosed 3 above.
4 [00253] As mentioned earlier, ensonification of the surface to be treated can be employed to augment the removal of microbial biofilm concomitantly with soak and rinse solutions. Apart 6 from the traditional use of ultrasound for biofilm removal, an additional modality that is within 7 the scope of the present compositions and methods is the use of ultrasound to assist enzymatic 8 activity. The introduction of a low energy, uniform ultrasound field into various enzyme-9 containing solutions can greatly improve their effectiveness by significantly increasing their reaction rate. The process is tuned so that cavitation does not result in reduction of the enzyme 11 activity, but rather results in its significant increase.
12 [00254] It has been established that the following specific features of combined 13 enzyme/ultrasound action are critically important: a) the effect of cavitation is several hundred 14 times greater in heterogeneous systems (solid-liquid) than in homogeneous, b) in water, maximum effects of cavitation occur at ¨50 C, which is the optimum temperature for many 16 industrial enzymes, c) cavitation effects caused by ultrasound greatly enhance the transport of 17 enzyme macromolecules toward substrate surface and, d) mechanical impacts, produced by 18 collapse of cavitation bubbles, provide an important benefit of "opening up" the surface of 19 substrates to the action of enzymes (Yachmenev V, Condon B, Lambert A, "Technical Aspects of Use of Ultrasound for Intensification of Enzymatic Bio-Processing: New Path to "Green 21 Chemistry", "Proceedings of the International Congress on Acoustics, 2007). Enzyme reaction 22 rates can be increased by more than an order of magnitude. In an example of specific enzyme 23 application, alpha-amylase reaction rates were increased with the use of ultrasound (Zhang Y, 24 Lin Q, Wei IN, and Zhu HJ, "Study on enzyme-assisted extraction of polysaccharides from Dioscorea opposite," Zhongguo Zhong Yao Za Zhi. 2008 Feb, 33(4): 374-377.).
For ultrasound-26 assisted enzyme-based treatment, the solution-based multi-step treatment previously disclosed, 27 can be modified to include ensonification of enzyme-containing treatment solutions and surfaces 28 under treatment.
29 [00255] Embodiments to Address Industrial Biofilms.
CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00256] There are numerous industrial biofilm treatment approaches that can be enabled by 2 the use of trehalase enzyme in combination with antimicrobials. These approaches involve both 3 creation of appropriate mixtures of trehalase with antimicrobials and other compounds, and 4 development of methods for delivery of these mixtures to the sites of biofilm presence.
[00257] With respect to treatment mixtures, the combination of trehalase with antimicrobials 6 can be used alone in aqueous or saline solution or can be added to compounds that maintain the 7 optimum pH range (buffer compositions), and metallic ion concentrations that can maximize the 8 hydrolysis rate of trehalose. Additionally, one or more combinations of trehalase with 9 antimicrobials can be added to compositions of dispersants, surfactants, detergents, other enzymes, and biocides that are delivered to the biofilm in order to achieve synergistic effects.
11 Also, trehalase in combination with antimicrobials can be used as a pretreatment step in various 12 protocols involving other biofilm treatment compounds and/or methods.
13 [00258] Also, combination of trehalase with antimicrobials can be immobilized on substrate 14 compounds in liquid suspensions, as discussed above, for use in industrial treatments, where the substrate compound may have an affinity for the target of treatment.
16 [00259] For oil pipelines, an oil-water emulsion containing trehalase in combination with 17 antimicrobials will provide a dosing opportunity to treatment of the biofilms within the pipeline.
18 These emulsion-borne mixtures can include combination of trehalase with antimicrobials alone, 19 or with additional conventional treatment compounds such as other biocides, surfactants, detergents, and dispersants as are well known in the prior art.
21 [00260] A specific treatment embodiment for pipelines involves the exploitation of annular 22 liquid flow geometries. The annular flow pattern of two immiscible liquids having very different 23 viscosities in a horizontal pipe (also known as "core¨annular flow") has been proposed as an 24 attractive means for the pipeline transportation of heavy oils since the oil tends to occupy the center of the tube, surrounded by a thin annulus of a lubricant fluid (usually water) (Bannwar 26 AC, "Modeling aspects of oil¨water core¨annular flows," Journal of Petroleum Science and 27 Engineering Volume 32, Issues 2-4, 29 December 2001, Pages 127-143.). A
thin water film can 28 be introduced between the oil and the pipe wall to act as a lubricant, giving a pressure gradient 29 reduction. In 8-inch diameter pipes, it has been shown that, under certain conditions, it is CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 possible to use very thin water films. For crude oils with viscosities exceeding 2000 mPas, stable 2 operation has proved feasible with as little as 2 % water (Oliemans RVA, Ooms G, Wu HL, 3 Duijvestijn A, "Core-Annular Oil/Water Flow: The Turbulent-Lubricating-Film Model and 4 Measurements in a 2-in. Pipe Loop," Middle East Oil Technical Conference and Exhibition, 11-14 March 1985, Bahrain.). In an embodiment of the present compositions and methods to 6 address delivery of trehalase with antimicrobials-containing solutions to the interior of oil 7 pipelines, the thin water film is replaced by a trehalase with antimicrobials in aqueous solution.
8 This combination of trehalase with antimicrobials in aqueous solution will be a flowing annular 9 layer immediately adjacent to the inner surface of the pipeline.
1002611 Another embodiment of the compositions and methods, addressing microbial biofilm 11 in the oil pipelines, comprises the exploitation of magnetic force to deliver trehalase in 12 combination with antimicrobials to the target treatment sites within pipelines. Specifically, 13 combination of trehalase with antimicrobials can be immobilized on a support structure 14 compound that exhibits either magnetic or preferably ferromagnetic properties. When this immobilized combination of trehalase with antimicrobials is released into pipeline flow, a 16 magnetic field exterior to the pipeline can be used to guide and retain the immobilized 17 combination of trehalase with antimicrobials in the target vicinity on the interior of the pipeline.
18 The magnetic field can be generated by magnetic or electromagnetic means well known in the 19 prior art. Optimization of this embodiment could include spatial and temporal variation of the generated magnetic field to achieve appropriate concentration of trehalase in combination with 21 antimicrobials at treatment sites in the presence of fluid flow.
Residual magnetism induced in 22 the pipeline wall can be diminished by methods well known in the prior art.
23 1002621 Dry dock removal of hull biofouling material, including biofilms, can use aqueous 24 solutions containing trehalase in combination with antimicrobials in rinse and/or soak protocols.
Application of hydrogel containing trehalase in combination with antimicrobials to ships' hull is 26 another means of ensuring sustained exposure of the biofilm for hydrolysis of the trehalose 27 component of the biofilm matrix and initial elimination of biofilm-embedded microorganisms.
28 This can be done prior to or at the time of additional biocide application. Further, the biofilm 29 preventive coatings, containing combination of trehalase with antimicrobials, can be CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 immobilized on marine surfaces. The solution-based, multi-step treatment that includes trehalase 2 in combination with antimicrobials discussed for medical device treatment can be used in the 3 marine surface applications, or it can be modified to use gel delivery of treatment compounds 4 instead of aqueous or saline solutions.
1002631 For HVAC systems the solution-based multi-step treatment method that includes 6 combination of trehalase with antimicrobials and other compounds, can be used as stated for 7 certain components such as cooling coils and drain pans, or modified so that treatment 8 compounds can be fed into HVAC ductwork in the form of aerosols.
9 1002641 Candidate industrial biocides for use along with trehalase in combination with antimicrobials include popular industrial biocide products on the market such as Ultra KleenTM
11 manufactured by Sterilex Corp., Hunt Valley, MD , the active ingredients of which comprise:
12 [00265] n-Alkyl(C14 60%, C16 30%, C12 5%, C18 5%) dimethylbenzylammonium chloride;
13 and 14 1002661 n- Alkyl(C12 68%, C14 32%) dimethylethylbenzylammonium chloride.
1002671 Another example is SWG Biocide manufactured by Albermarle Corp., Baton Rouge, 16 LA, the active ingredients of which comprise sodium bromosulfamate and sodium 17 chlorosulfamate. Candidates may also be found among the wider generic categories of industrial 18 biocides comprising: glutaraldehyde, quaternary ammonium compounds (QACs), blends of Gut 19 and QACs, Amine salts, Polymeric biguanide, benzisothiazolone, blend of methyl isothiazolones, and acrolein (Handbook of Biocide and Preservative Use, Edited by H. W.
21 Rossmoore, Chapman and Hall, 1995).
22 1002681 For treatment of biofilms associated with food processing, storage, and transport 23 systems, conventional enzyme treatments can be augmented with the use of trehalase in 24 combination with antimicrobials. This can be done in the context of the solution-based multi-step procedure. In addition, the present compositions and methods include the use of trehalase in 26 combination with antimicrobials and other enzymes in one-step procedure, given that added 27 enzymes are not proteolytic. Also, ultrasound-assisted enzyme-based cleaning is applicable with 28 the use of trehalase in combination with antimicrobials.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1002691 Biofilms are found in the household environment on many surfaces including the 2 inside surfaces of plumbing and drainpipes, on the surfaces of sinks, bathtubs, tiling, shower 3 curtains, shower heads, cleaning sponges, glassware, toothbrushes, and toilets. Aqueous- or 4 saline- based solutions containing trehalase in combination with antimicrobials can be used alone or in proper combination with other biofilm treatment products tailored to the applicable surfaces 6 in cleaning procedure. For example, certain compounds used for plumbing treatment would be 7 inadmissible for treating toothbrushes. The aforementioned solution-based multi-step procedure 8 easily can be applied to many household surfaces with the exception of the internal surfaces of 9 plumbing.
1002701 Test Results. There now follows a summary of various in vitro test results using 11 Trehalase to determine Trehalase potential in gram-positive and gram-negative bacteria and 12 determine the effect of Trehalase in combination with the antibiotics ceftazidime (CAZ), 13 gentamicin (GENT) and tobramycin (TOB) both with grown biofilms, added at the beginning of 14 experiments, and added to an early 24-hour grown and late 48-hour grown preformed biofilms during a 24-hour exposition. The effect of Trehalase in gram-positive bacteria (MRSA and 16 MSSA) S. aureus and in gram-negative bacteria P. aeruginosa is also summarized. Following 17 this summary is a more detailed explanation of the experimental process with supporting graphs 18 and charts for the results. The tests were conducted and summarized by the Drug Discovery and 19 Development Pharmaceutical Services Company Aptuit, LLC in Verona, Italy.
[00271] It is well known that Gram-positive and Gram-negative bacteria use a ubiquitous 21 multifunctional sugar, i.e., disaccharide trehalose, as a general stress response metabolite and 22 osmoprotectant, to form biofilm as a protective cover against harmful environmental factors, and 23 to preserve integrity of the bacterial cells for survival in a hazardous environment (including the 24 milieu of the human body). The results from the testing show that the enzyme Trehalase, highly specific for degrading disaccharide trehalose (one substrate ¨ one enzyme), can be effectively 26 used for prevention and treatment of microbial biofilms, overcome bacterial resistance to 27 antimicrobials, and increase the effectiveness of existing treatment modalities. Due to structural 28 differences of Gram-positive and Gram-negative bacteria, the effect of applied enzyme Trehalase 29 can have some special features in both types of bacteria.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1002721 Because Trehalose is a structural element in the cell wall of Gram-positive bacteria, 2 the use of the external enzyme Trehalase can affect not only biofilm formation, but also can exert 3 certain effects on the bacterial cell wall and cell membrane, increasing their permeability to 4 antibiotics, and possibly decreasing antimicrobial resistance. Gram-negative bacteria has the intrinsic enzyme Trehalase present in the outer membrane space (acidic Trehalase) that degrades 6 trehalose released from the cytosol for recycling, and metabolizes trehalose taken from outside 7 the cell for intracellular utilization. In Gram-negative bacteria, the application of external 8 enzyme Trehalase may require more time for addressing the biofilm formation, and taking longer 9 to exert an effect on the cell membranes, but still influencing their permeability to antibiotics and decreasing antimicrobial resistance in many cases.
11 1002731 The results of in vitro studies performed by Aptuit, LLC
demonstrated some 12 differences in the effect of the enzyme Trehalase (alone and in combination with antibiotics) on 13 microbial biofilms produced by Gram-positive and Gram-negative bacteria.
The enzyme tested 14 in the study was an enzyme of mammalian origin, i.e., pig kidney Trehalase, produced by "Sigma-Aldrich" (USA), which in some tests was later purified by a dialysis procedure 16 developed by Aptuit/Verona S.r.l. as will be explained briefly below.
17 [00274] Various parameters that were evaluated in each part of the analysis include: 1) 18 Biofilm mass formation (% inhibition ¨ by Crystal Violet staining method); 2) Bacterial cell 19 viability (% inhibition ¨ by Resazurin assay); and 3) Bacterial cell growth (by Colony Counting ¨ CFU/ml plating).
21 [00275] One in vitro test was an exploration of the Trehalase potential in gram-negative 22 bacteria and an exploration of Trehalase Potential in Gram-Negative Bacteria (P. aeruginosa 23 PA01 ¨ reference strain), and which showed an effect of Trehalase alone added to the media 24 (TSB) at the beginning of a 72 hour experiment. Trehalase alone inhibited biofilm mass formation in a dose-dependent manner. From various doses (0.023, 0.046, and 0.092 U/well) the 26 highest effect was seen with 0.092 U/well. The "U" corresponds to the International Units for 27 the enzyme in each testing well. This dose was further used in subsequent experiments.
28 Compared to a control, Trehalase alone inhibited the biofilm mass formation during 72 hours of 29 biofilm growth, with the most significant inhibition noted at the initial (4 hour) stage of biofilm CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 development at 83.3%. In an early (24 hour) mature biofilm inhibition was 82.4%, and in fully 2 mature (48 hour) biofilm inhibition was at 91.0%. In an old stage (72 hour of growth), the 3 biofilm formation was still inhibited by about 50.0%. It appears that Trehalase alone did not 4 affect the bacterial cell viability and growth at various stages of the biofilm development (4 hours, 24 hours, and 48 hours), as confirmed by Resazurin assessment and bacterial counting 6 (CFU/ml), i.e., colony-forming unit per milliliter. At the same stages of biofilm development (4 7 hours, 24 hours, and 48 hours), no effect on biofilm formation and cell viability was seen with 8 solvent present in the Sigma Trehalase preparation (Glycerol/water 50/50 solution and 1%
9 Triton-X100) used in the testing.
1002761 A next series of tests explored the effect of Trehalase in combination with the 11 antibiotics Ceftazidime (CAZ) and Tobramycin (TOB) in 1/4 and 1/8 sub-MIC (Minimum 12 Inhibitory Concentration) doses in 24 hour to 48 hour grown biofilms that were added at the 13 beginning of the experiments. Trehalase alone demonstrated continuing inhibitory effect on the 14 biofilm mass formation with longer exposition time (about 65% inhibition in a 48 hour grown biofilm) with no effect on bacterial cell viability and growth in both 24 hour and 48 hour grown 16 biofilms. Both antibiotics (CAZ and TOB) in sub-MIC doses (1/8 and 1/4 MIC), introduced to 17 the media at the beginning of experiment, triggered the Biofilm Induction Response (BM) in 24 18 hour grown and in 48 hour grown biofilm, increasing biofilm mass formation by 1.2 to 1.3 19 (CAZ) and 1.3 to 1.7 fold (TOB) compared to the Control level, i.e., the level of biofilm mass formed by P. aeruginosa PA01 in the absence of antibiotics. When Trehalase was added to 21 antibiotics, it abrogated the biofilm induction response "Blit" to both antibiotics (CAZ and TOB) 22 in the 24 hour grown biofilm down to the Control level, with an additional reduction (about 23 30%) in the biofilm mass formation only for the Trehalase/TOB
combination. With a longer 24 exposition time in the 48 hour grown biofilm, Trehalase added to antibiotics, demonstrated higher effect, abrogating biofilm induction response "Blit" to both antibiotics down to the 26 Control level, with an additional reduction of biofilm mass formation by about 65% for the 27 Trehalase/CAZ combination and by about 70% for the Trehalase/TOB
combination (p<0.001;
28 p<0.001 compared to the Control). Tobramycin alone (at a dose of 1/4 MIC) inhibited cell 29 viability (by Resazurin assay) by about 25% in both the 24 hour and 48 hour grown biofilms.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 The Tobramycin/Trehalase combination enhanced this inhibitory effect up to 45% only in the 24 2 hour grown biofilm. Ceftazidime alone at a dose of 1/8 and 1/4 MIC
inhibited cell viability 3 (Resazurin assay) by about 25% to 50% only with the longer exposition, i.e., in the 48 hour 4 grown biofilm. The CAZ/Trehalase combination did not enhance this effect.
1002771 A next series of tests explored the effect of Trehalase alone and in combination with 6 the antibiotics Ceftazidime (T+CAZ) and Tobramycin (T+TOB) in 1/4 MIC
doses, added to the 7 early (24 hour grown) preformed biofilm during the 24 hours of further exposition. When added 8 to the early (24 hour grown) pre-formed biofilm, Trehalase alone inhibited further biofilm mass 9 formation by about 30% (p=0.08) (during the 24 hour exposition). Both antibiotics (CAZ and TOB) in sub-MIC doses (1/4 MIC) added to the early (24 hour grown) preformed biofilm, 11 triggered "BIR", increasing biofilm mass formation by about 1.6 (CAZ) and about 1.4 (TOB) 12 compared to the Control level (i.e. the level of biofilm mass formed by P. aeruginosa PA01 in 13 the absence of antibiotics). Trehalase in combination with antibiotics, added to 24 hour 14 preformed biofilm, abrogated "BIR" to both antibiotics, and additionally slightly reduced biofilm mass formation by about 10% (T+CAZ) and about 37% (T+TOB) compared to the Control level.
16 Trehalase alone and in combination with both antibiotics (T+CAZ and T+TOB) as added to a 24 17 hour preformed biofilm did not inhibit bacterial cell viability and growth by Resazurin assay and 18 Colony counting (CFU/ml).
19 1002781 For the Gram-negative pathogen P. aeruginosa PA01, trehalase alone added at the beginning of the experiment significantly inhibited the biofilm mass formation during the 72 21 hours of biofilm growth, with the highest inhibition noted at the initial (4 hour) stage of biofilm 22 development at 83.3%. In the early (24 hour) mature biofilm, the inhibition was 82.4%. In the 23 fully mature (48 hour) biofilm, the inhibition was 91.0%. In the old stage of growth (72 hour), 24 the biofilm formation was still inhibited by about 50.0%. Trehalase alone did not appear to affect bacterial cell viability and growth at various stages of the biofilm development (4 hour, 24 26 hour, 48 hour, and 72 hour), as confirmed by Resazurin assay and Bacterial Cell Counting 27 (CFU/ml). Trehalase in combination with the antibiotics cephalosporin Ceftazidime and 28 aminoglycoside Tobramycin in sub-MIC doses (1/8 and 1/4 MIC), was effective in the 24 hour 29 to 48 hour grown biofilms, first by abrogating the Biofilm Induction Response (BIR) to both CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 antibiotics, and second by continuing inhibition of biofilm mass formation with longer 2 exposition time, with the additional reduction of biofilm mass by about 65% (T+CAZ) and about 3 70% (T+TOB) (p<0.001; p<0.001 compared to the Control), which occurred in the 48 hour 4 grown biofilm. Trehalase slightly potentiated the inhibitory effect of Tobramycin on biofilm cell viability (by Resasurin assessment) in the 24 hour grown biofilm by about 45%
inhibition for the 6 Trehalase/Tobramycin combination, compared to about 25% inhibition for the Tobramycin 7 alone.
8 [00279] These tests show positive results for the use of Trehalase as an adjuvant to antibiotics 9 for the prevention and treatment of biofilm-based chronic infections caused by Gram-negative pathogens, including drug-resistant bacteria. The Biofilm Induction Response "BlR" to sub-11 MIC doses of antibiotics is a known phenomenon and is considered to be the first step in 12 antimicrobial resistance in Gram-positive and Gram-negative bacteria, and is important in 13 clinical practice during the "adjustment" period of antibiotic use, both at the beginning and the 14 end of the treatment course.
[00280] In all of these tests, a Trehalase product from Sigma-Alrich was used as derived from 16 porcine kidney with a buffered aqueous glycerol solution of greater than or equal to 1.0 17 units/milligram protein. An example product can be acquired from Sigma-Alrich as:

Product Number: T8778 21 CAS Number: 9025-52-9 22 MDL: MFCD00132462 TEST SPECIFICATION
Appearance (color) Colorless to light yellow Appearance (form) Liquid Appearance (turbidity) Clear to slightly hazy CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 units/mg protein - one unit will convert 1.0 > 1.0 micromole of Trehalose to 2.0 micromoles of glucose per minute at pH 5.7 and at 37 C
(liberated glucose determined at pH 7.5) alpha-Galactosidase < 1%
alpha-Glucosidase < 1%
13-Glucosidase <5%
Invertase <1%
Amylase <2%
mg protein/ml (BCA) 0.5 ¨ 10.0 3 [00281] There now follows greater details about tests that show the effect of Trehalase in 4 gram-positive bacteria (MRSA and MSSA S.aureus). A first test showed the effect of Trehalase on initial biofilm formation by MRSA S.aureus ATCC 25923 (lab strain).
Trehalase alone (at a 6 dose of 0.092 U/well) was added to the media as a Trypticase soy broth (TSB) at the beginning 7 of the experiment, and inhibited the initial biofilm mass growth (p<0.001 compared to the 8 Control), most significantly (about 85% to about 90%) at the very early stages (3 hour and 7 9 hour) of biofilm formation, and about 67% in the early (28 hour) mature biofilm. It inhibited viability of bacterial cells in the biofilm (about 84%, about 81%, and about 63% correspondingly 11 by Resazurin assay), and reduced the bacterial cell growth in the 24 hour biofilm (about 1.5 log 12 by CFU/ml compared to the Control). Trehalase did not affect bacterial cell viability and growth 13 in the population of planktonic cells in the supernatant removed after 24 hours of biofilm growth.
14 [00282] A second test showed the effect of Trehalase in combination with the antibiotics Ceftazidime (CAZ) and Gentamicin (GENT) in sub-MIC doses (1/4 and 1/8 MIC) in early (24 16 hour grown) biofilm formed by MRSA S. aureus ATCC 25923 [MIC Ceftazidime on S. aureus 17 ATCC 25923=8 g/mL by CLSI guidelines]; [MIC Gentamicin on S. aureus ATCC 25923=2 18 g/mL (by CLSI guidelines]. When Trehalase (at a dose 0.092 U/well) in combination with both 19 antibiotics (CAZ and GENT) in sub-MIC (1/8 and 1/4 MIC) concentrations, was added to the CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 media (TSB) at the beginning of experiment, it demonstrated a synergistic effect on all 2 parameters in the study in the 24 hour grown biofilm.
3 [00283] There was a synergistic effect of the Trehalase in combination with Ceftazidime.
4 Biofilm mass formation was inhibited by about 48% (T), by about 15% to 20% (CAZ), and about 60% (T+CAZ). Biofilm cell viability (by Resazurin assay) was inhibited by about 62% (T), by 6 about 13% (CAZ in both sub-MIC doses), and by about 88% to 90% (T+CAZ in both sub-MIC
7 doses). Biofilm cell growth (CFU/mL) was reduced by about 1.67 log ¨ 1.77 log by T+CAZ in 8 both sub-MIC doses (p<0.001, p<0.001, p<0.001 compared to the Control, Trehalase alone and 9 Ceftazidime alone correspondingly). There was no effect on planktonic cells from supernatant removed after 24 hours of incubation was recorded.
11 1002841 There was a synergistic effect of Trehalase in combination with Gentamicin. The 12 synergistic effect of Trehalase in combination with Gentamicin (T+GENT) in both sub-MIC
13 doses 1/8 and 1/4 MIC) in 24 hour grown biofilm was significantly higher than in combination 14 with Ceftazidime, demonstrating. The inhibition of biofilm mass formation by about 90% to 92% (p<0.001, compared to the Control) and the full (100%) inhibition of bacterial cell viability 16 (by Resazurin assay). It also completely abrogated bacterial cell growth in both populations of 17 the cells by about 3.73 log in biofilm cells, and by about 3.31 log in planktonic cells from 18 supernatant removed after 24 hour of incubation (down to the "limit of detection," i.e., mean ¨
19 12500 CFU/mL) (p<0.001, p<0.001, compared to Control).
[00285] A series of tests were conducted to show the effect of Trehalase in combination with 21 the antibiotics Ceftazidime (CAZ) and Gentamicin (GENT) on early (24 hour grown) and late 22 (48 hour grown) preformed biofilms formed by MRSA S. aureus ATCC 25923 [Antibiotic 23 concentrations used in the experiment: Ceftazidime 2 [tg/mL = 1/4 MIC;
Gentamicin ¨ 0.5 24 [tg/mL = 1/4 MIC]. Trehalase (at a dose 0.092 U/well) in combination with both antibiotics (CAZ and GENT) in sub-MIC (1/4 MIC) doses, added to the early (24 hour grown) and late (48 26 hour grown) preformed biofilms, demonstrated synergistic effect on all parameters in the study 27 during the next 24 hour of exposition, with the higher effect on the late preformed biofilm. The 28 combination of trehalase with a sub-MIC dose (1/4 MIC) of Gentamicin (GENT) appeared to be 29 the most efficacious.
CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00286] Trehalase alone (T) and in combination with both antibiotics, added to the early (24 2 hour) preformed biofilm, significantly inhibited further biofilm growth (during 24 hour 3 exposition) by about 48% (T), by about 56% (T+CAZ), and by about 54%
(T+GENT) [p<0.001, 4 p<0.001, p<0.001, compared to the Control and antibiotics alone]. The inhibitory effect was higher in the late (48 hour) preformed biofilm: about 80% (T), about 70%
(T+CAZ), and about 6 83% (T+GENT) [p<0.001, p<0.001, p<0.001, compared to the Control and antibiotics alone].
7 [00287] Trehalase alone, added to the early (24 hour) and late (48 hour) preformed biofilms, 8 inhibited bacterial cells viability (by Resazurin assay) approximately to the same extent of about 9 48% (T). Trehalase in combination with both antibiotics showed a robust inhibition of cell viability approximately to the same extent in both early and late preformed biofilms: by about 11 70% to 80% (T+CAZ) and about 70% to 76% (T+GENT). Trehalase alone slightly inhibited 12 biofilm cell growth in the early preformed biofilm (about 0.96 log), and showed higher inhibition 13 in the late preformed biofilm (about 1.6 log, p<0.05 compared to the Control).
14 [00288] Trehalase in combination with Ceftazidime (T+CAZ) inhibited biofilm cell growth in both early and late preformed biofilms (p<0.001), with some higher effect of combination 16 T+CAZ in the early preformed biofilm (about 1.96 log) than in the late preformed biofilm (about 17 1.64 log) [p<0.01, compared to control], and by about 1.53 log [p<0.01]
compared with CAZ
18 alone in early preformed biofilm. Trehalase in combination with Gentamicin (T+GENT) 19 significantly inhibited biofilm cell growth in both early and late preformed biofilms (by about 3.06 log and about 3.49 log correspondingly, p<0.001, p<0.001 compared to the Control). It also 21 significantly inhibited biofilm cell growth compared to inhibition by Gentamicin alone, i.e., by 22 about 2.61 log (p<0.01) in the early preformed biofilm, and by about 3.26 log (p<0.001) in the 23 late preformed biofilm.
24 [00289] A next series of tests evaluated a purified (dialyzed) Trehalase on bacterial growth, cell viability and biofilm mass formation in early biofilm (24 hour grown) and early preformed 26 biofilm (preformed for 24 hours) produced by S. aureus ATCC 25923 [experimental dialysis of 27 Sigma Trehalase using "Amicon - R Ultra - 15 Centrifugal Filter Devices"
(Sigma-Aldrich), 28 method modified by Aptuit].

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00290] In the early (24 hour grown) biofilm, dialyzed Trehalase (at a dose 0.092 U/well) 2 added at the beginning of experiment, inhibited the biofilm mass formation by about 72%
3 (p<0.001, compared to Control), with no effect on biofilm cell viability (by Resazurin assay) and 4 growth (by colony counting ¨ CFU/mL). No inhibitory effect on biofilm mass formation, biofilm cell viability and growth was detected with solvent (potassium phosphate buffer) in 6 dialyzed Trehalase. Dialyzed Trehalase (at a dose of 0.092U/well) added to 24 hour preformed 7 biofilm, inhibited bacterial cell growth (by colony counting): T -1.36E+08 CFU/mL (p<0.001, 8 compared to the Control ¨ 1.06E+09 CFU/mL). Potassium phosphate buffer (solvent in dialyzed 9 Trehalase) did not show any inhibitory effect on bacterial cell growth in 24 hour preformed biofilm.
11 [00291] These series of tests show that the dialyzed porcine kidney Trehalase (prepared with 12 potassium phosphate buffer as a solvent) was confirmed to be at least as active as the Sigma 13 Trehalase enzyme (prepared with its solvent containing: 50% glycerol /water solution + 1%
14 Triton ¨ X100 and 25 mM potassium phosphate, pH 6.5). The performed dialysis reduced to the minimum the possibility of the inhibitory effect of Sigma Trehalase solvent on biofilm formation 16 and viability of the bacterial cells.
17 [00292] Tests were conducted to show the effect of dialyzed Trehalase on biofilm formed by 18 Gram-positive Bacteria (lab strains and clinical isolates: MRSA S.
aureus ATCC25923, 19 S.aureus ATCC33591, 3¨Belgium, and VRE/VSE E. faecalis ATCC29212; II-I851165; E. cocco 14) in 24 hour grown biofilm. Due to some negative effect of potassium phosphate buffer on 21 VRE/VSE E. faecalis ATCC29212, II-I851165, E.cocco 14, the following studies were performed 22 only on MRSA and MSSA S. aureus laboratory strains and clinical isolates.
23 [00293] Results showed the effect of Trehalase alone and in combination with the antibiotics 24 Gentamicin and Vancomycin in sub-MIC doses (1/4 MIC) in the early (24 hour grown) biofilms formed by the lab strains and clinical isolates of S. aureus, regarded as the good (high), medium, 26 and poor (low) biofilm producers. All strains had the same sensitivity (MIC) to Vancomycin, 27 but a different sensitivity to Gentamicin.
28 [00294] The test showed the effect of Trehalase alone and in combination with antibiotics 29 Gentamicin and Vancomycin in sub-MIC doses (1/4 MIC) in the early (24 hour grown) biofilm CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 produced by MRSA S. aureus ATCC25923, which is a lab strain known as a "good (strong) 2 biofilm producer" (Gentamicin: MIC= 0.125 g/m1; Vancomycin: MIC=2.0 [tg/m1). There is 3 strong reproducibility of the data generated with S. aureus ATCC25923 (similar activity among 4 different experiments). In a 24 hour grown biofilm, Trehalase alone, at a dose of 0.092 UFwell, significantly inhibited biofilm mass formation by about 90% (p<0.001, compared to Control) and 6 decreased biofilm cell viability by about 40% (by Resazurin assay). Both antibiotics Gentamicin 7 (GENT) and Vancomycin (VAN) in sub-MIC doses (1/4 MIC) triggered the biofilm induction 8 response (FUR), that resulted in increased biofilm mass by two-fold (GENT) and 1.8¨fold (VAN) 9 (p<0.001, p<0.001, compared to the Negative Control), with no effect on cell viability and growth. Trehalase in combination with antibiotics, the abrogated biofilm induction response to 11 both antibiotics and further significantly decreased biofilm mass formation to the same degree as 12 Trehalase alone did: by about 90% (for both combinations T+GENT and T+VAN, p<0.001, 13 p<0.001, compared to the Control), and also inhibited biofilm cell viability by about 50%
14 (T+GENT) and by about 30% (T+VAN) (by Resazurin assay).
1002951 Tests also showed the effect of Trehalase alone and in combination with the 16 antibiotics Gentamicin and Vancomycin in sub-MIC doses (1/4 MIC) in the early (24 hour 17 grown) biofilm produced by MSSA S. aureus ATCC33591, known as a "medium biofilm 18 producer" (Gentamicin: MIC= 2.0 g/m1; Vancomycin: MIC= 2.0 [tg/m1).
Trehalase alone, at a 19 dose 0.092 UFwell, significantly inhibited biofilm mass formation by about 67% (p<0.001, compared to the Control), with no effect on biofilm cell viability (by Resazurin assay) and 21 growth (by colony counting as CFU/mL). Neither Gentamicin (GENT), nor Vancomycin (VAN) 22 in sub-MICc doses (1/4/MIC) triggered biofilm induction response. They both even showed a 23 slight reduction in biofilm mass formation: about 10% (GENT) and about 20% (VAN) compared 24 to the Control. Trehalase in combination with both antibiotics, significantly inhibited biofilm mass formation to the same degree as Trehalase alone did (by about 67% for both combinations 26 T+GENT and T+VAN, p<0.001, p<0.001, compared to the Control). Trehalase in combination 27 with Gentamicin (T+GENT) significantly inhibited biofilm cell viability (about 80% by 28 Resazurin assay), and significantly reduced bacterial cell growth (p<0.05, compared to negative 29 Controls, by colony counting as CFU/mL) in populations of both biofilm cells and planktonic CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 cells removed from supernatant after 24 hour of biofilm growth. No such effects were recorded 2 for Trehalase in combination with Vancomycin.
3 [00296] The tests showed the effect of Trehalase alone and in combination with antibiotics 4 Gentamicin and Vancomycin in sub-MIC doses (1/4 MIC) on biofilm produced by S. aureus 3-Belgium, clinical isolate, known as a "poor (low) biofilm producer"
(Gentamicin: MIC= 32.0 6 g/m1; Vancomycin: MIC= 2.0 [tg/m1). Trehalase alone or in combination with sub-MIC (1/4 7 MIC) doses of Gentamicin and Vancomycin did not induce a decrease in terms of biofilm mass 8 formation, biofilm cell viability (by Resazurin assay) and bacterial cell growth (CFU/mL).
9 [00297] Further tests were conducted to explore the MIC (Minimum Inhibitory Concentration) determination with selected S. aureus strains (MSSA Oxford and MSSA ATCC
35556, and 11 MRSA ATCC 25923) in the presence and absence of Trehalase [in vitro assay: Gentamicin, 12 Vancomycin, Ciprofloxacin - MIC in broth according to CSLI guidelines].
Addition of 13 Trehalase resulted in significant MIC value reduction with Gentamicin (up to 16-fold and 14 33-fold with clinical and reference isolates). No significant effect on Vancomycin MIC and Ciprofloxacin MIC was observed. There was testing of Trehalase on Clinical Isolates (S. aureus 16 strains: Oxford, ATCC35556, and ATCC 25923 as the reference strain) at a time-point: 24 hours 17 of biofilm growth.
18 1002981 Trehalase alone (at a dose 0.092 UFwell), added to the media at the beginning of 19 experiment, significantly reduced biofilm mass formation with all three S. aureus strains tested:
about 80% (ATCC25923), about 90% (ATCC35556), and about 50% (Oxford) (p<0.001, 21 p<0.001, p<0.05 correspondingly, compared to the Control). In the presence of Trehalase, the 22 biofilm cell viability (by Resazurin assay) was inhibited with all tested S. aureus strains: by 23 about 40% (ATCC25923), about 25% (ATCC35556), and about 60% (Oxford).
Biofilm cell 24 growth (by colony counting as CFU/mL) was significantly reduced (p<0.05 compared to Control) in biofilms formed by S. aureus ATCC25923 (lab reference strain) and S. aureus 26 Oxford (clinical isolate). No effect on planktonic cell growth was recorded. Overall, Trehalase 27 added to the media at the beginning of experiment with the early (24 hour grown) biofilm, 28 induced a reduction in biofilm mass formation and inhibition of viability of all three selected 29 MSSA and MRSA strains at a 24 hour time-checking point.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1002991 Further tests showed the effect of Trehalase, added during the initial biofilm growth 2 (up to 24 hours) and after 24 hours of initial biofilm growth (up to 24 hours of further 3 incubation) on a catheter segment (14-gauge Teflon intravenous catheter) : in vitro assay with S.
4 aureus XEN 29, adapted by Kadurugamuwa et al., 2003). The bioluminescence signal on catheter was detected with the IVIS Lumina image system, and bacterial count (both biofilm and 6 planktonic cells) was evaluated by colony counting (CFU/ml, agar plating). The addition of 7 Trehalase at the beginning of the initial 24 hour biofilm growth, induced a significant reduction 8 in biofilm mass formation on a catheter segment (inner and outer surfaces) as demonstrated by 9 significantly lower intensity of bioluminescence signal with S. aureus XEN 29 + Trehalase 0.092 UI, compared to the Negative Control (Blank), Positive Control (S. aureus XEN
29), and Vehicle 11 (S. aureus XEN 29 + buffer content 25 mM potassium phosphate, pH 6.5).
Bacterial cell growth 12 (CFU/ml) was significantly reduced in both biofilm cells and population of planktonic cells 13 (p<0.05, p<0.05, compared to Positive Control). There was no effect of Trehalase added after 24 14 hours of biofilm growth on catheter segment in terms of bioluminescence signal intensity and bacterial cell growth (CFU/ml), compared to Positive Control and Vehicle.
16 [00300] There now follows further details of the various experimental designs and materials 17 and methods used to conduct many of the tests explained above. Various charts and tables that 18 reflect the tests and conclusions drawn from these tests are also explained with reference to the 19 drawing figures.
[00301] A number of the tests explained above evaluated the potential synergistic effect of a 21 combination between trehalase and three antibiotics (at sub-MIC
concentrations) on bacterial 22 growth, viability and biofilm mass in early (24 hours) and late (48 hours) preformed biofilms 23 produced by S. aureus ATCC25923 and P. aeruginosa PA01. The experimental design in this 24 test for a first strain included:
26 Strain: P. aeruginosa PA01 27 Temperature: 35 + 2 C
28 Incubation Conditions: Static 29 Time Points: 24-48 hours preformed biofilm CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Treatment: (added to well containing a suspension of P. aeruginosa PA01 ¨107 CFU/ml) 2 Solvent Content: 50% glycerol containing 1% TritonTm X-100 and 25 mM
potassium 3 phosphate, pH 6.5 4 Trehalase Content: 0.092 UI
6 Antibiotic Concentration:
7 - Ceftazidime 0.25 ug/ml (1/4 MIC) 8 - Ceftazidime 0.25 ug/ml + Trehalase 0.092 UI
9 - Tobramycin 0.06 ug/ml (1/4 MIC) - Tobramycin 0.06 ug/ml + Trehalase 0.092 UI

12 Parameters Assessed:
13 Biofilm mass formation (staining with crystal violet 1%) 14 Cells viability (incubation with resazurin) Cell growth by colony counting (CFU/mL) 18 1003021 The experimental design in this test for the second strain included:
19 Strain: S. aureus ATCC25923 Temperature: 35 + 2 C
21 Incubation Conditions: Static 22 Time Points: 24-48 hours preformed biofilm 23 Treatment: (added to well containing a suspension of 5'. aureus ATCC25923 ¨107 CFU/ml) 24 Solvent Content: 50% glycerol containing 1% TritonTm X-100 and 25 mM
potassium phosphate, pH 6.5 26 Trehalase Content: 0.092 UI
27 Antibiotic Concentration:
28 - Ceftazidime 2 ug/ml (1/4 MIC) 29 - Ceftazidime 2 ug/ml + Trehalase 0.092 UI

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 - Gentamicin 0.5 ug/ml (1/4 MIC) 2 - Gentamicin 0.5 ug/ml + Trehalase 0.092 UI
3 Parameters Assessed:
4 Biofilm mass formation (staining with crystal violet 0.06%) Cells viability (incubation with resazurin) 6 Cell growth by colony counting (CFU/mL) 9 1003031 FIGS. 3 and 4 are the tables of the conclusions for these first and second strains.
100304] Another series of tests evaluated the dialyzed trehalase on bacterial growth, viability 11 and biofilm mass in early biofilm (24 hours growth) and early preformed biofilm (preformed for 12 24 hours) produced by S. aureus ATCC25923 (first strain) and P.
aeruginosa PA01 (second 13 strain).
14 1003051 The dialysis of trehalase used Amicon Ultra-15 centrifugal filter devices with the following procedure:
16 [00306] 1) Added up to 15 mL of sample (1,600 [IL of Trehalase SIGMA +
13,400 [IL buffer 17 potassium phosphate to the AmiconR Ultra filter device;
18 1003071 2) Placed capped filter device into centrifuge rotor (at 4,000 x g; T=4 C) for 30 19 minutes;
[00308] 3) Removed filtrate (bottom) and exchanged buffer suspension (filter device);
21 [00309] 4) Repeated steps 2 and 3 twice;
22 1003101 5) As a last step recovered 250 of ultrafiltrate Trehalase and re-suspended it with 23 1,350 [IL of buffered suspension to obtain 1,600 tL of dialyzed trehalase; and 24 [00311] 6) Read spectrum of bottom filtrate.
[00312] As noted before, the dialysis procedure of Sigma Trehalase was introduced by using 26 Amicon Ultra-

15 Cenrifugal Filter Device. As a result of this procedure, 1,600 of dialyzed 27 trehalase containing 5 UI (International Units of activity) was in buffer solution. This activity 28 was the same as in Sigma Trehalase (1,600 L, containing solvent) before the dialysis. The 29 preparation of a Trehalase dose of 0.092 UI is used as 0.092 UFwell. The volume of each well CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 was 200[tL and included 100 L Dialyzed Trehalase (or Trehalase Sigma) and 100 L of bacterial 2 suspension of P. aeruginosa or S. aureus. To prepare a total volume 100 L
of Trehalase 3 (representing 0.092 UFwell), the test used 40 L of Trehalase (5 UI
activity) + 60 L of TSBG
4 (media), so that the activity of Trehaalase was 0.092 UI/100 L, i.e.
0.92 UP mL, close to ¨
lUI/mL (1mL = 1000 L).
6 1003131 In these tests, dialyzed (purified) trehalase in a dose 0.92 UI/mL of media, inhibited 7 biofilm formation by S. aureus by 72% in 24hr-grown biofilm. Sigma Trehalase is sold as a 8 liquid solution of Trehalase (as a proteinous substance in a solvent, but without the amount of 9 protein by weight), only having activity in UIs. In the original tests, the concentration is:
=or>0.4 units/mg. One unit will convert 1.0 mole of trehalose to 2 moles of glucose per min 11 at pH 5.7 at 37 A C (liberated glucose determined at pH 7.5). This concentration as =or>0.4 12 units/mg may represent Trehalase enzyme activity as = or > 0.4 units in lmg of protein (i.e. dry 13 weight of enzyme itself). The original assay: 2.0 ¨ 6.0 mg/ml protein basis (BCA) in a buffered 14 aqueous glycerol solution. Foreign activity (including a-galactosidase, invertase, a- and 13-glucosidase, and amylase) was < or = 1%, confirming that this enzyme is highly specific only for

16 degradation of trehalose. The article by Reiko Ishihara et. al., "Molecular cloning, sequencing

17 and expression of cDNA encoding human trehalase", Gene 202 (1997) 69-74), has the specific

18 activity of human trehalase expressed in E. coli, introduced in units/mg of protein (i.e. protein

19 from lysed bacterial cells).
[00314] The experimental design for the first strain on the early biofilm included:

22 Strain: S. aureus ATCC25923 23 Temperature: 35 + 2 C
24 Incubation Conditions: Static Time Points: 24 hours 26 Treatment: (added to well containing a suspension of 5'. aureus ATCC25923 ¨107 CFU/ml) 27 Solvent 1: 50% glycerol containing 1% Tritonlm X-100 and 25 mM potassium phosphate, 28 pH 6.5 29 Solvent 2: 25 mM potassium phosphate, pH 6.5 CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 SIGMA Trehalase: 0.092 UI
2 Dialyzed Trehalase: 0.092 UI
3 Parameters Assessed:
4 Biofilm mass formation (staining with crystal violet 0.06%) Cells viability (incubation with resazurin) 6 Cell growth by colony counting (CFU/mL) 9 [00315] The experimental design for the first strain on the preformed biofilm included:
Strain: S. aureus ATCC25923 11 Temperature: 35 + 2 C
12 Incubation Conditions: Static 13 Time Points: Preformed biofilm for 24 hours, 24 hours of further growth 14 Treatment: (added to well containing a suspension of 5'. aureus ATCC25923 ¨107 CFU/ml) Solvent 1: 50% glycerol containing 1% Tritonlm X-100 and 25 mM potassium phosphate, 16 pH 6.5 17 Solvent 2: 25 mM potassium phosphate, pH 6.5 18 SIGMA Trehalase: 0.092 UI
19 Dialyzed Trehalase: 0.092 UI
Parameters Assessed:
21 Biofilm mass formation (staining with crystal violet 0.06%) 22 Cells viability (incubation with resazurin) 23 Cell growth by colony counting (CFU/mL) [00316] The experimental design for the second strain on the early biofilm included:
26 Strain: P. aeruginosa PA01 27 Temperature: 35 + 2 C
28 Incubation Conditions: Static 29 Time Points: 24 hours CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Treatment: (added to well containing a suspension of P. aeruginosa PA01 ¨107 CFU/ml) 2 Solvent 1: 50% glycerol containing 1% TritonTm X-100 and 25 mM potassium phosphate, 3 pH 6.5 4 Solvent 2: 25 mM potassium phosphate, pH 6.5 SIGMA Trehalase: 0.092 UI
6 Dialyzed Trehalase: 0.092 UI
7 Parameters Assessed:
8 Biofilm mass formation (staining with crystal violet 0.06%) 9 Cells viability (incubation with resazurin) Cell growth by colony counting (CFU/mL) 12 [00317] The experimental design for the second strain on the preformed biofilm included:
13 Strain: P. aeruginosa PA01 14 Temperature: 35 + 2 C
Incubation Conditions: Static 16 Time Points: Preformed biofilm for 24 hours, 24 hours of further growth 17 Treatment: (added to well containing a suspension of P. aeruginosa PA01 ¨107 CFU/ml) 18 Solvent 1: 50% glycerol containing 1% TritonIm X-100 and 25 mM potassium phosphate, 19 pH 6.5 Solvent 2: 25 mM potassium phosphate, pH 6.5 21 SIGMA Trehalase: 0.092 UI
22 Dialyzed Trehalase: 0.092 UI
23 Parameters Assessed:
24 Biofilm mass formation (staining with crystal violet 0.06%) Cells viability (incubation with resazurin) 26 Cell growth by colony counting (CFU/mL) CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00318] FIGS. 5 and 6 are summary tables for the test results of the biofilm produced by S.
2 aureus ATCC25923 (FIG. 5) and the biofilm produced by P. aeruginosa PA01 (FIG. 6).
3 [00319] Tests were also conducted to explore the dialyzed trehalase potential in gram-positive 4 bacteria (reference strains and clinical isolates). The experiment included:
1003201 Step 1: The MIC (Minimum Inhibitory Concentration) activity determination in S.
6 aureus ATCC25923, ATCC33591, and XEN29 in combination with Trehalase;
based on the 7 data, ATCC33591 and XEN29 strains were replaced with ATCC35556 and Oxford strains.
8 [00321] Step 2: Evaluate the effect of Trehalase on biofilm formation by different clinical 9 isolates :
1003221 Phase 1: Screening of clinical isolates for biofilm formation;
11 1003231 Phase 2: Testing Trehalase on selected clinical isolates.
12 [00324] Step 3: Verify the effect of Trehalase on biofilm growth on Teflon catheter surface:
13 [00325] Phase 1: Effect of Trehalase added during biofilm growth (up to 24 hours);
14 1003261 Phase 2: Effect of Trehalase (added after 24 hours of initial biofilm growth) during the next 24 hours from addition).
16 [00327] The MIC determination with selected S. aureus strains proceeded as a first step. The 17 materials and methods included:
18 1003281 S. aureus strains: Oxford; ATCC35556 and ATCC25923 (as reference strain) 19 1003291 In vitro assay: MIC in broth (according to CLSI guidelines) [00330] Treatment group (for each strain):
21 1) Negative control: blank 22 2) Positive control: bacterial growth 23 3) Trehalase: bacteria +/- trehalase 0.092 UI
24 4) Gentamicin: compound +/- trehalase 0.092 UI
5) Vancomycin: compound +/- trehalase 0.092 UI
26 6) Ciprofloxacin: compound +/- trehalase 0.092 UI
27 1003311 Antibiotic concentration: two-fold series dilution from 16 to 0.0078 [tg/mL
28 [00332] Time-point: 24 hours 29 [00333] Incubation: T 37 C; static condition CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00334] The results for the MIC determination with selected S. aureus strains are shown in 2 FIG. 7. The addition of Trehalase resulted in the MIC value reduction with Gentamicin (up to 3 16-fold and 33-fold with clinical and reference isolates). No significant effect on Vancomycin 4 and Ciprofloxacin was observed.
1003351 The screening of clinical isolates proceeded as a second step (phase 1) and included 6 the following materials and methods:
7 [00336] S. aureus strains:
8 [00337] Oxford; PK2; 3226; IH-1018129; ATCCBAA1556; ATCC49230; ATCC35556 9 1003381 S. aureus ATCC25923 as the reference strain 1003391 Medium: TSB enriched with 1% glucose (TSBG) 11 1003401 In vitro assay format: 96 well ¨ MTP (Microtiter Plate) 12 [00341] Samples: 4 samples for each bacterial strain 13 [00342] Time-point: 24 hours of biofilm growth 14 1003431 Incubation: T 37 C; static condition 1003441 Read-out: Biomass (0.1% safranin staining) 16 [00345] The results of the screening of clinical isolates are shown in FIG. 8. The S. aureus 17 Oxford and ATCC35556 as the strains were selected for the next steps (both MSSA).
18 1003461 In the next phase (phase 2), the testing of the Trehalase on clinical isolates included 19 the following materials and methods:
[00347] S. aureus strains: Oxford; ATCC35556 and ATCC25923 as the reference strain 21 [00348] Medium: TSB enriched with 1% glucose (TSBG) 22 1003491 In vitro assay format: 96 well ¨ MTP
23 1003501 Treatment Groups (treatment added during the growth of biofilm):
24 Negative control: blank Positive control: S. aureus strains 26 Vehicle: S. aureus strains + buffer content 25 mM potassium phosphate, pH 6.5 27 Trehalase: S. aureus strains + Trehalase 0.092 UI
28 [00351] Samples: 4 samples for each bacterial strain 29 [00352] Time-point: 24 hours of biofilm growth CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00353] Incubation: T 37 C; static condition 2 [00354] Read-out: Biomass (crystal violet), cell viability (resazurin), planktonic and biofilm 3 bacterial count (agar plating) 4 [00355] The results of the Trehalase testing on clinical isolates are shown in FIGS. 9A, 9B
and 9C and FIGS. 10A, 10B and 10C. Overall, the addition of Trehalase during the 24 hours 6 biofilm growth induced a reduction in biomass and viability of selected MSSA strains.
7 [00356] The next step (Step 3) of experiment explored the effect of Trehalase during the 24-hr 8 initial biofilm growth (added at the start of experiment) and after preliminary (24-hr) biofilm 9 growth on catheters. Bacterial biofilm was developed on the catheter segments (14-gauge Teflon intravenous catheter) by incubating individual segments into tubes containing a S. aureus Xen 29 11 suspension in the exponential phase of growth. The bioluminescence signal on catheter 12 segments was detected with the IVIS Lumina image system, and bacterial count CFU/ml for 13 biofilm cells and planktonic cells was evaluated (agar plating). The Step 3 - phase 1 explored the 14 effect of Trehalase added during initial biofilm growth (up to 24 hours) and included the following materials and methods:
16 [00357] Bacterial strain: S. aureus XEN29 (suspension ¨107 CFU/ml) 17 [00358] Medium: TSB enriched with 1% glucose (TSBG) 18 [00359] In vitro assay: Biofilm formation on catheter (adapted by Kadurugamuwa et al., 2003) 19 [00360] Samples: 3-4 samples for each treatment condition [00361] Treatment:
21 Negative control: blank (catheter without bacterial suspension) 22 Positive control: control infection S. aureus XEN29 (catheter with bacterial 23 suspension) 24 Vehicle: S. aureus XEN29 + buffer content 25 mM potassium phosphate, pH 6.5 Trehalase: S. aureus XEN29 + Trehalase 0.092 UI
26 [00362] Time-point: Treatment added during the incubation ¨ biofilm growth for 24 hours 27 [00363] Incubation: T 37 C; static condition 28 [00364] Read-out: Bioluminescence signal and biofilm and planktonic cells bacterial count 29 CFU/ml (agar plating) CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00365] The results for exploring the effect of Trehalase added during initial biofilm growth 2 (up to 24 hours) on catheter segments are shown in FIGS. 11A, 11B and 11C. As a second 3 phase, the effect of Trehalase (added after preliminary 24-hr biofilm growth) during the 4 following 24 hours of biofilm growth on catheter segments was explored and used the following materials and methods:
6 [00366] Bacterial strain: S. aureus XEN29 (suspension ¨107 CFU/ml) 7 [00367] Medium: TSB enriched with 1% glucose (TSBG) 8 [00368] In vitro assay: Biofilm formation on catheter (adapted by Kadurugamuwa et al., 2003) 9 [00369] Samples: 3-4 samples for each treatment condition [00370] Treatment:
11 Negative control: blank (catheter without bacterial suspension) 12 Positive control: control infection S. aureus XEN29 (catheter with bacterial 13 suspension) 14 Vehicle: S. aureus XEN29 + buffer content 25 mM potassium phosphate, pH 6.5 Trehalase: S. aureus XEN29 + Trehalase 0.092 UI
16 [00371] Time-point: Treatment added after 24 hours of initial bacterial incubation ¨ biofilm 17 growth for further 24 hours 18 [00372] Incubation: T 37 C; static condition 19 [00373] Read-out: Bioluminescence signal and biofilm and planktonic cells counting (CFU/ml - agar plating) 21 [00374] FIGS. 12A, 12B and 12C show the results from the testing of Trehalase added after 22 24 hours biofilm growth (up to 24 hours). The addition of Trehalase at the beginning of 23 experiment (during the initial 24 hours of incubation) induced a significant reduction in biofilm 24 mass and biofilm cells and planktonic cells growth of 5'. aureus XEN29 on the catheter. There was not as much effect of the Trehalase when added after 24 hours biofilm growth in terms of 26 bioluminescence signal and CFU.
27 1003751 These tests prompted further study and analysis of the use of Trehalase with other 28 antimicrobials and ingredients of potential over-the-counter (OTC) products besides the listed 29 antibiotics. For example, further analysis and study determined that Trehalase may be combined CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 with silver in an effective amount so that the Trehalase operates to break down the biofilm and 2 help the host cells, for example, macrophages, to obtain access to kill the bacteria and eliminate 3 them from the human body together with any accumulated silver. The breakdown of the biofilm 4 with the added Trehalase allows the silver to operate in a more effective manner against infectious pathogens with an absence of toxicity and immunogenicity for humans and animals. It 6 is known that silver exhibits low toxicity in the human body and there is minimal risk due to 7 clinical exposure by inhalation, ingestion and dermal application. It is possible to use colloidal 8 silver preparations and silver sulphadiazine or the more popular silver nitrate. This is especially 9 effective when it can be used as coatings for indwelling catheters and cardiac devices in combination with the Trehalase. With the emergence of antibiotic-resistant strains such as 11 CA-MRSA and HA-MRSA as flesh-eating bacteria, there is renewed interest in using silver as 12 an antibacterial agent and it has been determined from the study that the Trehalase may make the 13 silver even more efficient and effective as an antibacterial agent with the breakdown of biofilm.
14 1003761 The silver source releases silver ions that are effective as an antimicrobial and based on the conformational changes in trans-membrane proteins, the Trehalase helps break down 16 more of the biofilm and parts of the matrix and may ease entry of silver ions into the cell.
17 Different compounds may be used both in medical devices, textiles and in ointments for 18 dermatological applications, including metallic silver that includes nanocrystalline forms in 19 silver coatings. Phosphate silver compounds have moderate ionizing capacity while the silver nitrate has very high ionizing capacity and is commonly used with some dermatological carriers.
21 Silver chloride has low ionizing capability while silver sulfate has a moderate ionizing 22 capability. The sulphadiazine complexes have high ionizing capacity and colloidal silver 23 preparations have moderate to high ionizing capacity. All these compounds may be used in 24 combination with the Trehalase in effective amounts, including ointments, sprays and other applications. Nanochemistry techniques can be used to produce micro-fine silver particles of 26 less than 20 nm diameter with increased solubility and release of silver ions at about 70 to 100 27 ppm. The disruption produced by the Trehalase facilitates the silver ion interaction. Typically, 28 the ionization of silver metal is proportional to the surface area of a particle that is exposed.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00377] It is possible to use a sustained silver release dressing on a tissue dressing in 2 combination with Trehalase. The silver nitrate should not exceed 1% in contact with living 3 tissue and at 0.5% has been effective in inhibiting P. aeruginosa. In combination with the 4 Trehalase, it is even more effective. Silver sulphadiazine combines the antibiotic properties of silver with sulphonamide and avoids the disadvantage related to silver nitrate and may be used at 6 1% in a cream base in combination with an effective amount of Trehalase.
It may be combined 7 with polyethylene glycol plus poly-2-hydroxyethyl methacrylate, liposomes, poly-L-leucine or 8 even cadaver skin. It may be combined with a lipocolloid formulation such as 1% to 5% silver 9 sulphadiazine at about 0.25 to 0.3% of Trehalase in a non-limiting example and up to 1% or 2%.
In a preferred embodiment, it should include 0.5 to 10.0 mg protein per ml based on the BCA
11 (bicinchoninic acid) and dry weight of the enzyme, and with 0.4 units in 1 mg of protein optimal 12 and with the range of 0.2 to 1Ø
13 [00378] In terms of the dressing, the silver could be about less than 10 mg per 100 cm2 to 14 more than 100 mg per 100 cm2 with an effective amount of Trehalase of a similar 0.5 mg to 10 mg/cm2 and with a proper enzyme unit activity. This amount may vary depending on the type of 16 Trehalase. Silver ions may be incorporated into a substance and released slowly with time as 17 with silver sulfadiazine or may come from ionizing the surface of a solid piece of silver as with 18 silver nanoparticles. Although the action is not positive, it is observed that some cells exposed to 19 silver (AO ions may have activated a stress response that lead to the condensation of DNA in the center of the cell and there is some cell membrane detachment from the cell wall, cell wall 21 damage and electron dense granules outside and sometimes inside the cell. Thus, the silver may 22 help inactivate proteins by binding the sulfur-containing compounds.
Further research and 23 development has observed that the silver in combination with the Trehalase may disrupt the 24 biofilm and provide enhanced structural change to help inactivate proteins even more. This is found to be effective with gram-negative bacteria that may sustain more structural damage than 26 gram-positive bacteria such as E.Coli for the gram-negative bacteria as compared to the gram-27 positive S. Aureus. The silver may lead to cell shrinkage and dehydration so that in combination 28 with the Trehalase it is even more effective. This makes sense since gram-positive bacteria have 29 a charge of peptidoglycan molecules in the bacterial cell wall and peptidoglycan is negatively CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 charged. Silver ions are positively charged and more silver may get trapped by peptidoglycan.
2 With the enhancement and effectiveness from the added Trehalase degrading the biofilm, this 3 mechanism may trap even more of the silver.
4 [00379] It is known that silver nitrate usually releases its silver ions immediately into solution while silver sulfadiazine gradually releases the majority of its silver ions over an extended period 6 of time, and thus, may provide a more steady supply of silver ions that would be more effective 7 perhaps in a wound cream or bandage. It is possible to use silver salts and other silver 8 components with silver nanoparticles. The nanoparticles may be spherical or rod-shaped and 9 triangular. Different sizes may be used such as 1 ug, 12.5 ug, 50 ug or 100 ug. It is possible that the silver nanoparticles or other silver may be applied to wound dressings and endotracheal tubes 11 as a coating, including surgical masks. It has been found that cotton fibers are more desirable in 12 some cases.
13 [00380] The amounts of Trehalase can vary but for one possible composition, silver nitrate at 14 55 ppm may be used in a wound gel and may vary from 50 to 60 ppm in one example and 40 to 60 ppm in yet another example. The gel may include a water-based product with glycerin, 16 carbomer, sodium chloride, silver nitrate and triethanolamine or any or their equivalents. An 17 equivalent amount of Trehalase may be added corresponding to in one example to 0.5 to 10 18 mg/ml and in yet another example, 0.2 to 0.6 units per mg of protein and for a specific amount, 19 and 0.4 units in 1 mg of protein. The ppm equivalence could be similar to the silver in yet another example. The Trehalase could be 0.5 to 10.0 mg protein/ml. It has also been determined 21 that the silver-killed bacteria may increase the antibacterial activity by the "zombies" effect as 22 noted in the article "Antibacterial Activity of Silver-Killed Bacteria:
The "Zombie" Effect,"
23 Scientific Reports, 2015, the disclosure which is hereby incorporated by reference in its entirety.
24 [00381] It is possible to use an antimicrobial composition such as disclosed in U.S. Patent No.
8,568,711, the disclosure which is hereby incorporated by reference in its entirety, and which 26 includes the silver ion and 80% water as a solvent including a hydrophilic polymer where the 27 silver content has the addition of the Trehalase also ranging from 0.0001 to 0.01 milliliter. It 28 should be understood that the activity units are to be acceptable units of USP per milligram.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1003821 In one example as a solution, it is possible to use 0.5 to 10 mg/mL on a protein basis 2 for the Trehalase and yet another example in an assay 2.0 to 6.0 mg/ml or about 0.4 units/mg.
3 One unit is defined as the amount of enzyme that may convert 1.0 umol of Trehalose to 2.0 4 umols of glucose per minute at a pH of about 5.7 at 37 Degrees C (the liberated glucose is determined at pH 7.5). Its physical form may be contained in 50% glycerol containing 1%
6 Triton X-100 and 25 mM potassium phosphate at a pH of about 6.5. The molecular weight 7 varies but could be about 80,000 Daltons and can range from 70,000 to 85,000 Daltons 8 depending on the origin of the Trehalase in an aspect. One technique that can be used to prepare 9 the Trehalase is described in an article from Yoneyama entitled, "Purification and Properties of Detergent-Solubilized Pig Kidney Trehalase," Archives of Biochemistry and Biophysics (1987), 11 the disclosure which is hereby incorporated by reference in its entirety.
12 [00383] It is possible that Trehalase as alpha, alpha-trehalase, EC 3.2.1.28 could be 13 solubilized from the brush border membrane of pig kidney cortex by Triton X-100 and sodium 14 deoxycholate in the presence of inhibitors of proteolytic enzymes. In this example, the kidney enzyme can be purified 3060-fold using gel filtration, ion exchange chromatography, Con A-16 Sepharose chromatography, phenyl-Sepharose CL-4B hydrophobic interaction chromatography.
17 Contaminant proteins can be absorbed as described in the article with 99% or greater purity 18 based on amino-terminal amino acid analysis. This purified enzyme in this example had a 19 specific activity of 278 units/mg protein, a molecular weight of about 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a glycoprotein and contained 2 mol of 21 glucosamine per mole of trehalase. The apparent Km for this trehalase was calculated to be 2.1 22 mM. This type of produced kidney trehalase was highly specific for trehalose and exhibited an 23 optimal pH of 5.9, and an isoelectric point is between about pH 4.7 and 4.4. Other details may 24 be found in the incorporated by reference article.
[00384] The Trehalase may also be combined with chlorhexidine gluconate or an equivalent 26 both as an antiseptic skin cleanser and as in an oral rinse. In one example, the chlorhexidine 27 gluconate is about 0.12% and in an oral rinse and may be used 3 to 4 times daily. The Trehalase 28 may be added in units as noted before and in a percentage if used for labeling at about 0.1% to 29 0.3% and in yet another example, 0.1% to 0.5% and up to 1% to 2% in yet another example. It CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 has been found that a small amount is effective to aid in an oral rinse for potential applications 2 and to help in breaking down some biofilm to make the chlorhexidine gluconate more effective.
3 As an antiseptic skin cleanser, great amounts of the Trehalase may be used and the higher end 4 range closer to 50 to 100 mg protein/ml and up to 1% to 7% and in another example about 4% in combination with 4% solution chlorhexidine gluconate. This use of the oral cleaner and skin 6 cleanser is excellent for the extremely-drug-resistant ()CDR) strains of klebsiella pneumoniae that 7 may have reduced susceptibility to chlorhexidine and with the added Trehalase, allows the 8 chlorhexidine to be more effective since the added Trehalase helps break apart the biofilm to 9 allow more effective chlorhexidine.
[00385] It is possible to use another decolonization measure such as mupirocin to prevent 11 Staphylococcus aureus skin and soft tissue infections (SSTI). It is also possible to use it on the 12 Acinetobacter baylyi adpl . It may be used in a water-based solution in one example and for a 13 skin cleanser could be mixed with an alcohol-based solution of 70% or a water-based solution 14 that may include other components such as some glycol. The incorporation of about 2 weight percent and 3 weight percent chlorhexidine in combination with a 1 to 4 weight percent 16 Trehalase at an effective enzyme activity is believed to be effective at that range for the 17 antiseptic skin cleanser. Concentrations may be about or greater than 1 [tg/m1 and in some 18 applications, concentrations up to 10 or even greater than 73 g/m1 in some cases as a wipe with 19 Trehalase.
[00386] In one example, it is possible to use 4% w/w for chlorhexidine gluconate and alcohol 21 at 4% w/w in water with a similar preparation of the Trehalase. Another possible application is 22 with denture cleaning tablets that includes a number of components such as a mild bleach, for 23 example, dilute sodium hypochlorite and may include other ingredients such as sodium 24 bicarbonate to alkalize the water and may include citric acid to help remove stains and sodium perborate, sodium polyphosphate, and potassium monopersulfate as a cleaning and bleaching 26 agent and EDTA. As a percent w/w, it may be added to about 1% to 5% for the Trehalase. It can 27 be added but at an effective 0.5 to 10 mg protein per ml, and about 0.2 to 1.0 units in a milligram 28 (mg) of protein based on a dry weight basis, and in an example, 0.4 units/mg. An example of 29 some the components that may be included with Trehalase include the following:

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 Chemical Name Proportion (% w/w) Potassium peroxymonosulfate sulfate < 10 Sodium carbonate peroxide 10 - < 30 Sodium carbonate 10 - < 30 Citric acid 10 - < 30 Malic acid < 10 Other ingredients classified as not hazardous to 100 according to NOHSC

[00387] It is possible to include Trehalase in mouthwashes, toothpastes, and cement 6 material for dental applications where the Trehalase is combined with chlorhexidine (as the 7 antimicrobial), or with essential oils (or their active fractions). For example, some mouthwashes 8 may include Chlorhexidine Digluconate at 0.06% w/v and Sodium Fluoride (250 ppm fluoride) 9 and include the added Trehalase in an amount equivalent to the optimal doses similar to that added to other compositions and treatments discussed above. Another example is a mouthwash 11 that may contain as active ingredients Eucalyptol at 0.092%; Menthol at 0.042%; Thymol at 12 0.064%, (these are active antimicrobial fractions of essential oils) and Methylsalicylate at 13 0.060%. These components may be labeled together on the bottle as Antiplaque/antigingivitis.
14 Another type of mouthwash contains as active ingredient Sodium fluoride 0.02% (0.01% w/v fluoride ion) along with all ingredients from the first type (but without their doses, just 16 mentioned as inactive ingredients). Trehalase can be added to all three mouthwashes in amount 17 equivalent to the "optimal" doses noted above. Trehalase may be added to a toothpaste, for 18 example, a type having an active ingredient as Sodium fluoride at 0.310%
w/w (1400 ppm 19 fluoride), or another toothpaste that has active ingredients such as Sodium fluoride at 0.24%
(0.15% w/v fluoride ion) and Triclosan at 0.30%, as antigingivitis, i.e., antimicrobial.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 [00388] As noted before, silver and the Trehalase is effective and it has also been found that 2 copper is effective. Hospital environments may act as a reservoir for biofilm-forming pathogens 3 that cause healthcare-associated infections (HCAIs), so that approaches to reducing 4 environmental microbial contamination in addition to cleaning, attracted attention over the last decade. Copper is well recognized as a powerful antimicrobial with rapid broad spectrum 6 efficacy against bacteria, viruses and fungi. In a novel cross-over study in an acute medical ward, 7 a toilet seat, a set of tap handles and a ward entrance door push plate, each containing copper, 8 were sampled for the presence of microorganisms and compared to equivalent standard, non-9 copper-containing items on the same ward, demonstrating a statistically significant decrease of microbial contamination on copper-covered surfaces (A.L. Casey, D. Adams, T.J.
Karpanen, 11 et.al. "Role of Copper in reducing hospital environment contamination", The Journal of Hospital 12 Infection, Vol.74, Issue 1, pages 72-77 (January 2010).
13 [00389] Extensive scientific information on the role of Antimicrobial Copper in reducing 14 transmission of infection and spread of antimicrobial resistance is introduced in "Scientific References" (http://www.antimicrobialcopper.org/uk/antimicrobial-resistance) in multiple 16 laboratory tests carried out under typical indoor conditions, it was demonstrated that 17 "antimicrobial copper" was effective against many pathogens, including those with drug-18 resistance: MRSA S. aureus, MDR Tubercle bacillus, MDR Acinetobacter baumannii, 19 Vancomycin-resistant enterococcus (VRE), Carbapenem-resistant Enterobacteriaceae (CRE), ESBL-producing Klebsiella pneumoniae, ESBL-producing E. coli.
21 [00390] This efficacy translates into the clinical environment, as demonstrated in a multi-22 center ICU trial in the US (MG Schmidt, HH Attaway, PA Sharpe, et. al., "Sustained Reduction 23 of Microbial Burden on Common Hospital Surfaces through Introduction of Copper", Journal of 24 Clinical Microbiology 2012, Vol. 50 No. 7 2217-2223). Six near-patient surfaces were upgraded to copper, and sampling was undertaken weekly over a period of 23 months. Over the 26 intervention period, the combined MRSA and VRE burdens were 96.8% lower on copper 27 surfaces than on comparable plastic, wood, metal, and painted surfaces and were 98.8% lower on 28 the bed rails, the most heavily burdened object. In this US trial, the bioburden reduction was 29 associated with a 58% reduction in infections (CD Salgado, KA Sepkowitz, JF John, JR Cantey, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 HH Attaway, KD Freeman, MG Schmidt, "Copper Surfaces Reduce the Rate of Healthcare-2 Acquired Infections in the Intensive Care Unit", Infection Control and Hospital Epidemiology 3 2013, 34(5), 479-486.).
4 [00391] The enzyme Trehalase can be immobilized on the copper-containing surfaces to prevent microbial biofilm formation by microorganisms accumulated on such surfaces during the 6 time periods between the cleaning procedures (frequency of cleaning such surfaces is usually 7 introduced in guidelines for maintenance of such surfaces). The enzyme Trehalase can also be 8 included in disinfecting solutions for such cleaning procedures (as described in previous 9 embodiments for prevention and treatment of biofilm-based infections on medical devices and medical equipment surfaces).
11 1003921 Waterborne infections contribute to transmission of infections in hospital settings, in 12 particular when used in medical equipment (surgical instruments, endoscope washer-disinfectors, 13 nebulizers, endotracheal tubes) disinfection procedures. For example, surgical instruments may 14 have high post-cleaning levels of carbohydrates (up to 352 g/cm2) and endotoxin (up to 25 373 EU/cm2), suggesting unrecognized issues with the quality of water used for the final rinse, and 16 showing the necessity to monitor the water quality used in instrument washers (M.J.Alfa, N
17 Olson, A Al-Fadhaly, "Cleaning efficacy of medical device washer in North American 18 healthcare facilities", The Journal of Hospital Infection, Vol.74, Issue 2, Pages 168-177 19 (February 2010). The development of microbial biofilm in washer-disinfectors, the type of biofilms and the nature of the bacteria within them, along with increasing antimicrobial 21 resistance in those pathogens is of a major concern (W.G. MacKay, A.T.
Leanord, C.L.
22 Williams, "Water, water everywhere nor any of a sterile drop to rinse your endoscope", The 23 Journal of Hospital Infection, Vol.51, Issue 4, pages 256-261 (August 2002). Growing resistance 24 of HCAIs to antimicrobials and biocides is on the rise. Various biocides show a wide range of their efficacy. For example, in a comparative study on polytetrafluoroethylene (PTFE) tubes, 26 contaminated by a liquid medium inoculated with Pseudomonas aeruginosa, using five different 27 alternative disinfectant solutions: two peracetic acid solutions (with and without an activator), 28 glutaraldehyde, orthophthaldehyde and succine dialdehyde, it was shown that repeated 29 treatments of a PTFE tube with a 2% glutaraldehyde solution induced an important accumulation CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 and/or fixation of protein, compared to peracetic-acid-based disinfectants, for which the 2 accumulation and/or fixation of protein remained low, but varied from one formulation to 3 another (L. Pineau, C. Desbuquois, B. Marchetti, D. Luu Duck, "Comparison of the fixative 4 properties of five disinfectant solutions", The Journal of Hospital Infection, Vol. 68, Issue 2, Pages 171-177 (February 2008).
6 1003931 In this context again, the enzyme Trehalase can be included in various formulations 7 of disinfection solutions, and can be used as directed in the previous embodiments for 8 composition and methods for treatment of biofilm-based infections on medical devices.
9 1003941 It should be understood that the copper may operate to short circuit the current in the cell membrane to disturb the "trans membrane potential" and can cause oxidative damage and 11 thus, "punch" a hole in the bacterium. It is possible to add Trehalase to Medicinal Copper, and 12 even possible to apply Trehalase and copper in a spray as an adhesive and spray it onto surfaces.
13 Besides copper, it is also possible to use Trehalase in combination with biofilm disruptors such 14 as xylitol, lactoferrin, and D-amino acids. Xylitol is a sugar alcohol used as a sweetener.
1003951 The bacterial biofilms are the major contributing factors to chronic wounds, such as 16 diabetic foot ulcers, pressure ulcers, and venous leg ulcers, providing increased bioburden to the 17 wounds and interfering with the healing process. Traditional methods of treatment have proven 18 ineffective, therefore therapy of non-healing wounds demands biofilm-targeted strategies.
19 1003961 Studies using a colony-drip-flow reactor biofilm model, demonstrated positive result of the combined treatment of bacterial biofilms with the innate immune molecule lactoferrin and 21 the rare sugar-alcohol xylitol against a clinical wound isolate; for both a single species biofilm 22 and a dual species biofilm, the lactoferrin/xylitol hydrogel in combination with the silver wound 23 dressing Acticoat TM had a statistically significant reduction in biofilm cell viability compared 24 to the commercially available wound hydrogel (Ammons MC, Ward LS, James GA, "Anti-biofilm efficacy of a lactoferrin/xylitol wound hydrogel used in combination with silver wound 26 dressings", Int Wound J, 2011 Jun 8(3); 268-73). In an in vitro biofilm model with a clinical 27 isolate of P. aeruginosa, subjected to treatment with either lactoferrin or xylitol alone or in 28 combination, it was shown that combined lactoferrin and xylitol treatment disrupted the structure 29 of the P. aeruginosa biofilm and resulted in a >2 log reduction in biofilm cell viability; in situ CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 analysis indicated that xylitol appeared to disrupt the biofilm structure and lactoferrin increased 2 the permeability of bacterial cells (Ammons MC, Ward LS, Fisher ST, Wolcott RD, James GA, 3 "In vitro susceptibility of established biofilms composed of a clinical wound isolate of 4 Pseudomonas aeruginosa treated with lactoferrin and xylitol", Int J
Antimicrob Agents, 2009 Mar, 33(3):230-6.).
6 1003971 As noted before, the enzyme trehalase can be used in combination with antimicrobials 7 (ex, silver gel or silver-based wound dressings) for chronic wounds treatment along with other 8 antimicrobials, or can be used in combination with lactoferrin or lactoferrin/xylitol to increase 9 effectiveness of these treatment modalities. As further noted before, to increase the healing process of such chronic wounds, trehalase in combination with antimicrobials can be used along 11 with antioxidants, anti-inflammatory and immune-modulating substances.
Trehalase may be 12 added in the preparation and amounts disclosed previously.
13 [00398] In many cases, chronic wounds have tendency to be additionally contaminated by 14 Gram-positive pathogens, such as S. aureus and Streptococci spp. that contribute to increased bioburden in mixed species biofilms. In these cases, Trehalase in combination with 16 polymicrobial antibiotics and/or natural antimicrobials (i.e., silver compounds as noted before) 17 can provide dual effect, targeting the biofilm structure and reducing viability of biofilm-forming 18 pathogens (including both biofilm-embedded cells and dispersed planktonic cells).
19 1003991 It is also possible to add units of Trehalase enzyme to an antibiotic ointment that may include one or more of bacitracin zinc, neomycin sulfate that may be equivalent to a neomycin 21 base and a polymyxin B sulfate. Although the amounts can vary in one example, 300 to 500 22 units of a bacitracin zinc may be added to 2.5 to 4.5 mg of the neomycin base and 4,000 to 6,000 23 polymyxin B units added as the polymyxin B sulfate together with units of Trehalase with 24 sufficient units of activity and in one example, 0.2 to 1.0 units per mg and around 0.4 units per milligram.
26 1004001 A sufficient amount of Trehalase may be added to an enzyme supplement that could 27 include protease as either 4.5, 6.0 and/or 3.0 such as respective amounts of 14,000 to 23,000 28 HUT for the protease 4.5, 2,000 to 4,000 HUT for the protease 6.0, and 13 to 19 SAPU for 29 protease 3.0 and in specific amounts about 19,000; 3,000; and 16. About 3,000 to 5,000 DU of CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 amylase may be added and in a specific amount 4,200, and 7 to 13 AGU of glucoamylase and in 2 a specific amount 10 AGU, 20 to 28 LU of lipase and in a specific amount 24 LU. 90 to 150 CU
3 of cellulase may be added (specific 120 CU) and 180 to 260 SU of invertase (specific amount 4 220 SU), and 500 to 600 DP for malt diastase (specific 550 DP). There may also be included about 80 to 120 GALU (100 GALU) for alpha-galactosidase and 1,600 to 2,400 HUT
of 6 peptidase (specific amount 2,000 HUT). Many of these quantities can vary and be supplemented 7 with other enzymes depending on the end use desired. The Trehalase can be supplemented in an 8 effective amount of 0.2 to 1.0 unit in 1 mg of protein. It may also be added with probiotics such 9 as a blend of lactobacillus acidophilus and biphobacterium lactis such as around 4 billion for the probiotic in one non-limiting example.
11 1004011 It is also possible to combine the Trehalase with antimicrobial peptides that may be 12 an alternative to small molecule antibiotics, including modifications of short antimicrobial 13 peptides. This could include lipopeptides and a combination of fatty acids with cationic 14 antimicrobial peptides that help change the confirmation of the antimicrobial peptides secondary structure in the presence of bacterial membranes.
16 [00402] It is possible assay Trehalase based on the technique described in Dahlquist, A., 17 (Assay of Intestinal Disaccharidases Analytical Biochemistry (1968)). An enzymatic assay of 18 Trehalase has been formulated as (EC 3.2.1.28) by Sigma-Andrich.

23 Principle: Trehalose + H20 Trehalase > 2 Glucose Conditions: T = 37 C, pH = 5.7, A340 nm, Light path = 1 cm 27 Method: Spectrophotometric Stop Rate Determination CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 The Reagents in this assay include:

3 A. 135 mM Citric Acid Buffer, pH 5.7 at 37 C, which is prepared 100 ml in deionized water 4 using Citric Acid, Free Acid, Monohydrate, as Sigma Prod. No. C-7129, corresponding to the citric acid monohydrate. It is adjusted to pH 5.7 at 37 C with 1 M NaOH.

7 B. 140 mM D-Trehalose Solution, which is prepared by 10 ml in Reagent A
using 8 D(+)Trehalose, Dihydrate, as Sigma Prod. No. T-5251 corresponding to D-(+)-Trehalose 9 dehydrate 11 C. 500 mM Tris Buffer, pH 7.5 at 37 C, which is prepared by 100 ml in deionized water using 12 Trizma Base, as Sigma Prod. No. T-1503. It is adjusted to pH 7.5 at 37 C
with 1 M HCl.

14 D. Trehalase Enzyme Solution. Immediately before use, prepare a solution containing 0.1 - 0.3 unit/ml of Trehalase in cold Reagent A.

17 E. Glucose Determination Vial. It is possible to use an ion exchange type as Sigma Stock No.
18 16-10 (Mono QHR 16/10), Glucose (HK) 10 Reagent. The contents are dissolved in 10 ml of 19 deionized water.

PROCEDURE:

27 Step 1: Pipette (in milliliters) the following reagents into suitable cuvettes:
28 Test Blank 29 Reagent A (Citrate Buffer) 0.3 0.3 CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Reagent D (Enzyme Solution) 0.1 0.1 3 Mix by inversion and equilibrate to 37 C using a suitably thermostatted spectrophotometer. Then 4 add:
Reagent B (D-Trehalose) 0.1 7 Immediately mix by inversion and incubate at 37 C for exactly 15 minutes.
Then add:

9 Reagent C (Tris Buffer) 0.5 0.5 Reagent B (D-Trehalose) 0.1 13 Step 2: Pipette (in milliliters) the following reagents into suitable cuvettes:

Test Blank 16 Reagent E (16-10) 3.0 3.0 18 Equilibrate to 37 C. Monitor the A340 until constant, using a suitably thermostatted 19 spectrophotometer. Record the initial A340nm for both Test and Blank.
Then add:
21 Test Solution 0.1 22 Blank Solution 0.1 24 Immediately mix by inversion and record the increase in A34onm until complete (approximately 5 minutes). Obtain the final A340nm for both the Test and Blank.

27 CALCULATIONS:

29 AA340nm Test ¨ A340 Test Final - A340nm Test Initial CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 2 AA34onm Blank = A34onm Blank Final - A34onm Blank Initial 4 Units/ml enzyme = (AA340nm Test - A34onm Blank) (1.0) (3.1)!
(6.22)(2)(15)(0.1)(0.1) 6 6.22 = Millimolar extinction coefficient of I3-NADH at 340nm 7 2 = Number of Glucose molecules per molecule of Trehalose 8 15 = Reaction time (in minutes) of Step 1 9 1.0 = Final volume (in milliliters) of Step 1 3.1 = Final volume (in milliliters) of Step 2 11 0.1 = Volume From Step 1 used in Step 2 12 0.1 = Volume (in milliliters) of enzyme used 14 Units/mg protein = units/ml enzyme / mg protein/ml enzyme 16 UNIT DEFINITION: One unit will convert 1.0 mole of trehalose to 2.0 moles of glucose 17 per minute at pH 5.7 at 37 C (liberated glucose determined at pH 7.5).

19 FINAL ASSAY CONCENTRATION: In a 0.50 ml reaction mix, the final concentrations are 135 mM citric acid, 28 mM D-trehalose, and 0.01 - 0.03 unit of trehalase.

23 1004031 As noted above, trehalase may be used in combination with antimicrobials for 24 treatment of industrial biofilms in pipelines, HVAC systems, ship hulls, farm equipment, and other metal surfaces, especially products produced from carbon steel, stainless steel, and related 26 metals. Industrial biofilms cause biofouling and biocorrosion, also known as microbiologically 27 influenced corrosion (MIC), that threatens the effective function and life of oil pipelines, water 28 utilities and gas lines, and other metallic components and equipment in these and related 29 industries because it reduces the service life of pipelines and equipment, leading to billions of CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 dollars of economic losses each year in the United States and other countries as noted in D. Xu, 2 R. Jia, Y. Li, T. Gu, Advances in the treatment of problematic industrial biofilms, World J
3 Microbiol Biotechnol (2017) 33:97; D. Xu, J. Xia, E. Zhou, D. Zhang, H.
Li, C. Yang, Q. Li, H.
4 Lin, X. Li, K. Yang, Accelerated corrosion of 2205 duplex stainless steel caused by marine aerobic Pseudomonas aeruginosa biofilm, Bioelectrochemistry, 113 (2017); R.
Jia, D. Yang, 6 H.H. Al-Mahamedh, T. Gu, Electrochemical Testing of Biocide Enhancement by a Mixture of d-7 Amino Acids for the Prevention of a Corrosive Biofilm Consortium on Carbon Steel, Ind. Eng.
8 Chem. Res. 56 (2017); and R. Jia, D. Yang, D. Xu, T. Gu, Mitigation of a nitrate reducing 9 Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by D-tyrosine, Sci. Rep. 7 (2017).
11 1004041 Sulfate reducing bacteria (SRB) biofilms are often involved in MIC because they use 12 the oxidant sulfate that is common in water and function as a terminal electron acceptor with 13 different carbon sources as electron donors as noted in T. Gu, R. Jia, T. Unsal, D. Xu, Toward a 14 better understanding of microbiologically influenced corrosion caused by sulfate reducing bacteria, J. Mater. Sci. Technol. 35 (2019) 631-636; D. Enning, J. Garrelfs, Corrosion of iron by 16 sulfate-reducing bacteria: new views of an old problem, Appl Env.
Microbiol. 80 (2014) 17 1226-1236; and A. Vigneron, E.B. Alsop, B. Chambers, B.P. Lomans, I.M.
Head, N. Tsesmetzis, 18 Complementary microorganisms in highly corrosive biofilms from an offshore oil production 19 facility, Appl Env. Microbiol. 82 (2016) 2545-2554. During various corrosion tests, scanning electron microscopy (SEM) images of SRB cells in a biofilm on carbon steel have shown the 21 pitting corrosion caused by SRB MIC, and the perforating pits caused by biocorrosion damage as 22 noted by D. Xu, T. Gu, Carbon source starvation triggered more aggressive corrosion against 23 carbon steel by the Desulfovibrio vulgaris biofilm, Int. Biodeterior.
Biodegrad. 91(2014) 74-81;
24 and S. Bhat, B. Kumar, S.R. Prasad, M.V. Katarki, Failure of a new 8-in pipeline from group gathering station to central tank farm, Mater. Perform. 50 (2011) 50-54.
26 1004051 Biocides are commonly used to treat biofilms. The biocide dosing used in more 27 traditional biofilm treatments, however, has a low efficacy against biofilms and involves high 28 operational costs and adverse environmental impacts as noted by P.A.
Rasheed, K.A. Jabbar, K.
29 Rasool, R.P. Pandey, M.H. Sliem, M. Helal, A. Samara, A.M. Abdullah, K.A. Mahmoud, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Controlling the biocorrosion of sulfate-reducing bacteria (SRB) on carbon steel using 2 ZnO/chitosan nanocomposite as an eco-friendly biocide, Corros. Sci. 148 (2019) 397-406. Tests 3 conducted in the past have found that a much higher biocide dosage is required to treat sessile 4 cells, which are embedded in a biofilm as compared to the amount of biocide necessary to treat planktonic cells suspended in a bulk liquid as noted by T.-F.C. Mah, G.A.
O'Toole, Mechanisms 6 of biofilm resistance to antimicrobial agents, Trends Microbiol. 9 (2001) 34-39. Some tests have 7 indicated that ten times the dosage of a biocide may be required.
Biofilms may have different 8 defense mechanisms, including diffusional limitations and a lowered metabolic rate to reduce 9 intakes as noted by Y. Li, R. Jia, H.H. Al-Mahamedh, D. Xu, T. Gu, Enhanced Biocide Mitigation of Field Biofilm Consortia by a Mixture of D-Amino Acids, Front.
Microbiol. 7 11 (2016) 896. The more well known biocides such as glutaraldehyde and tetrakis hydroxymethyl 12 phosphonium sulfate (THPS) have been favored in the oil and gas industry, not only because 13 they have broad-spectrum efficacy, but also because they are readily biodegradable and reduce 14 adverse impacts on the environment as noted by D. Enning, R. Smith and S. Desai, Comparing the effects of THPS and Glutaraldehyde batch biocide treatment on microbial corrosion in 16 circulating flow loops, ExxonMobil Upstream Research Company, June 5, 2015 (at 17 www.exxonmobil.com); T. M. Williams and L. E. Cooper, The Environmental Fate of Oil and 18 Gas Biocides, Corrosion, March 9-13, 2014, 3876.
19 1004061 Because biofilms can have such harmful and long-lasting effects, researchers are working hard to find biocide enhancers that work synergistically with existing biocides for 21 biofilm mitigation as noted by H. Lam, D.-C. Oh, F. Cava, C.N. Takacs, J. Clardy, M.A. de 22 Pedro, M.K. Waldor, D-amino acids govern stationary phase cell wall remodeling in bacteria, 23 Science. 325 (2009) 1552-1555; I. Kolodkin-Gal, D. Romero, S. Cao, J.
Clardy, R. Kolter, R.
24 Losick, D-Amino Acids Trigger Biofilm Disassembly, Science. 328 (2010) 627-629; and G.A.
Kahrilas, J. Blotevogel, P.S. Stewart, T. Borch, Biocides in Hydraulic Fracturing Fluids: A
26 Critical Review of Their Usage, Mobility, Degradation, and Toxicity, Environ. Sci. Technol. 49 27 (2015) 16-32.
28 [00407] These biocide enhancers may improve the efficacy of a biocide, even though the 29 enhancer itself may not be biocidal in its function as noted by R. Jia, D. Yang, D. Xu, T. Gu, CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion 2 using ciprofloxacin enhanced by D-tyrosine, Sci. Rep. 7 (2017) 6946; and R. Jia, D. Yang, W.
3 Dou, J. Liu, A. Zlotkin, S. Kumseranee, S. Punpruk, X. Li, T. Gu, A sea anemone-inspired small 4 synthetic peptide at sub-ppm concentrations enhanced biofilm mitigation, Int. Biodeterior.
Biodegrad. 139 (2019) 78-85.
6 1004081 D-amino acids have been found to enhance the effect and function of THPS and other 7 non-oxidizing biocides against biofilm growth in both oil field, e.g., pipelines, and cooling water 8 systems such as HVAC and related systems as noted by D. Xu, Y. Li, T. Gu, D-Methionine as a 9 biofilm dispersal signaling molecule enhanced tetrakis hydroxymethyl phosphonium sulfate mitigation of Desulfovibrio vulgaris biofilm and biocorrosion pitting, Mater.
Corros. 65 (2014) 11 837-845; and R. Jia, D. Yang, Y. Li, D. Xu, T. Gu, Mitigation of the Desulfovibrio vulgaris 12 biofilm using alkyldimethylbenzylammonium chloride enhanced by d-amino acids, Int.
13 Biodeterior. Biodegrad. 117 (2017) 97-104.
14 1004091 For example, when treating biofilm growth in a water cooling tower, the combination of 50 ppm (w/w) of a D-amino acid mixture and 15 ppm of THPS have the same effect on some 16 biofilm growth as a treatment using 30 ppm THPS alone as noted by R.
Jia, Y. Li, H.H.
17 Al-Mahamedh, T. Gu, Enhanced Biocide Treatments with D-amino Acid Mixtures against a 18 Biofilm Consortium from a Water Cooling Tower, Front. Microbiol. 8 (2017) 1538. Non-19 biocidal Peptide A has also been found to enhance biocides because the combination of 100 nM
(180 ppb by mass) Peptide A and 100 ppm THPS led to an additional 2-log reduction in sessile 21 Sulfate Reducing Bacteria (SRB) cell count compared to a treatment using 100 ppm THPS alone 22 as noted by R. Jia, D. Yang, W. Dou, J. Liu, A. Zlotkin, S. Kumseranee, S. Punpruk, X. Li, T.
23 Gu, A sea anemone-inspired small synthetic peptide at sub-ppm concentrations enhanced biofilm 24 mitigation, Int. Biodeterior. Biodegrad. 139 (2019) 78-85. In an example, the number of amino acid residues, such as in a 14-mer peptide has been synthesized with its sequence inspired by the 26 Equinatoxin II protein secreted by the sea anemone that secretes its protein to keep its exterior 27 surfaces clean in the marine ecosystem. Its marine origin and usage at a low dosage makes it 28 practical.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1004101 It has been known that Trehalose is a disaccharide that plays an essential role in 2 biofilm formation, while Trehalase as an enzyme catalyzes the conversion of trehalose to glucose 3 as noted by E.J. Hehre, T. Sawai, C.F. Brewer, M. Nakano, T. Kanda, Trehalase:
4 stereocomplementary hydrolytic and glucosyl transfer reactions with alpha-and beta-D-glucosyl fluoride, Biochemistry. 21(1982) 3090-3097. The action of trehalase against trehalose 6 component of microbial biofilm was disclosed in the inventors' work disclosed in commonly 7 assigned European Patent No. EP 2717926 B1 issued May 24, 2017, and the U.S. Patent No.
8 10,420,928, issued September 24, 2019, which disclosed the use of trehalase for preventing and 9 treating biofilms, the disclosures which are hereby incorporated by reference in their entirety.
100411] The following are the test results from different experiments that tested trehalase as a 11 biocide enhancer to enhance THPS and mitigate SRB biofilm and biocorrosion. It was found 12 that trehalase enhanced the THPS and aided in preventing a pure-strain SRB biofilm from being 13 established, and showed efficacy to abate carbon steel corrosion by the SRB biofilm. In the 14 experiments, C1018 carbon steel coupons were exposed to an ATCC 1249 medium and inoculated with SRB in anaerobic vials at 37 C for 7 days. After the 7-day incubation, the SRB
16 biofilms and surface morphologies were examined under a scanning electron microscope (SEM), 17 a confocal laser scanning microscope (CLSM), and an infinite focus microscope (IFM).
18 Planktonic cell count, sessile cell count, and coupon weight loss and corrosion pit depth were 19 also measured.
[00412] For this experiment, D. vulgaris (ATCC 7757) was cultured in a modified Baar's 21 medium (ATCC 1249 medium) with 200 ppm Fe2 . The composition of the Baar's medium is 22 shown in greater detail in Table 1.

Composition of ATCC 1249 Medium Component I MgSO4 2.0 g Sodium Citrate 5.0 g CaSO4.2H20 1.0 g CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1.0 g Distilled Water 400 mL
Component II K2HPO4 0.5 g Distilled Water 200 mL
Component III Sodium Lactate 3.5 g Yeast Extract 1.0 g Distilled Water 200 mL
Component IV (NH4)2Fe(504)2.6H20 1.38 g (filter-sterilized) Distilled Water 200 mL

3 [00413] The culture medium's pH was adjusted to 7.0 using a hydrochloric acid (5% v/v) 4 solution and a sodium hydroxide solution (5% w/w) before autoclaving. The culture medium and lab tools, such as utensils and 50 mL anaerobic vials, were sterilized in an autoclave for 20 6 minutes at 121 C. A 10,000 ppm (w/w) THPS stock solution was filter-sterilized using a 0.22 7 [tm Stericup, such as from Millipore of Bedford, MA. One hundred ppm (w/w) of the amino 8 acid, L-cysteine, was added to the culture medium as an oxygen scavenger.
After autoclaving, 9 the medium was sparged with filter-sterilized nitrogen to deoxygenate for at least one hour. All tests were carried out in an anaerobic chamber. Chemicals for this experiment were obtained 11 from Sigma-Aldrich of St. Louis, MO, and Fisher Scientific of Pittsburgh, PA.
12 1004141 C1018 carbon steel coupons were used as test samples for testing corrosion and each 13 coupon had a 1 cm x 1.2 cm exposed surface area. All other surfaces were coated with Teflon.
14 The chemical composition of the C1018 carbon steel used to produce the test samples as steel coupons is given in Table 2.

17 Composition of C1018 Carbon Steel CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 Element C Mn P Cu S Ni Fe Amount 0.20 1.40 0.04 0.55 0.04 0.012 balance (wt%) 3 [00415] Before each test, the carbon steel coupons were abraded with 180 and 600 grit 4 abrasive papers progressively, then cleaned with 100% isopropanol, and dried under UV light for at least 20 minutes. The Trehalase used in this experiment was of prokaryotic origin from 6 Megazyme, Inc. of Bray, Wicklow, Ireland. In this experiment, its specific activity was ¨300 7 U/mg (40 C, pH 5.5 on trehalase).
8 [00416] Three replicate carbon steel coupons, a 40 mL culture medium, and 0.4 mL D.
9 vulgaris seed culture were added to each 50 mL anaerobic vial with different combinations of trehalase and THPS. The vials were then incubated at 37 C for 7 days. The test matrix as the 11 treatment used for these experiments is shown in Table 3.

Test Matrix for Testing THPS Treatment of D. vulgaris Enhanced by Trehalase Parameter Condition Bacterium D. vulgaris Temperature 37 C
Culture medium ATCC 1249 medium pH 7.0 Incubation time 7 days Biocide THPS
Biocide enhancer Trehalase CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 No treatment 1 ppm trehalase alone 3 ppm trehalase alone ppm trehalase alone 30 ppm trehalase alone Treatments 50 ppm THPS alone 50 ppm THPS + 1 ppm trehalase 50 ppm THPS + 3 ppm trehalase 50 ppm THPS + 10 ppm trehalase 50 ppm THPS + 30 ppm trehalase 100 ppm THPS alone Analysis Weight loss, pit depth, SEM images of biofilm, SEM images of pits, CLSM images.
Coupon C1018 carbon steel Volume 50 mL vial, each filled with 40 mL
medium 3 .. [00417] Vials containing modified Postgate's B (MPB) medium as a SRB
growth medium 4 especially adapted for oil and gas systems and obtained from Biotechnology Solutions of 5 Houston, Texas were used to count SRB cells in a liquid sample using the most probable number 6 (MPN) technique. MPB was formulated specifically to support the growth of SRB as noted in 7 Modified Postgate's B Media (MPB) - BTS - Biotechnology Solutions, (n.d.).
8 https://biotechnologysolutions.com/modified-postgates-b-media-mpb/
(accessed April 23, 2019).
9 The biogenic sulfide created by SRB reacts with Fe2+ in the MPB medium.
This reaction 10 precipitates iron sulfide (FeS), which has a characteristic ink black color and can be seen as 11 black color in the presence of SRB in the vials. After the 7-day D.
vulgaris incubation, the metal 12 coupons were removed from the vials and rinsed in a pH 7.4 phosphate buffered saline (PBS) 13 .. solution three times to remove loosely attached planktonic cells and any culture medium. Sessile CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 cells on a coupon were removed by brush to move the biomass into a 10 mL
PBS solution in a 2 disposable plastic weighing dish. The brush, the coupon, and 10 mL PBS
solution were vortexed 3 together in a 50 mL conical tube for 30 seconds. The lVfPN vials were incubated at 37 C for 14 4 days and any black color noted.
[00418] For planktonic cell counting, 1 mL of broth from a 50 mL anaerobic vial was diluted 6 with a 9 mL PBS buffer in a 50 mL conical tube. The tube was vortexed for 30 seconds before 7 the diluted cell suspension was counted using MPN. The blackened tubes were considered 8 positive test results and converted to cell numbers per mL using a standard 3-tube MPN table.
9 An example of this method is taught in an example described in the lVfPN
Method, https://www.jlindquist.com/generalmicro/102di13.html. In this work, MPN assays were done in 11 triplicates with three rolls of vials to obtain the averaged planktonic cell counts and sessile cell 12 counts. Sessile cell counts (cells/cm2) were calculated from their corresponding cell counts 13 (cells/mL) in cell suspension samples based on the exposed surface area (cm2) of the coupon 14 from which the sessile cells were harvested.
[00419] A hemocytometer was used to count cells in a liquid in a sample based upon a cell 16 suspension under an optical microscope at 400x magnification. This technique was considered 17 more accurate than lVfPN to enumerate planktonic and sessile cell counts when the cell count was 18 larger than 5 x 104 cells/mL as noted by R. Jia, D. Wang, P. Jin, T.
Unsal, D. Yang, J. Yang, D.
19 Xu, T. Gu, Effects of ferrous ion concentration on microbiologically influenced corrosion of carbon steel by sulfate reducing bacterium Desulfovibrio vulgaris, Corros.
Sci. (2019). For 21 lower cell counts, MPN was used. It should be understood that some cells may not have been 22 distinguishable from artifact particles in the background liquid under microscopic views.
23 D. vulgaris cells were considered motile and thus easily distinguishable from other stationary 24 particles. The hemocytometer was used to count the planktonic and sessile cells, except for the 50 mL anaerobic vial treated with 100 ppm THPS, which had the smallest SRB
cell count, and 26 thus, was considered too low to count with the hemocytometer and MPN was used.
27 [00420] After a 7-day incubation at 37 C, the biofilm and corrosion products on the coupon 28 surfaces were removed using a fresh Clarke's solution according to ASTM
G1-03 with the ratio 29 of 1000 mL hydrochloric acid having a specific gravity of 1.19, and with

20 grams antinomy CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trioxide (Sb203) and 50 grams stannous chloride (SnC12) following a procedure as described by 2 S. Wade, Y. Lizama, Clarke' solution cleaning used for corrosion product removal: effects on 3 carbon steel substrate, in: Adelaide, Australia, 2015. Each coupon was cleaned in a weighing 4 dish containing 20 mL of fresh Clarke's solution for 1 minute. After cleaning, the coupons were rinsed in a 250 mL beaker of deionized water and then followed by an isopropanol rinse and 6 dried. Coupons were weighed before and after cleaning using a high accuracy mass balance 7 having a 0.1 mg readability. The difference between the initial weight and final weight was 8 reported as a weight loss.
9 1004211 An infinite focus microscope (IFM) as a Model ALC13, Alicona Imaging GmbH, Graz, Austria, was used to measure the pit depth on the coupon surfaces. IFM
is a rapid non-11 contact optical 3D measurement device, which combines the low depth of field of an optical 12 microscope with vertical scanning, traversing across the surface of the sample, to provide high 13 resolution of field topographical images as noted by H. Schroettner, M.
Schmied, S. Scherer, 14 Comparison of 3D Surface Reconstruction Data from Certified Depth Standards Obtained by SEM and an Infinite Focus Measurement Machine (IFM), Microchim. Acta. 155 (2006) 279-284.
16 After cleaning using the fresh Clarke's solution and rinsing with deionized water and 17 isopropanol, the coupon surface was scanned under IFM and the pit depth data obtained using 18 the IFM.
19 1004221 A scanning electronic microscope (SEM) as an example Model JSM-6390, JEOL
from Tokyo, Japan, was used to observe the biofilm morphology on a coupon surface after

21 incubation. Coupons were sequentially dehydrated with 25% (v/v), 50%, 75%, and 100%

22 isopropanol sequentially for 10 minutes at each concentration. The coupons were then dried in a

23 critical point dryer, in this example, a Model CPD 020 from Balzers Union, Liechtenstein, using

24 supercritical CO2 supplied by a siphoning type of gas cylinder. The coupons were then coated with palladium to provide conductivity using a sputter coater, such as Model Hummer 6.2 sputter 26 coater from Anatech, Hayward, CA, for biofilm viewing under the SEM. To observe corrosion 27 pits, the coupons were cleaned with the Clarke's solution for 1 minute and rinsed with deionized 28 water and isopropanol. The coupons were transferred to the vacuum chamber for storage before 29 being observed under the SEM.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 1004231 A confocal laser scanning microscope (CLSM) such as a Model LSM
510 from Carl 2 Zeiss, Jena, Germany, was used to detect live and dead cells in a biofilm on the coupons, which 3 were rinsed with the pH 7.4 phosphate buffered solution to remove treatment chemicals, the 4 medium and planktonic cells, before staining. Example dyes that were used to stain the biofilms include the Live/Dead BacLightTM Bacterial Viability Kit L7012 from Life Technologies of 6 Grand Island, NY, SYTO 9. A green fluorescent stain and propidium iodide and a red 7 fluorescent stain may be used. When observed under CLSM, live cells would show up as green 8 dots at an excitation wavelength of 488 nm and dead cells as red dots at 559 nm.
9 1004241 The black FeS color of an SRB broth or an SRB biofilm indicated SRB growth. The colors of 50 mL anaerobic vials changed from pitch black without treatment to being clear with 11 the 100 ppm THPS treatment after 7 days of incubation, in the two rounds of tests, one with 12 initial pH 5.5 and another pH 7Ø With the treatment of THPS or the combination of THPS plus 13 the trehalase, the colors of anaerobic vials and coupon surfaces were lighter than without 14 treatment or with trehalase alone treatment for both initial pH
conditions.
1004251 Referring now to FIGS. 13 and 14, there are shown bar charts indicating the 16 planktonic cell counts for an initial pH 5.5 (FIG. 13) and pH 7.0 (FIG.
14) after a 7-day 17 incubation in 50 mL anaerobic vials with different treatments. The average planktonic cell 18 counts for pH 5.5 (FIG. 13) were 3.0 x 107 cells/mL (no treatment); 2.5 x 107 cells/mL (1 ppm 19 trehalase); 1.8 x 107 cells/mL (3 ppm trehalase); 2.5 x 107 cells/mL (10 ppm trehalase); 3.0 x 107 cells/mL (30 ppm trehalase); 3.3 x 106 cells/mL (50 ppm THPS); 3.0 x 106 cells/mL (50 ppm 21 THPS + 1 ppm trehalase); 1.4 x 106 cells/mL (50 ppm THPS + 3 ppm trehalase); 1.4 x 106 22 cells/mL (50 ppm THPS + 10 ppm trehalase); 1.1 x 106 cells/ mL (50 ppm THPS + 30 ppm 23 trehalase); and 7.8 x 104 cells/mL (100 ppm THPS). The average planktonic cell counts for pH
24 7.0 (FIG. 14) were 2.3 x 107 cells/mL (no treatment); 2.3 x 107 cells/mL
(1 ppm trehalase); 3.5 x 107 cells/mL (3 ppm trehalase); 3.0 x 107 cells/mL (10 ppm trehalase); 1.8 x 107 cells/mL (30 26 ppm trehalase); 3.8 x 106 cells/mL (50 ppm THPS); 3.5 x 106 cells/mL (50 ppm THPS + 1 ppm 27 trehalase); 1.3 x 106 cells/mL (50 ppm THPS + 3 ppm trehalase); 1.3 x 106 cells/mL (50 ppm 28 THPS + 10 ppm trehalas); 7.8 x 105 cells/mL (50 ppm THPS + 30 ppm trehalas); and 7.8 x 104 29 cells/mL (100 ppm THPS). These results show that the planktonic cell counts for the same CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 treatment did not have a large difference for the two different initial pH conditions. With the 2 treatment of 50 ppm THPS, the planktonic cell count decreased by almost 1-log compared with 3 no treatment control. With the combination of 50 ppm THPS + 3 ppm trehalase and 50 ppm 4 THPS + 30 ppm trehalase, there was a 2.4-fold (0.38-log) reduction and 3.0-fold (0.48-log) reduction of planktonic cell counts as compared to the 50 ppm THPS treatment alone, 6 respectively, for initial pH 5.5. For initial pH 7.0, the planktonic cell count reductions for 50 ppm 7 THPS + 3 ppm and 50 ppm THPS + 30 ppm trehalase are 2.9-fold (0.47-log) and 4.9-fold (0.69-8 log), respectively. With the 100 ppm THPS treatment, the average planktonic cell count was 7.8 9 x 104 cells/mL, which reflects a 48.8-fold (1.7-log) reduction compared with the 50 ppm THPS
treatment alone.
11 [00426] These results are clearly evident in these bar charts. In the bar chart of FIG. 13, the 12 planktonic cell counts of SRB were obtained from the hemocytometer readings, except for the 13 100 ppm THPS, which had a low reading and required lVfPN. After the 7-day incubation in vials 14 with different treatments for initial culture medium pH 5.5, the better results were shown with the 50 ppm THPS and the 3, 10 and 30 ppm of trehalase.
16 [00427] The test results shown by FIG. 14 also show the planktonic cell counts of SRB from 17 hemocytometer readings, except for 100 ppm THPS, after the 7-day incubation in vials with 18 different treatments for an initial culture medium pH 7.0, and similar positive results with the 50 19 ppm THPS and 3, 10 and 30 ppm of Trehalase. The sessile cell counts may be more important than planktonic cell counts in biofilm treatment studies because biofilms are usually directly 21 responsible for MIC and biofouling, rather than planktonic cells.
22 1004281 Referring now to FIGS. 15 and 16, the SRB sessile cell counts from hemocytometer 23 readings, except for 100 ppm THPS, after a 7-day incubation in anaerobic vials with different 24 treatments for two initial pH conditions are illustrated. The sessile cell counts for pH 5.5 are shown in the chart of FIG. 15 and were 2.3 x 107 cells/cm2 (no treatment); 2.5 x 107 cells/cm2 (1 26 ppm trehalase); 2.0 x 107 cells/cm2 (3 ppm trehalase); 2.5 x 107 cells/cm2 (10 ppm trehalase); 3.5 27 x 107 cells/cm2 (30 ppm trehalase); 4.6 x 106 cells/cm2 (50 ppm THPS);
4.0 x 106 cells/cm2 (50 28 ppm THPS + 1 ppm trehalase); 1.4 x 106 cells/cm2 (50 ppm THPS + 3 ppm trehalase); 1.0 x 106 29 cells/cm2 (50 ppm THPS + 10 ppm trehalase); 8.9 x 105 cells/cm2 (50 ppm THPS + 30 ppm CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 trehalase); and 3.8 x 105 cells/cm2 (100 ppm THPS). Based on these sessile cell counts, the 2 trehalase alone treatment did not decrease sessile cell count. The sessile cell count had a roughly 3 1-log reduction for the 50 ppm THPS alone treatment compared with the no treatment as the 4 control. However, with the combinations of 50 ppm THPS + 3 ppm trehalase and 50 ppm THPS
+ 30 ppm trehalase, the sessile cell count decreased 3.3-fold (0.52-log) reduction and 5.2-fold 6 (0.72-log) reduction, respectively.
7 [00429] As shown in the bar chart of FIG. 16, the results from SRB
sessile cell counts after 8 the 7-day incubation with initial culture medium of pH 7.0 were 3.3 x 107 cells/cm2 (no 9 treatment); 3.0 x 107 cells/cm2 (1 ppm trehalase); 3.8 x 107 cells/cm2 (3 ppm trehalase); 3.0 x 107 cells/cm2 (10 ppm trehalase); 2.3 x 107 cells/cm2 (30 ppm trehalase); 3.9 x 106 cells/cm2 (50 ppm 11 MPS); 3.0 x 106 cells/cm2 (50 ppm THPS + 1 ppm trehalase); 8.0 x 105 cells/cm2 (50 ppm 12 THPS + 3 ppm trehalase); 7.3 x 105 cells/cm2 (50 ppm THPS + 10 ppm trehalase); 6.8 x 105 13 cells/cm2 (50 ppm THPS + 30 ppm trehalase); and 3.8 x 105 cells/cm2 (100 ppm THPS) 14 respectively. With the combinations of 50 ppm THPS + 3 ppm trehalase and 50 ppm THPS + 30 ppm trehalase, the sessile cell counts decreased by 4.9-fold (0.69-log) and 5.7-fold (0.76-log), 16 compared with 50 ppm THPS alone treatment, respectively.
17 [00430] Referring now to FIG. 17, a bar chart shows the test results of the weight loss of 18 C1018 carbon steel coupons after 7-day incubation with different treatments. The coupon weight 19 loss is indicative of general corrosion process. The weight loss was 1.9 mg/cm2 for no treatment control. For trehalase alone treatment, the weight losses were 1.6 mg/cm2; 1.5 mg/cm2; 1.8 21 mg/cm2; and 1.4 mg/cm2 corresponding to 1 ppm, 3 ppm, 10 ppm, and 30 ppm trehalase, 22 respectively, indicating that trehalase alone had some effect on the weight loss in a dose-23 dependent manner. With the 50 ppm THPS alone treatment, the weight loss was reduced to 1.0 24 mg/cm2. The combination of 50 ppm THPS + 30 ppm trehalase treatment further reduced the weight loss to 0.7 mg/cm2, achieving an extra 30% reduction in weight loss compared with the 26 50 ppm THPS alone treatment (i.e. additional 30% reduction in general corrosion process).
27 [00431] Referring now to FIG. 18, a bar chart shows the test results of measuring the 28 corrosion pit depth (indicative of pitting corrosion) on C1018 carbon steel coupons after a 7-day 29 incubation with different treatments. On the no treatment control coupon surface, the maximum CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 pit depth reached 15.9 [tm after 7 days. With the 30 ppm trehalase alone treatment, the maximum 2 pit depth slightly changed to 16.4 [tm. With the 50 ppm THPS alone treatment, the maximum pit 3 depth decreased to 9.0 [tm. The maximum pit depth decreased further by 54% to 4.1 [tm when 50 4 pm THPS + 30 ppm trehalase was used. The maximum pit depth was 4.3 [tm when the dosage of THPS increased to 100 ppm. The 30 ppm trehalase reduced the THPS dosage from 100 ppm to 6 50 ppm to achieve the same maximum pit depth reduction. The average coupon weight loss and 7 the pit depth data with different treatments are shown in Table 5.

11 Average Weight Loss and Pit Depth Data in Different Treatments After 7-day Incubation Pit depth ([tm) Weight loss (mg/cm2) No treatment 15.9 1.9 1 ppm trehalase 15.8 1.6 3 ppm trehalase 18.2 1.5 10 ppm trehalase 15.5 1.8 30 ppm trehalase 16.4 1.4 50 ppm THPS 9.0 1.0 50 ppm THPS + 1 ppm trehalase 8.0 1.0 50 ppm THPS + 3 ppm trehalase 4.0 0.8 50 ppm THPS + 10 ppm trehalase 4.4 0.7 50 ppm THPS + 30 ppm trehalase 4.1 0.7 100 ppm THPS 4.3 0.5 [00432] The confocal laser scanning microscope (CLSM) was used to visualize biofilms on 16 coupons as shown in the images of FIGS. 19A through 19K. These images of SRB biofilms on CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 C1018 carbon steel coupons were made after the 7-day biofilm prevention test. The no treatment 2 image is shown in FIG. 19A; 1 ppm trehalase (FIG. 19B); 3 ppm trehalase (FIG. 19C); 10 ppm 3 trehalase (FIG. 19D); 30 ppm trehalase (FIG. 19E); 50 ppm THPS (FIG.
19F); 50 ppm THPS + 1 4 ppm trehalase (FIG. 19G); 50 ppm THPS + 3 ppm trehalase (FIG. 19H); 50 ppm THPS + 10 ppm trehalase (FIG. 191); 50 ppm THPS + 30 ppm trehalase (FIG. 19J); and 100 ppm THPS
6 (FIG. 19K).
7 [00433] The live cells (in green color in the original images and noted in the drawings by the 8 lighter shaded areas, with some indicated by "G") densely covered the no treatment control 9 coupon and the coupons that had treatments at 1 ppm, 3 ppm, 10 ppm and 30 ppm trehalase alone, which were retrieved after 7 days of incubation (FIGS. 19A through 19E). With the 11 treatment of 50 ppm THPS alone, many sessile cells were killed, and mostly dead cells (in red 12 color in the original images and noted in the drawings by the lighter shaded areas, with some 13 indicated by "R") show up (FIG. 19F). However, there were still a few live cells on the coupon 14 surface. With the enhancement of trehalase, 50 ppm THPS + 30 ppm trehalase (FIG. 19J) killed most sessile cells and less biofilm biomass was left on the coupon surface.
With the 100 ppm 16 THPS alone treatment (FIG. 19K), almost all sessile cells were dead.
There was mostly dead 17 biofilm biomass left on the coupon surface.
18 1004341 Referring to images shown in FIGS. 20A through 21K, there are shown surface 19 morphologies of SRB biofilms and corrosion pits on the surfaces of the different coupons. The various SEM images of SRB biofilms on C1018 carbon steel coupons after the 7-day biofilm 21 prevention test are illustrated with no treatment (FIG. 20A); 1 ppm trehalase (FIG. 20B); 3 ppm 22 trehalase (FIG. 20C); 10 ppm trehalase (FIG. 20D); 30 ppm trehalase (FIG. 20E); 50 ppm THPS
23 (FIG. 20F); 50 ppm THPS + 1 ppm trehalase (FIG. 20G); 50 ppm THPS + 3 ppm trehalase (FIG.
24 20H); 50 ppm THPS + 10 ppm trehalase (FIG. 201); 50 ppm THPS + 30 ppm trehalase (FIG.
20J); and 100 ppm THPS (FIG. 20K).
26 1004351 Abundant sessile cells are seen on the coupons having the no treatment control, 1 27 ppm, 3 ppm, 10 ppm and 30 ppm trehalase (FIGS. 20A through 20E). With the 50 ppm THPS
28 alone treatment and the combination treatment of 50 ppm THPS + 1 ppm, 3 ppm, 10 ppm 29 trehalase (FIGS. 20F through 201), fewer sessile cells are seen in the SEM images, but many CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 sessile cells were still seen. With the enhancement of 30 ppm trehalase (FIG. 20J) for 50 ppm 2 THPS, sessile cells were seen less numerous than with 50 ppm THPS alone treatment. FIG. 20K
3 shows that 50 ppm THPS + 30 ppm trehalase treatment appeared to be nearly comparable to that 4 of 100 ppm THPS treatment. The parallel polishing lines on the base metal were seen on coupon surfaces (FIGS. 20J and 20K).
6 1004361 Referring now to FIGS. 21A thorugh 21K, there are shown corrosion pit images on 7 the coupon surfaces with different treatments in the biofilm prevention test. SEM images of 8 corrosion pits on carbon steel surfaces after the 7-day biofilm prevention test are shown with no 9 treatment (FIG. 21A); 1 ppm trehalase (FIG. 21B); 3 ppm trehalase (FIG.
21C); 10 ppm trehalase (FIG. 21D); 30 ppm trehalase (FIG. 21E); 50 ppm THPS (FIG. 21F); 50 ppm THPS +
1 ppm 11 trehalase (FIG. 21G); 50 ppm THPS + 3 ppm trehalase (FIG. 21H); 50 ppm THPS + 10 ppm 12 trehalase (FIG. 211); 50 ppm THPS + 30 ppm trehalase (FIG. 21J); and 100 ppm THPS (FIG.
13 21K).
14 1004371 Obvious pits are seen on the no treatment control (FIG. 21A) and the trehalase alone (FIGS. 21B through 21E), coupon surfaces. The 50 ppm THPS alone treatment and 50 ppm 16 THPS + 1 ppm, 3 ppm, 10 ppm trehalase (FIGS. 21F through 211) led to smaller and fewer pits 17 on coupon surfaces. With the treatment of the combination of 50 ppm THPS
+ 30 ppm trehalase, 18 fewer and shallower pits can be found in FIG. 21J and the polishing lines were revealed. It 19 achieved the similar pitting corrosion prevention outcome to that of 100 ppm THPS (FIG. 21K).
The SEM pits in FIGS. 21A through 21K are consistent with the SEM biofilm images in FIGS.
21 20A thorugh 20K.
22 1004381 All performed tests verify the positive use of trehalase with non-oxidizing biocides, 23 such as THPS, against the sulfate reducing bacteria (SRB) that are often found responsible for 24 recalcitrant biofilm formation and microbiologically infuenced corrosion (MIC) problems.
Biocides are used to treat SRB biofilms and MIC, but SRB gradually develop resistance to 26 biocides, that with prolonged use, ultimately requires constant increase in biocide concentration 27 and/or frequency of use, that raise operational and environmental problems in addition to 28 increased costs. In these tests, trehalase was tested as biocide enhancer to enhance tetrakis CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 hydroxymethyl phosphonium sulfate (THPS) to mitigate Desulfovibrio vulgaris biofilm and 2 biocorrosion on carbon steel in ATCC 1249 culture medium at 37 C for 7 days.
3 [00439] In summary, the sessile cell counts showed that 50 ppm (w/w) THPS
+ 30 ppm (w/w) 4 trehalase led to an extra 5.2-fold (0.72-log) or 5.7-fold (0.76-log) sessile cell reduction compared with the 50 ppm THPS alone treatment with an initial culture medium pH 5.5 or 7.0, 6 respectively. The treatment of 50 ppm (w/w) THPS + 30 ppm (w/w) trehalase led to an extra 7 54% and 30% in pit depth reduction and weight loss reduction compared with the 50 ppm THPS
8 alone treatment. The treatment of 50 ppm THPS + 30 ppm achieved the same maximum pit 9 depth reduction as the 100 ppm THPS alone treatment. The CLSM (confocal laser scanning microscopy) and SEM (scanning electron microscopy) images confirmed weight loss and pit 11 depth reduction results clearly indicating that trehalase significantly enhanced the THPS
12 mitigation of D. vulgaris biofilm and biocorrosion on carbon steel.
13 [00440] The composition and method for treating biofilm formation and growth on a substrate 14 is provided. The composition includes 1 ppm to 1,000 ppm of Trehalase, and in an example, about 3 ppm to 50 ppm, and in another example, about 3 ppm to 30 ppm of Trehalase, and 1 ppm 16 to 10,000 ppm of at least one biocide, and in an example, about 50 to 100 ppm of the biocide, 17 including a preferred non-oxidizing biocide tetrakis hydroxymethyl phosphonium sulfate 18 (THPS). The composition is effective for reducing or eliminating biofilm formation or biofilm 19 growth or both, as well as eradicating established, recalcitrant biofilms, such as biofilms comprising sulfate reducing bacteria (SRB). As previously mentioned, biofilms that include 21 SRB are known to cause MIC or biofouling or both. A method for treating the biofilm formation 22 and growth on a substrate is provided and the method may be implemented in a variety of ways 23 and is effective for reducing or preventing biofilm formation or biofilm growth or both, as well 24 as eradicating established, recalcitrant biofilms, such as biofilms that include SRB that may lead to MIC, biofouling or both. As used throughout the description, unless clear from the context or 26 otherwise specified, the units of ppm (parts per million) are based on mass (w/w). For example, 27 a 1 ppm biocide solution would have one mg of biocide per one kg of solution, in an example.
28 The term aqueous solution as used herein, unless otherwise specified, refers to a solution in 29 which the solvent is water, including, but not limited to, water containing various salts such as CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 magnesium sulfate, sodium citrate, calcium sulfate, ammonium chloride, dipotassium phosphate, 2 sodium lactate, and ammonium iron sulfate and ocean and sea water or brackish water or other 3 sources of fresh water. The compositions and methods may include at least one biocide as THPS
4 and may include 1 ppm to 10,000 ppm THPS, but in other embodiments, the biocide may be 5 ppm to 5,000 ppm THPS, including 10 ppm to 1,000 ppm THPS, including 20 ppm to 500 ppm 6 THPS, including 25 ppm to 100 ppm THPS, and also including 30 ppm to 50 ppm THPS, and 30 7 ppm to 100 ppm THPS, and 50 ppm to 100 ppm THPS. Different chemical additives may be 8 added, including dispersants, surfactants and combinations thereof. In an example, the trehalase 9 may include 3 ppm to 50 ppm trehalase, or 3 ppm to 30 ppm of trehalase, or 10 ppm to 30 ppm of trehalase, or 3 ppm to 30 ppm of trehalase, or 3 ppm to 100 ppm of trehalase.
11 1004411 Based on physico-chemical properties and mechanism of action against biofilm-12 forming bacteria, trehalase can be widely used as a "green" biocide enhancer for prevention and 13 treatment of microbial biofilms and mitigation of biocorrosion caused by various 14 microorganisms. The term "microorganisms" as used herein, unless otherwise specified, shall mean any and all microorganisms capable of colonizing biofilm-vulnerable surfaces and/or 16 causing microbiologically influenced corrosion, or both, either directly or indirectly. Examples 17 of microbes that generally colonize and cause damage to pipelines in the gas and oil industries 18 include, but are not limited to, Enterobacter and Citrobacter bacteria (e.g., E. dissolvens, E.
19 ludwigii, C. farmeri and C. amalonaticus); Eubacterium and Clostridium bacteria (e.g., Clostridium butyricum, Clostridium algidixylanolyticum, Anaeorfilum pentosovorans, 21 Bacteroides sp., Acinobacter sp., Propionibacterium sp.); sulfate reducing bacteria including, but 22 not limited to, Desulfovibrionales (e.g., Desulfovibrio desulfuricans, Desulfovibrio vulgaris, 23 Desulfovibrio aminophilus); nitrate reducing bacteria; nitrite reducing bacteria;
24 Desulfobacterales, and Syntrophobacterales; thiosulfate reducing anaerobes (e.g., Geotoga aestuarianis, Halanaerobium congolense, Sulfurospirillum sp.);
tetracholoroethene degrading 26 anaerobes (e.g., Sporomusa ovata); triethanolamine degrading bacteria (e.g., Acetobacterium 27 sp.); denitrifiers (e.g., Acidovorax sp., Pseudomonas sp.); xylan degrading bacteria; Nitrospirae;
28 Halomonas spp.; Idiomarina spp.; Marinobacter aquaeolei; Thalassospira sp.; Silicibacter sp.;
29 Chromohalobacter sp.; Bacilli (e.g., Bacillus spp., Exiguobacterium spp.); Comamonas CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 denitrificans; Methanobacteriales; Methanomicrobiales; Methanosarcinales.
Examples of 2 microbes that generally colonize and cause damage to pipelines in other industries include, but 3 are not limited to, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus 4 ("MRSA"), Escherichia coli, Enterococcus fecalis, Pseudomonas aeruginosa, Aspergillus, Candida, Clostridium difficile, Staphylococcus epidermidis, and Acinobacter sp.
6 1004421 As noted before, the biocide is a non-oxidizing biocide such as THPS. Other biocides 7 may be different agents that exhibit various biocidal or antimicrobial properties. For example, in 8 certain embodiments of the compositions and methods disclosed herein, the at least one biocide 9 is selected from the group consisting of tetrakis hydroxymethyl phosphonium sulfate (THPS), glutaraldehyde, dibromonitriloproprionamide (DBNPA), polyhexamethylene biguanide (PHIMB), 11 methylene bis(thiocyanate) (MB T), 2-(thiocyanomethylthio)benzothiazole (TCMTB), bronopol, 12 2-bromo-2-nitro-1,3-propanediol (BNPD), tributyl tetradecyl phosphonium chloride (TTPC), 13 taurinamide and derivatives thereof, phenols, quaternary ammonium salts, quinaldinium salts, 14 lactones, organic dyes, thiosemicarbazones, quinones, carbamates, urea, salicylamide, carbanilide, guanide, amidines, imidazolines, p-hydroxybenzoate esters, isopropanol, propylene 16 glycol, formaldehyde, iodine and solutions thereof, povidone-iodine, hexamethylenetetramine, 17 noxythiolin, 1-(3-chloroally1)-3,5,7-triazo-1-azoniaadamantane chloride, taurolidine, taurultam, 18 N-(5-nitro-2-furfurylidene)-1-amino-hydantoin, 5-nitro-2-furaldehyde semi carbazone, 3,4,4'-19 trichlorocarbanilide, 3,4',5-tribromosalicylanilide, 3-trifluoromethy1-4,4'-dichlorocarbanilide, 8-hydroxyquinoline, thymol, chlorhexidine, benzalkonium chloride, cetylpyridinium chloride, 21 silver sulfadiazine, silver nitrate, bromine, isothiazolones, polyoxyethylene (dimethylimino) 22 ethylene (dimethylimino) ethylene dichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-5-23 triazine (terbuthylazine), and combinations thereof.
24 [00443] The addition of trehalase to non-oxidizing biocide THPS
demonstrates the high efficacy of this combination for mitigation of a recalcitrant D vulgaris biofilm and biocorrosion 26 and strong implications for biocide use reduction, environmental harm diminishing, and 27 operational cost savings in the oil and gas industries, the industrial water systems, and various 28 other industrial sectors. Preferred biocides for use in combination with trehalase are those non-29 oxidizing biocides and those biocides that have chemical compatibility with trehalase, and CPST Doc. 281421.1 Date Recue/Date Received 2020-07-28 CA Application CPST Ref: 40102/00001 1 approved by the FDA and/or EPA (such as cleaner-sanitizers and disinfectants for hard surfaces, 2 porous and non-porous materials, agriculture and food processing industry premises and 3 equipment), including but not limited to, benzalkonium chloride (a/k/a alkyl dimethyl benzyl 4 ammonium chloride) and quaternary ammonium compounds, glutaraldehyde and glutaraldehyde/quaternary ammonium compound combinations, chlorhexidine, 6 polyhexamethylene biguanide, silver nanoparticles and iodophors.
7 [00444] The composition and method treats the formation and growth of a biofilm that 8 includes sulfate reducing bacteria on a substrate. The substrate can be metal such as carbon steel 9 or stainless steel, a metal alloy, nylon, plastic, composite material, wood, glass, ceramic, porcelain, a painted surface, a rock or soil. The substrate may be a holding vessel, pipe, 11 underground rock formations, underground soil formations, a ship hull, a well bore, 12 infrastructure, a beam, a trough, a girder, sheeting, prefabricated structures, and underwater 13 structures. The biocide and trehalase, in one example, are in a solution that is added to a medium 14 that contacts the substrate or added to the substrate as a solution alone. The medium could be an oil, an aqueous solution, a hydraulic fracturing fluid, a fuel, carbon dioxide, a natural gas, an 16 oil/water mixture, a fuel/water mixture, water containing salts, ocean or sea water, brackish 17 water, sources of fresh water, lakes, rivers, streams, boggs, ponds, marshes, run-off from the 18 thawing of snow or ice, springs, groundwater, aquifers, precipitation, different liquids at ambient 19 temperature and hydrophobic but soluble in organic solvents, hexanes, benzene, toluene, chloroform, diethyl ether, vegetable oils, petrochemical oils, crude oil, refined petrochemical 21 products, volatile essential oils, fossil fuels, gasoline, mixtures of hydrocarbons, jet and rocket 22 fuels, biofuels, and different combinations. The medium could be an oil/water mixture.
23 1004451 The previous description of the disclosed embodiments is provided to enable any 24 person skilled in the art to make or use the disclosed embodiments.
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the principles defined 26 herein may be applied to other embodiments without departing from the scope of the disclosure.
27 Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is 28 to be accorded the widest scope possible consistent with the principles and novel features as 29 defined by the following claims.

CPST Doc: 281421.1 Date Recue/Date Received 2020-07-28

Claims (9)

THAT WHICH IS CLAIMED IS:

1. A method for treating the formation and growth of a biofilm comprising sulfate reducing bacteria on a substrate, comprising:
contacting the substrate with a composition comprising 3 ppm to 30 ppm of trehalase and 50 to 100 ppm of a non-oxidizing biocide.

2. The method of claim 1 wherein the non-oxidizing biocide comprises tetrakis hydroxymethyl phosphonium sulfate (THPS).

3. The method of claim 1 wherein the non-oxidizing biocide comprises a biocide selected from the group consisting of glutaraldehyde, dibromonitriloproprionamide (DBNPA), polyhexamethylene biguanide (PHMB), methylene bis(thiocyanate) (MBT), 2-(thiocyanomethylthio)benzothiazole (TCMTB), bronopol, 2-bromo-2-nitro-1,3-propanediol (BNPD), tributyl tetradecyl phosphonium chloride (TTPC), taurinamide and derivatives thereof, phenols, quaternary ammonium salts, quinaldinium salts, lactones, organic dyes, thiosemicarbazones, quinones, carbamates, urea, salicylamide, carbanilide, guanide, amidines, imidazolines, p-hydroxybenzoate esters, isopropanol, propylene glycol, formaldehyde, iodine and solutions thereof, povidone-iodine, hexamethylenetetramine, noxythiolin, 1-(3-chloroallyl)-3,5,7-triazo-1-azoniaadamantane chloride, taurolidine, taurultam, N-(5-nitro-2-furfurylidene)-1-amino-hydantoin, 5-nitro-2-furaldehyde semicarbazone, 3,4,4'-trichlorocarbanilide, 3,4',5-tribromosalicylanilide, 3-trifluoromethyl-4,4'-dichlorocarbanilide, 8-hydroxyquinoline, thymol, chlorhexidine, benzalkonium chloride, cetylpyridinium chloride, silver sulfadiazine, silver nitrate, bromine, isothiazolones, polyoxyethylene (dimethylimino) ethylene (dimethylimino) ethylene dichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-S-triazine (terbuthylazine), and combinations thereof.

4. The method of claim 1 wherein the substrate is one of a metal, metal alloy, nylon, plastic, composite material, wood, glass, ceramic, porcelain, a painted surface, a rock or soil.

5. The method of claim 1 wherein the substrate is selected from the group consisting of a holding vessel, pipe, underground rock formations, underground soil formations, a ship hull, a well bore, infrastructure, a beam, a trough, a girder, sheeting, prefabricated structures, and underwater structures.

6. The method of claim 1 wherein the substrate is a pipeline.

7. The method of claim 1 wherein the trehalase and non-oxidizing biocide are in a solution and added to a medium that contacts the substrate.

8. The method of claim 7 wherein the medium is selected from the group consisting of an oil, an aqueous solution, a hydraulic fracturing fluid, a fuel, carbon dioxide, a natural gas, an oil/water mixture, a fuel/water mixture, water containing salts, ocean or sea water, brackish water, sources of fresh water, lakes, rivers, streams, boggs, ponds, marshes, run-off from the thawing of snow or ice, springs, groundwater, aquifers, precipitation, different liquids at ambient temperature and hydrophobic but soluble in organic solvents, hexanes, benzene, toluene, chloroform, diethyl ether, vegetable oils, petrochemical oils, crude oil, refined petrochemical products, volatile essential oils, fossil fuels, gasoline, mixtures of hydrocarbons, jet and rocket fuels, biofuels, and combinations thereof.

9. The method of claim 7 wherein the medium comprises an oil/water mixture.