Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
  • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
  • A01N43/38—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings condensed with carbocyclic rings
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
  • A01N43/84—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms six-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/34—Alcohols
  • A61K8/345—Alcohols containing more than one hydroxy group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/69—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing fluorine
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
  • C08K5/00—Use of organic ingredients
  • C08K5/16—Nitrogen-containing compounds
  • C08K5/34—Heterocyclic compounds having nitrogen in the ring
  • C08K5/3412—Heterocyclic compounds having nitrogen in the ring having one nitrogen atom in the ring
  • C08K5/3415—Five-membered rings
  • C08K5/3417—Five-membered rings condensed with carbocyclic rings
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
  • C09D5/16—Antifouling paints; Underwater paints
  • C09D5/1606—Antifouling paints; Underwater paints characterised by the anti-fouling agent
  • C09D5/1612—Non-macromolecular compounds
  • C09D5/1625—Non-macromolecular compounds organic
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D7/00—Features of coating compositions, not provided for in group C09D5/00; Processes for incorporating ingredients in coating compositions
  • C09D7/40—Additives
  • C09D7/60—Additives non-macromolecular
  • C09D7/63—Additives non-macromolecular organic
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/26—Organic compounds containing nitrogen
  • C11D3/28—Heterocyclic compounds containing nitrogen in the ring
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
  • C11D7/22—Organic compounds
  • C11D7/32—Organic compounds containing nitrogen
  • C11D7/3281—Heterocyclic compounds
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F1/00—Treatment of water, waste water, or sewage
  • C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F2303/00—Specific treatment goals
  • C02F2303/20—Prevention of biofouling
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W10/00—Technologies for wastewater treatment
  • Y02W10/30—Wastewater or sewage treatment systems using renewable energies
  • Y02W10/37—Wastewater or sewage treatment systems using renewable energies using solar energy

Definitions

• Microbial biofilms cause systemic infections in humans and costly marine and industrial related damage and inefficiency. They cost billions of dollars yearly in equipment damage, product contamination, energy losses and medical infections.
• biofilms can be associated with tissues (e.g., inner ears, teeth, gums, lungs, heart valves and the urogenital tract) and on indwelling medical devices (e.g., contact lenses, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, urinary catheters, and voice prostheses).
• tissues e.g., inner ears, teeth, gums, lungs, heart valves and the urogenital tract
• indwelling medical devices e.g., contact lenses, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, urinary catheters, and voice prostheses.
• An estimated 80% of all microbial infections involve biofilms.
• Biofilms are extremely difficult to remove with existing technology because they can withstand high temperature (>150°C), biocides, anti-infective compounds including anti-biotics, and host immune responses. Also, the huge doses of antimicrobials required to rid systems of biofilm bacteria are environmentally undesirable and medically impractical. Thus, there is an immediate need for safe and effective products that combat biofilms.
• the present disclosure provides a composition for inhibiting biofilm formation, growth or survival comprising a compound having a structure:
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C 2 H 4 , CO, SO 2 , O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CNR A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , or
• the present disclosure provides a method for inhibiting biofilm formation, growth or survival, the method comprising applying or administering a composition comprising an anti-biofilm compound to a surface susceptible to biofilm formation, the anti-biofilm compound having a structure:
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C 2 H 4 , CO, SO 2 , O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CNR A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , or
• the present disclosure provides an article of manufacture comprising a compound having a structure:
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C2H 4 , CO, SO2, O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CNR A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , or CONR
• an object of this invention is to protect biologic and non- biologic surfaces from biofilm formation.
• the present invention is based on the finding that reef fish have developed ways to prevent biofilm formation on their surfaces and under the skin barrier.
• the present invention involves isolating bacteria from the surfaces of such coral reef fish (e.g., Sparisoma ninidae and Lutjanus purpureus), wherein the surfaces remain relatively free of macro-fouling.
• a probiotic microbial community present on the mucosal surfaces of the reef fish was found to provide broad protection against microbial settlement, infections, and macro-fouling.
• the present invention further demonstrates that the probiotic microbial community protects the fish from colonization of pathogens by producing antibacterial substances, making the environment unsuitable for foreign bacteria, or producing signaling molecules inhibiting the attachment of foreign bacteria.
• the effect is based at least in part on a compound of the structure:
• the present invention involves isolating bacteria from the mucosal surface of the fish and finding that their extracts can be used develop novel anti-biofilm forming agents.
• natural compounds such as those provided in the present invention
• it is also advantageous to use an organism that produces an anti- biofilm signaling substance instead of an antibacterial substance. Bacteria are unable to develop resistance against a signaling molecule, thus extending the lifetime of the drug.
• Another advantage of using an organism that naturally has inhibitory effects on biofilm formation is that the biotechnological process to collect the substance is not likely to be hindered by genetic reversion because the wild type producer was not a result of genetic manipulation.
• the effect is based at least in part on a compound of the structure:
• FIG. 1 This figure shows the growth curves for isolates P4-4, P5-2, and P3-2.
• FIG. 5 This figure is a phylogenetic tree showing relatedness of strain P4-4.
• Isolate P4-4 is distantly related (94.85%) to P. immobilis and (94.73%) to P. phenylpyruvicus.
• FIG. 7 The figures show confocal laser scanning microscopic images of S. aureus biofilm stained with congo red.
• the negative control biofilm developed thick and tightly adhered (FIG. 7A).
• Table 1 Table 1 presents the culture collection catalogue of fish isolates.
• Table 2 Table 2 lists the morphological characteristics of the fish skin isolates.
• Table 3 Table 3 lists the physiological characteristics of the isolates from the skin of fish.
• Table 4 Table 4 provides a summary of antagonistic activity of isolates against reference strains for the extract test and streak test that yielded positive results.
• Table 5 Table 5 provides a summary of activity of living cells of isolates (P4-4, P5-2, P2-1 , P3-2) against bacterial and eukaryotic fouling.
• Table 6 Table 6 provides a summary of extract activity of isolates (P4-4, P5-2, P2-1 , P3-2) against bacterial and eukaryotic fouling.
• Table 7 Table 7 supplies information on the taxonomic affiliation of the isolates.
• Table 8 Table 8 provides a summary of DNA GC content for isolates (P4-4, P3-2, P3-1 , P5-2) and closest related species.
• Table 9 Table 9 provides DNA-DNA hybridization data for isolates (P4-4, P3-2, P3-1 , P5-2) and closest related species.
• Table 10 provides the Ames test results fro P3-2 crude supernatant. DETAILED DESCRIPTION OF THE DISCLOSURE
• biofilm refers to a population of microorganisms
• anti-fouling refers to counteracting or preventing the building up of deposits on underwater surfaces.
• anti-fouling agent refers to compound used to protect underwater surfaces from attaching organisms and includes, in one embodiment, a compound having the structure:
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C 2 H 4 , CO, SO 2 , O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CN R A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B ,
• anti-fouling coating refers to a coating labeled and formulated for application to submerged stationary structures and their appurtenances to prevent or reduce the attachment of marine or freshwater biological organisms.
• Anti-fouling coatings are used to protect articles against infestation, especially ships' hulls, screens, nets, constructions, quaysides, signaling equipment and articles which come into contact with sea water or brackish water.
• these coatings include a compound having the structure:
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C 2 H 4 , CO, SO 2 , O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CNR A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , or
• antimicrobial refers to a substance that destroys or inhibits the growth of microorganisms.
• biocide refers to a chemical which can kills or inhibits the growth of living organisms such as bacteria, fungi, molds, and slimes.
• biodeterioration refers to the deterioration of materials of economic importance by microorganisms.
• fouling refers to an accumulation of marine organism deposits on a submerged surface.
• hull refers to the body or frame of a ship or boat.
• the terms “inhibit”, “inhibiting” and “inhibition” refer to stopping, preventing, reducing or eliminating the growth or functioning of an organism or part of an organism. Such inhibition may adversely affect the physiological and/or morphological characteristics of a target organism.
• indwelling refers to a medical device placed or implanted within the body, such as a catheter or pacemaker.
• the term "isolate” when used as a verb refers to a process of separating a particular species, strain, or substance from a mixture, sample or biological specimen. Such a process may further involve characterizing the separated species, strain or substance.
• the term "isolate" when used as a noun refers to a particular species, strain, or substance separated from a mixture, sample or biological specimen.
• the term “medical device” refers to an instrument, apparatus, implement, machine, contrivance, implant, in vitro reagent, or other similar or related article, including any component, part or accessory, which is intended for use in the diagnosis of disease or other conditions, or in the cure, mitigation, treatment, or prevention of disease, in man or other animals, or intended to affect the structure or any function of the body and which does not achieve its primary intended purposes through chemical action and which is not dependent upon being metabolized for the achievement of its primary intended purposes.
• target organism refers to any organism for which inhibition is desired. Such organisms include but are not limited to bacteria, photosynthetic eukaryotic organisms, and non-photosynthetic eukaryotic organisms. II. Fish Microflora.
• Marine eukaryotes have developed natural means for preventing colonization of bacteria and higher organisms. They developed two strategies to protect against biofouling: the secretion of signaling compounds and housing of probionts. Both strategies interfere with signals regulating biofilm formation.
• Fish possess bacterial populations on or in their skin, gills, digestive tract, and light-emitting organs.
• the internal organs (kidney, liver, and spleen) of healthy fish may contain bacteria, but there is debate on whether or not muscle is actually sterile.
• the numbers and taxonomic composition of the bacterial populations often reflect those of the surrounding water.
• the role of the bacteria includes the ability to degrade complex molecules (therefore exercising a potential benefit in nutrition), to produce vitamins and polymers, and to be responsible for the emission of light by the light- emitting organs of deep-sea fish.
• the surface of fish consists of skin, scale and mucus.
• the microorganisms that inhabit the slime and external surfaces of healthy marine fish include, Pseudomonas, Vibrio, Achromobacter, Flavobacterium/Cytophaga, Moraxella, Micrococcus, Acinetobacter, Photobacterium, Bacillus, and Aeromonas (Hansen and Olafsen, 1999). Bacteria may assist the fish in locomotion and protection against pathogens. Bacteria associated with a fast moving cornetfish (Fistularia commersonii) were hydrophobic and produced drag-reducing slime (Hansen, 1999), which allow for the fish to travel faster in the water.
• the bacteria may be selected for by the fish if they are beneficial for the fish.
• Bacteria isolated from the skin of a healthy turbot differed from the flora in the surrounding water (Hansen and Olafsen, 1999).
• the microorganisms may be selected for because of the specific sugars in the fish mucus (Hansen and Olafsen, 1999).
• the colonization of bacteria begins with a chemotactic attraction of the bacteria to the mucus, followed by penetration and adhesion to receptors in the mucus or epithelial cells (Hansen and Olafsen, 1999).
• fish mucus can have inhibitory effects on bacteria. The inhibition can be caused by immunoglobulins, lysozyme, and continuous shedding (Hansen and Olafsen, 1999).
• a biofilm is an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance matrix.
• Biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical devices, industrial or potable water system piping, or natural aquatic systems.
• Biofilms are composed primarily of microbial cells and Extracellular Polymeric
• EPS Substances
• Noncellular materials such as mineral crystals, corrosion particles, clay or silt particles, or blood components, depending on the environment in which the biofilm has developed, may also be found in the biofilm matrix.
• EPS may account for 50%, 60%, 70%, 80%, and even up to 90% of the total organic carbon of biofilms and can be considered the primary matrix material of the biofilm.
• EPS may vary in chemical and physical properties, but it is primarily composed of polysaccharides. Different organisms produce differing amounts of EPS and that the amount of EPS increases with age of the biofilm.
• EPS is highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding and prevents desiccation in some natural biofilms.
• EPS may associate with metal ions, divalent cations, other macromolecules (such as proteins, DNA, lipids, and even humic substances). EPS production is known to be affected by nutrient status of the growth medium; excess available carbon and limitation of nitrogen, potassium, or phosphate promote EPS synthesis. Slow bacterial growth will also enhance EPS production. EPS may also contribute to the antimicrobial resistance properties of biofilms by impeding the mass transport of antimicrobials through the biofilm, probably by binding directly to these agents (Donlan 2002).
• the development of a biofilm occurs in distinct stages. Once a conditioning film develops on the surface, planktonic bacteria attach to the surface, proliferate, excrete EPS, communicate, and build complex structures (Donlan and Costerton, 2002).
• the structures are composed of single-species and multi-species bacterial microcolonies that take the form of towers, mushroom shapes, and streamers with water channels running through them (Donlan and Costerton, 2002). Proximity of cells within the microcolony (or between microcolonies) provides an ideal environment for creation of nutrient gradients, exchange of genes, and quorum sensing.
• microcolonies may be composed of multiple species, the cycling of various nutrients (e.g., nitrogen, sulfur, and carbon) through redox reactions can readily occur in aquatic and soil biofilms.
• Organisms composing the biofilm may also have a marked effect on the biofilm structure. Number of component organisms may affect the thickness of the biofilm. Structure may also be influenced by the interaction of particles of non-microbial components from the host or environment.
• the bacteria in the biofilm are phenotypically different than their planktonic form, showing a decrease in growth rate and different gene expression (Donlan and Costerton, 2002). Bacteria in a biofilm are protected from grazing and secondary environmental stresses such as, ultraviolet (UV) exposure, desiccation, and temperature shifts (Nystrom et al, 1992). More nutrients are made available to bacteria in a biofilm, however, diffusion of nutrients slows considerably deep in the biofilm (Anderl et al, 2003). Signaling molecules are produced when the cells reach a critical density and the biofilm is formed.
• UV ultraviolet
• signals are used for bacterial communication and formation of the microcolony architecture (Stoodley et al, 1999, Stoodley et al, 2001 , Donlan and Costerton, 2002, and O'Toole et al, 2000) or cell detachment from biofilms.
• Biofilm cells may be dispersed either by shedding of daughter cells from actively growing cells, detachment as a result of nutrient levels or quorum sensing, or shearing of biofilm aggregates (continuous removal of small portions of the biofilm) because of flow effects.
• the pattern and development of a biofilm is not only regulated by QS.
• Biofilms also provide an ideal niche for the exchange of extrachromosomal DNA (plasmids). Conjugation (the mechanism of plasmid transfer) occurs at a greater rate between cells in biofilms than between planktonic cells.
• Biofilms develop preferentially on inert surfaces, or on dead tissue, and occur commonly on medical devices and fragments of dead tissue such as sequestra of dead bone; they can also form on living tissues, as in the case of endocarditis. Characteristics of biofilms involved in infectious disease processes include a) detachment of cells or biofilm aggregates may result in bloodstream or urinary tract infections or in the production of emboli, b) cells may exchange resistance plasmids within biofilms, c) cells in biofilms have dramatically reduced susceptibility to antimicrobial agents, d) biofilm-associated gram-negative bacteria may produce endotoxins, and e) biofilms are resistant to host immune system clearance (Donlan 2002).
• Tissue associated infections include native valve endocarditis (NVE), otitis media (OM), chronic bacterial prostatitis, cystic fibrosis (CF), and periodontitis.
• NVE native valve endocarditis
• OM otitis media
• CF cystic fibrosis
• Biofilms on indwelling medical devices can occur on central venus catheters, urinary catheters, prosthetic heart valves, contact lenses, and intrauterine devices (lUDs). Medical devices such as dental unit equipment and waterlines can be a source of infections.
• NVE is caused by the interaction between the vascular endothelium and bacteria or fungi in the bloodstream.
• the microorganisms involved include Streptococci sp., Staphylococci sp., Candida, and Aspergillus sp. (Donlan and Costerton, 2002).
• OM is a disease of the middle ear with an inflamed mucoperiosteal lining.
• the microorganisms involved include Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa (Donlan and Costerton, 2002).
• Chronic bacterial prostatitis is a bacterial infection of the prostate gland by E. coli, Klebsiella sp., Proteus sp., Serratia sp., Pseudomonas aeruginosa, and Enterococcus faecalis, Bacteroides sp., Gardnerella sp., and Corynebacterium sp. (Donlan and Costerton, 2002).
• CF is a chronic lethal single gene disorder with symptomatic infection of the lower respiratory system by P. aeruginosa, Staphylococcus aureus and Burkholderia cepacia.
• Periodontal diseases are infections of the supporting tissues of teeth that range from mild to chronic.
• the bacteria involved include, Actinomyces naeslundii, Bacteroides forsythus, B. intermedius, B. pneumosintes, Eubacterium brachy, E. timidum, Fusobacterium nucleatum, Haemophilus aphrophilus, Lactobacillus spp., Peptostreptococcus micros, Porphyromonas gingivalis, Pseudomonas anaerobius, Selenomonas sproda, and Wolinella recta (Donlan and Costerton, 2002).
• Typical animal diseases where bacterial biofilms are believed to be involved based on histopathologic and ultrastructural appearance of the bacteria within tissue include: mastitis (Streptococcus agalactiae, Staphylococcus aureus), pneumonia (Mannheimia haemolytica, Pasteurella multockia), liver abscess (Fusobacterium necrophorum), lymphadenitis (Corynebacterium pseudotuberculosis, Streptococcus spp.), enteritis (Escherichia coli, Salmonella spp.) and wound infections (Staphylococcus aureus, Pseudomonas aeruginosa).
• indwelling medical devices e.g., ocular lenses, dental implants, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, coronary stents, vascular bypass grafts, pacemakers, peritoneal dialysis catheters, prosthetic joints, central nervous system shunts, tympanostomy tubes, urinary catheters, and voice prostheses
• indwelling medical devices e.g., ocular lenses, dental implants, central venous catheters and needleless connectors, endotracheal tubes, intrauterine devices, mechanical heart valves, coronary stents, vascular bypass grafts, pacemakers, peritoneal dialysis catheters, prosthetic joints, central nervous system shunts, tympanostomy tubes, urinary catheters, and voice prostheses
• other devices used in the health-care environment have been shown to harbor biofilms, resulting in measurable rates of device-associated infections.
• Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative bacteria or yeast. Bacteria commonly isolated from these devices include the gram-positive Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus viridans; and the gram-negative Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Biofilms may be composed of a single species or multiple species, depending on the device and its duration of use in the patient.
• Device-related infection results from the introduction of organisms, primarily bacteria, during the device insertion or implantation procedure, or from attachment of bloodborne organisms to the newly inserted device and their subsequent propagation on its surface.
• the organisms first attach to the device surface through the secretion of polymers (polysaccharides) or the extension of fibrils, which anchor the bacteria to the surface.
• cell division of the bacteria produces sister cells that form microcolonies and create a protective barrier commonly known as biofilm or bioslime. Once this barrier is formed, the bacteria can propagate within the biofilm and release substantial amounts of bacterial cells into the surrounding fluids and tissues.
• the infections that ensue can be difficult to treat, because the body's macrophages and antibiotics are unable to reach the primary source of the infecting bacteria. Often, effective treatment requires removal of the offending device.
• Urinary catheter infections pose the greatest risk of the indwelling medical devices infections, the organisms involved include Staphylococcus aureus, S. epidermidis, P. aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, and Candida albicans (Donlan and Costerton, 2002).
• Urinary catheter infections can be caused by Acinetobacter calcoaceticus, Enterobacter aerogenes, E. coli, Enterococcus faecalis, K pneumoniae, M morganii, P. aeruginosa, P. mirabilis, Providencia stuartii, Proteus vulgaris, and S. epidermidis (Donlan and Costerton, 2002).
• the organisms that may colonize prosthetic heart valves include, Streptococci sp., S. aureus, gram-negative coccobacilli, or fungi (Donlan and Costerton, 2002).
• Organisms that can attach to contact lenses include P. aeruginosa, S. aureus, S. epidermidis, Serratia sp., E. coli, Proteus sp., and Candida sp. (Donlan and Costerton, 2002).
• lUDs can be contaminated with S. epidermidis, Enterococcus sp. Lactobacillus plantarum, Corynebacterium sp., Micrococcus sp., Candida albicans, and S. aureus (Donlan and Costerton, 2002).
• the anti-biofilm substances produced by the isolates of the present invention can be used on the surface of or within these devices to provide long term protection against bacterial colonization and reduce the incidence of device-related infections.
• These substances can also be incorporated as an anti-biofilm forming agent, in combination with an antibiotic, into coatings for indwelling medical devices. Coatings will sufficiently kill or inhibit the initial colonizing bacteria and prevent device-related infection as long as the substance is presented in an inhibitory concentration at the device-microbe interface.
• the effect is based at least in part on a compound of the structure:
• the medical devices which are amenable to coatings of the subject anti- biofilm substances generally have surfaces composed of thermoplastic or polymeric materials such as polyethylene, Dacron, nylon, polyesters, polytetrafluoroethylene, polyurethane, latex, silicone elastomers and the like.
• Devices with metallic surfaces are also amenable to coatings with the anti-biofilm substances.
• Such devices for example bone and joint prosthesis, can be coated by cement mixture containing the subject anti-biofilm substances.
• the anti-biofilm substances leach from the cement into the surrounding prosthesis surface environment.
• TDMAC tridodecylmethyl ammonium chloride
• a medical device having a polymeric surface such as polyethylene, silastic elastomers, polytetrafluoroethylene or Darcon
• TDMAC precoated catheters are commercially available; for example, arterial catheters coated with TDMAC are available from Cook Critical Care, Bloomington, Ind.
• the device carrying the absorbed TDMAC surfactant coated can then be incubated in a solution of the anti- biofilm substance for one hour or so, washed in sterile water to remove unbound anti-biofilm substance and stored in a sterile package until ready for implantation.
• a further method useful to coat the surface of medical devices with the subject antibiotic combinations involves first coating the selected surfaces with benzalkonium chloride followed by ionic bonding of the anti-biofilm substance composition. See, e.g., Solomon, D. D. and Sherertz, R. J., J. Controlled Release 6:343-352 (1987) and U.S. Pat. No. 4,442,133.
• Alternative methods and reagents provided in U.S. Pat. Nos. 4,107,121 , 4,442,133, 4,678,660 and 4,749,585, 4,895,566, 4,917,686, 4,952,419, and 5,013,30, can be used to coat devices with the anti-biofilm substances of the present invention.
• the anti-biofilm agent of the present invention can be directly incorporated into the polymeric matrix of the medical device at the polymer synthesis stage or at the device manufacture stage.
• the anti-biofilm agent can also be covalently attached to the medical device polymer.
• the second premise is that white blood cells (WBC), antibodies, and antibiotics are unable to penetrate the biofilm EPS matrix well (Donlan and Costerton, 2002).
• WBC white blood cells
• the third deals with the physiological change of the bacteria. A change in phenotype can allow for the bacteria to enhance their activity against the immune response even if WBC and antibiotics can penetrate (Donlan and Costerton, 2002 and Artini, 2003).
• the fourth phenomenon is that some bacteria deep in the biofilm have a reduced growth rate which makes them more resistant to certain agents (Anderl and Costerton, 2003 and Donlan and Costerton, 2002).
• bacteria in biofilms may avoid the immune responses by mimicking human tissue with EPS material (McClane and Montgomeryzner, 1999 and Vuong et al, 2003). For these reasons biofilm infections typically show recurring symptoms, after cycles of antibiotic therapy, until the sessile population is surgically removed from the body. VII. Industrial Biofilm Damage.
• Biofilms in industrial systems cause severe clogging, contamination, and biodeterioration. Bacterial contamination of the water distribution systems can occur if biofilms are sloughed off naturally or removed by treatment (Piriou et al, 1997). Biofilms in drinking water piping systems accommodate Heliobacter pylori, Mycobacterium spp., and protozoa infected with Legionella pneumophila (Storey et al, 2003 and Obst et al, 2003). This results in decreased water quality and increased treatment costs and health risks. Biofilms in pipes carrying water or other liquids cause reduced flow and increased resistance to flow. Formation of biofilms on probes, sensors, screens and filters results in reduced efficiency.
• Microbial induced corrosion is the deterioration of materials caused by microorganisms under anaerobic or aerobic conditions. MIC is associated with localized, underdeposited, pitting corrosion, and accounts for 15% to 30% of the corrosion-related pipeline failures in the gas and nuclear industries alone. It is also a major cause of failures in the water treatment and chemical industries, and is also associated with corrosion failures, blockage and souring in gas and oil production and storage. To prevent MIC many chemicals are used commercially, but few of them have been tested extensively before they were released. Hexavalent Chromium (Cr(VI)) was used as a component of anti-corrosive coatings (chrome plating and spray coatings). The Occupational Safety and Health Administration (OSHA) recognizes that Cr VI is a hazardous potential lung carcinogen, it can cause permanent eye damage, and it can cause skin and nasal ulcers (Anon, 2003d).
• Cr VI Hexavalent Chromium
• Biofilm formation and other multicellular-like activities that cover a wide range of processes such as, swarming, bioluminescence, virulence, and dispersal (Greenberg 2003, Yarwood et al, 2003, Rasmussen et al, 2000, and Stoodley et al, 2001 ) are a result of bacterial communication, referred to as quorum sensing (QS).
• QS quorum sensing
• Numerous bacteria communicate intercellularly, to regulate the transcription of multiple target genes in concert with their cell density, through the production of one or more diffusible signal molecules.
• bacteria multiply, reach a critical number of cells, and then produce signaling molecules (autoinducers).
• the process of detachment of bacteria from the biofilm occurs when some cells in the interior of a cluster use QS to revert to their planktonic form, an opening is created and the planktonic cells are allowed to disperse (Davies, 2003, Greenberg, 2003, Kjellenberg, 2003, Purevdorj and Stoodley, 2003, Sauer, 2003, Sauer et al, 2002, Stoodley et al, 2001 , and Yarwood et al, 2003). Dispersal is thought to occur naturally when conditions change favoring the planktonic form and/or cell clusters reach a size greater than 40 m x 10 m (Sauer 2003, Sauer et al, 2002, and Davies, 2003).
• the Luxl/LuxR QS system used by Gram-negative bacteria produces acylated homoserine lactones (AHLs) and quinolones (Pesci et al, 1999) that freely diffuse in and out of each cell.
• AHLs acylated homoserine lactones
• quinolones Pani et al, 1999
• the AHL concentration is proportional to cell density.
• the autoinducer interacts with the LuxR protein that binds DNA promoter elements and activates the transcription and expression of QS related genes (Federle et al, 2003).
• Luxl/LuxR quorum-sensing systems including the genera Agrobacterium, Aeromonas, Burkholderia, Chromobacterium, Citrobacter, Enterobacter, Erwinia, Hafnia, Nitrosomonas, Obesumbacterium, Pantoea, Pseudomonas, Rahnella, Ralstonia, Rhodobacter, Rhizobium, Serratia, Vibrio, Xenorhabdus, and Yersinia (Hentzer and Givskov, 2003).
• Gram-positive bacteria use an oligopeptide/two-component QS system.
• the signaling molecules involved are autoinducing peptides and lactones (butyrolactone) (Federle et al, 2003 and Newman et al, 2003).
• AIPs typically consist of 5-17 amino acids (Federle et al, 2003).
• ATP-binding cassette (ABC) transporters usually process the precursor peptides and export them as autoinducers (Bassler, 1999).
• This QS system differs from the Luxl/LuxR system used by Gram-negative bacteria because cell-surface oligopeptide transporters are needed to secrete AIP into the environment because the Gram-positive bacterial cell membrane is not permeable to AIPs.
• Bacteria that produce AIPs include S. aureus, S. epidermidis, Streptococcus gordonii, Streptococcus pyogenes, and Streptococcus pneumoniae (Bassler, 2003).
• AI-2 signaling has been adapted by the different bacteria that use it to influence virulence, biofilm formation, and a variety of niche-specific behaviors.
• AI-2 signaling is involved with virulence factors in Streptococcus pyogenes Vibrio harveyi, and Staphylococcus aureus. (Federle et al, 2003, Manefield et al, 2000, Novick, 2003).
• AI-2 is involved in the mixed-species biofilm formation between two oral bacteria, Streptococcus gordonii and Porphyromonas gingivalis (Federle et al, 2003) as well as S. aureus (Cramton et al, 1999).
• AI-2 is also associated with tight adherence to intestinal epithelia by E. coli (Federle et al, 2003). IX. Antimicrobials.
• Antimicrobial classification can be based on bacterial spectrum (broad versus narrow), route of administration (injectable versus oral versus topical), type of activity (bactericidal versus bacteriostatic), or chemical structure.
• Antibiotics are produced by microorganisms and have antagonistic effects on other microorganisms. They target metabolism, cell wall synthesis, protein synthesis, nucleic acid synthesis, cell membrane permeability or transport (Johnson et al, 2002). These include bacteriostatic drugs that inhibit growth or bacteriocidal drugs that kill the microorganism.
• Bacteriocins are substances produced by bacteria that kill closely- related species without rupturing cell walls and membranes. The aim of antimicrobial chemotherapy is to harm the microorganism but not be toxic to the host.
• Antimetabolites are structural analogs of normal metabolites that inhibit the action of specific enzymes (Johnson et al, 2002).
• Cell wall synthesis inhibitors may inhibit trans-peptidation, inhibit the synthesis of peptidoglycan, act in the cytoplasm, in the membrane, or in the cell wall.
• Cell wall synthesis inhibitors include -lactam drugs: penicillins, cephalosporins, carbapenems. Penicillins inhibit the trans- peptidation enzymes involved in peptidoglycan synthesis for Gram-positive and Gram-negative bacteria. Cephalosporins and Carbapenems have mechanisms of action similar to penicillin (Johnson et al, 2002).
• the cephalosporin antibiotics was isolated from a fungus of the Cephalosporilum genus, that inhibited Gram-positive and Gram-negative bacteria, isolated from seawater near a sewage outlet in Cagliari, Sardinia, Italy (Mascaretti, 2003). Protein synthesis inhibitors are known as broad- spectrum antibiotics that require bacterial growth to be effective. Protein synthesis inhibitors include aminoglycosides, macrolides, lincomycins, tetracyclines, chloramphenicol, and griseofulvin. Aminoglycosides are bacteriocidal for Gram- negative bacteria and bind to the 30S ribosomal subunit and they may irreversibly block translation initiation and/or cause mRNA misreading.
• Bacteriostatic macroglides and lincomycins bind to the 23S RNA in the 50S ribosomal subunit and block translation. Tetracylines are bacteriostatic and bind to the ribosomal subunit preventing aminoacyl tRNA from binding the acceptor site. Chloramphenicol binds to the SOS ribosomal subunit and inhibits peptide-bond formation thus making it bacteriostatic for Gram-positive and Gram-negative bacteria.
• Griseofulvin is a fungistatic drug that inhibits protein assembly and is active against fungi with chitin in the cell walls (Johnson et al, 2002). Nucleic acid synthesis inhibitors inhibit DNA or RNA synthesis. Ethambutol inhibits mycobacterial mycolic acid biosynthesis (Johnson et al, 2002). Cytoplasmic membrane inhibitors alter plasma membrane osmotic properties or lipid synthesis in the fungal membrane (Johnson et al, 2002).
• Bacteriocins are proteinaceous toxins produced by bacteria that inhibit the growth of a narrow range of bacteria. Bacteriocins include lactic acid produced by lactiobacilli are part of the intestinal floral of healthy fish (Jankauskiene, 2002), mutacins produced by Streptococcus mutans an indigenous oral bacteria (Parrot et al, 1989), and colicins produced by Escherichia coli and other members of Enterobacteriaceae (Morency et al, 1995). Bacteriocins may inhibit the growth of pathogens in the surrounding environment. Anti-viral compounds inhibit viral replication. These compounds target viral nucleic acid replication, host-cell receptor recognition, the penetration and uncoating process, or specific enzyme functions (Johnson et al, 2002). The agents are inhibitors of herpesviruses, retroviruses, or other viruses including influenza virus.
• the bacterial isolates of the present invention and their extracts can be used as antibacterial or bactericidal agents to remove disease-causing organisms from external surfaces. They can be used in different products such as soaps, detergents, health and skincare products and household cleaners.
• the antibacterial agents of the present invention can be used alone, or in combination with other antimicrobial agents.
• Fouling is an undesirable growth of biological material on a surface immersed in water. Fouling usually starts with adhering and spreading of populations of bacteria over faces that are in contact with water. The bacteria pioneers are followed by numerous different algae, invertebrate larvae, hydroids, bryozoans, sponges, tunicates, echinoderms, cnidarians, and coelenterates (McDougall, 1943, Holmstrom et al, 1996 and Hadfield, 2003).
• fouling creates many problems. Fouling results in increased drag, weight and corrosion for marine structures; decreased aesthetic appearance of the marine structure; and increased maintenance costs associated with removal of the fouling and repair of the structure. Settlement of the foulers on the hulls of boats creates an increase in drag. Just a small amount of fouling can lead to an increase of fuel consumption of up to 40%, and possibly as much as 50%, since the resistance to movement will be increased. Vessel bottoms not protected by anti-fouling systems may gather 150 kg of fouling per square meter in less than six months of being at sea. On a Very Large Crude Carrier with 40,000 square meter underwater areas, this would add up to 6,000 tonnes of fouling. The cost related to fouling is estimated at 6 billion USD for 2002.
• Marine fouling occurs not only on marine vessels such as ship's hulls and drive systems, but also on other structures exposed to sea water.
• Such structures may include: pilings, marine markers, undersea conveyances like cabling and pipes, fishing nets, bulkheads, cooling towers, and any device or structure that operates submerged.
• Plants have been shown to produce compounds that reduced biofilms by 65- 75% (Pasmore, 2003). Algae secrete anti-fouling substances on their surfaces. Cross-kingdom signaling molecules, brominated furanones, produced by the red algae, Delisea pulchra, is a molecular analog of AHLs that prevented biofilm formation, reduce the settlement of barnacles, and control the development of the fertilized eggs of the fouling alga Ulva. (Rasmussen et al, 2000, Rice et al, 1999, Muir, 2000, Kjelleberg, 1997, and Givskov et al, 1999).
• Changing a hydroxyl group to an acetate group on the furanone increased the anti-fouling activity by orders of magnitude (Clare, 1995).
• the eelgrass, Zostra marina produced zosteric acid, a sodium salt of a sulfated phenolic acid, that is an anti-fouling agent against barnacles and tubeworms (Geiger et al, 2001 ).
• the sulphate ester group on zosteric acid may be responsible for the anti-fouling activity.
• Octocorals (Dendronephthya sp. and Sinularia sp.) produced anti-fouling trigonelline and diterpenoid lipids (Kawamata et al, 1994 and Mizobuchi et al, 1994).
• the whip coral, Leptogorgia virgulata produced two anti-fouling lipids, pukalide and epoypukalide against barnacles (Rittschof et al, 1985).
• a sea pansy, Renilla renijormis produced a group of anti-fouling diterpene compound called renillafoulins (Rittschof et al, 1985).
• the anti-fouling activity is attributed to small oxygen containing rings known as lactones and furans (Clare, 1995).
• the bryozoan, Zoobotryon pellucidum produced toxic tribromogramine that inhibits larvae settlement (Clare, 1995).
• the sponge, Mycale microsigmatosa, and the gorgonian, Phyllogorgia dilate prevented the attachment of barnacles in situ (Pereira et al, 2002).
• the sponge, Protophlitaspongia aga produced a pyrimidine derivative, 3,4,5,6-tetrahydro-6-hydroxymethyl-3,6-dimethyl-4-pyrimidinecarboxylic acid and zooanemonin that are active against the barnacle, Balanus amphitrite and .alpha.
• the housing of probionts is another way plants and animals are able to prevent the colonization of common fouling organisms such as, algal spores, bacteria, invertebrate larvae, and fungi. Signaling molecules produced by bacteria can inhibit the attachment of the target fouling organisms providing protection for the host. Bacteria isolated from rock surfaces, marine animals, and marine algae inhibited vertebrate larva by 10%, 30%, and 74% respectively (Holmstrom et al, 1996).
• P. tunicata a dark green pigmented marine bacterium, isolated from C. intestinalis, produced a compound inhibitory against the largest range of organisms, including various bacteria (including Bacillus subtilis), green algal spores (U. lactuca), red algal spores (Po ⁇ ysiphonia sp.), sea squirt larvae (C.
• P. tunicata is a facultatively anaerobic rod, oxidase-positive, and motile by a sheathed polar flagellum that exhibited non- fermentative metabolism and required sodium ions for growth (Holmstrom et al, 2002). It was not capable of using citrate, fructose, sucrose, sorbitol and glycerol but it was able to utilize mannose and maltose and hydrolyses gelatin (Holmstrom et al,
• the substance produced by P. tunicata has three active components, two are anti-biofilm and anti-fouling proteins and the third is a toxic low molecular weight compound (Clare, 1995).
• Inhibition of QS system can occur by the inhibition of signal generation, inhibition of signal dissemination (degradation or importing), and inhibition of signal reception (Hentzer et al, 2003).
• AHL signal generation can be inhibited by competitive inhibition with analogs of the amino donor used in the generation of the homoserine lactone ring.
• Inhibition of signal dissemination can be observed with the degradation of signaling molecules by enzymes produced by a Bacillus species (Hentzer et al, 2003).
• Some bacteria including the S. typhimurium and E. coli, are able to import extracellular AI-2 into the cell by transporters, thus eliminating the signal from the environment and rendering it nonfunctional (Federle and Bassler,
• Anti-fouling paints that contain copper, TBT, and other toxic additives have historically been produced to protect marine surfaces from biofouling. These paints containing the additives are usually formulated to expose the toxic materials embedded within the coating structure to the environment. It is this exposure that allows the toxic materials to leach into the marine environment, thus reducing attachment by the marine organisms. However, these additives have a generally adverse effect upon the marine environment.
• TBT based coatings are the most effective yet also the most harmful. According to the Environmental Protection Agency (EPA) (Anon, 2003a), TBT is an endocrine disrupting chemical that causes reproductive problems in aquatic animals.
• EPA Environmental Protection Agency
• Endocrine system disruptors are chemicals that activate or block hormones, interfering with the normal system (Watts, 2000). Since the eighties imposex, the development of male sexual characteristics, has been reported. TBT caused deformities of imposex in gastropods (Prosobranchia) (Marisa cornuarietis) (Anon, 2003a), the mud snail llyanassa obsolete (Gooding, 2003), the whelk Buccinum undatum (Svavarsson et al, 2001 ) and impacted the clam Ruditapes decussates (Coelho et al, 2002).
• TBT also caused reduced growth rate in the blue mussel (Mytilus edulis), shell hardening in the oyster (Crassostrea gigas), mortality in Rainbow trout (Salmo gairdneri), and impacted a variety of other organisms (Anon, 2003b).
• High levels of TBT found in marine mammals is evidence that TBT is bio-accumulating in the food chain.
• TBT suppresses the immune system in mammal. Consequently, the Marine Environment Protection Committee (MEPC) of the International Maritime Organization (IMO) has approved a resolution to phase out and eventually prohibit the use of toxic organotin derivatives in anti-fouling paint.
• MEPC Marine Environment Protection Committee
• IMO International Maritime Organization
• the bacterial isolates and/or extracts of the present invention can be incorporated into marine coatings to limit undesirable marine fouling.
• the anti-fouling paints of this disclosure offer significant advantages over previous attempts to solve marine fouling problems.
• the disclosed method relies on living cells and/or extracts to prevent biofouling.
• the coatings of this invention can be formulated so as not to contain toxic materials (such as heavy metals), and still retain their efficacy. This avoids the environmental concerns associated with the use of heavy metal biocides.
• the anti-fouling paint of the present disclosure may further contain binders(s), pigment(s), solvent(s) and additive(s).
• Solvents carry the solid components of paint and are used to obtain the desired viscosity and correct consistency.
• the solvent include, but not limited to, aromatic hydrocarbons such as xylene and toluene; aliphatic hydrocarbons such as hexane and heptane, esters such as ethyl acetate and butyl acetate; amides such as N-methylpyrrolidone and N,N- dimethylformamide; alcohols such as isopropyl alcohol and butyl alcohol; ethers such as dioxane, THF and diethyl ether; and ketones such as methyl ethyl ketone, methyl isobutyl ketone and methyl isoamyl ketone.
• the solvent may be used alone or in combination thereof.
• the binder or resin is one of the most important components of paint. It is the basic solid film former that remains after the solvent has evaporated and which binds the pigment particles together into a cohesive paint film.
• the binder determines many of the necessary film properties such as adhesion, gloss level, hardness, abrasion resistance, flexibility, speed of drying and durability.
• binders include, but not limited to, alkyd resin, acrylic or vinyl emulsions, polyurethane resins, epoxy resins, silicone based resins, acrylic resins and inorganic silicate based resins.
• binders which have been used in anti-fouling coatings are vinyl resins, particularly a vinyl chloride/vinyl acetate copolymer, and rosin.
• the paint composition can contain one or more pigments.
• the pigments used in paint are normally present as fine solid particles that are dispersed, but not soluble, in the binder and solvent.
• Examples of pigments include, but not limited to, titanium dioxide, cuprous oxide, iron oxide, talc, aluminium flakes, mica flakes, ferric oxide, cuprous thiocyanate, zinc oxide, cupric acetate meta-arsenate, zinc chromate, zinc dimethyl dithiocarbamate, zinc ethylene bis(dithiocarbamate) and zinc diethyl dithiocarbamate.
• Additive ingredients may optionally be incorporated into the coating composition of the present invention thus prepared.
• the additive ingredients are dehumidifiers, wetting/dispersing agents, anti-settling agents, anti- skinning agents, drying/curing agents, anti-marring agents and additives ordinarily employed in coating compositions as stabilizers and anti-foaming agents.
• any antibiotic which is toxic to gram negative organisms and which is relatively insoluble in seawater can be used with an anti-fouling marine paint.
• U.S. Pat. No. 4,678,512, U.S. Pat. No. 4,286,988, U.S. Pat. No. 4,675,051 , U.S. Pat. No. 4,865,909 and U.S. Pat. No. 5,143,545 describe methods for preparing marine anti-fouling paints.
• the anti-fouling coatings so produced can be used for the submersible surfaces of boat hulls, pilings, buoys, floating or emplaced offshore platforms, submergence vehicles, navigational aids, and any marine structures where marine biofouling may be a problem.
• St. George's University Microbiology Depository meets the following requirements: (1 ) it has a continuous existence; (2) it exists independently of the control of the depositor; (3) it possesses the staff and facilities sufficient to examine the viability of a deposit and store the deposit in a manner which ensures that it is kept viable and uncontaminated; (4) it provides for sufficient safety measures to minimize the risk of losing biological material deposited with it; (5) it is impartial and objective; (6) it can furnish samples of the deposited material in an expeditious and proper manner; and (7) it will promptly notify depositors of its inability to furnish samples, and the reason why.
• Some embodiments include a compound represented by Formula 1 :
• Id is optionally substituted indolyl
• A is a bond, CH 2 , CH 2 OH, C2H 4 , CO, SO2, O, or S
• Z is CO, SO 2 , CHOH, CH 2 , or CNR A
• X-Y is: X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A , and Y is CR 3 R 4 or OCR 3 R 4 ; or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 , and Y is R 6
• R 1 , R 2 , and R 3 are independently R A
• R 4 is R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , or CONR
• the compound may comprise a salt, prodrug, tautomer, alternate solid form, non-covalent complex, analog, derivative or combination thereof of the structure according to Formula 1 .
• a compound or chemical structural feature such as aryl when referred to as being “optionally substituted,” it includes a feature that has no substituents (i.e. be unsubstituted), or a feature that is "substituted,” meaning that the feature has one or more substituents.
• substituted has the broadest meaning known to one of ordinary skill in the art, and includes a moiety that replaces one or more hydrogen atoms attached to a parent compound or structural feature.
• the substituent may be an ordinary organic moiety known in the art, which may have a molecular weight (e.g. the sum of the atomic masses of the atoms of the substituent) of 15 g/mol to 50 g/mol, 15 g/mol to 100 g/mol, 15 g/mol to 150 g/mol, 15 g/mol to 200 g/mol, 15 g/mol to 300 g/mol, or 15 g/mol to 500 g/mol.
• a molecular weight e.g. the sum of the atomic masses of the atoms of the substituent
• the substituent comprises: 0-30, 0-20, 0- 10, or 0-5 carbon atoms; and 0-30, 0-20, 0-10, or 0-5 heteroatoms independently selected from: N, O, S, Si, F, CI, Br, or I; provided that the substituent comprises at least one atom selected from: C, N, O, S, Si, F, CI, Br, or I.
• substituents include, but are not limited to, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, hydroxy, alkoxy, aryloxy, acyl, acyloxy, alkylcarboxylate, thiol, alkylthio, cyano, halo, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxyl, trihalomethanesulfonyl, trihalome
• molecular weight is used with respect to a moiety or part of a molecule to indicate the sum of the atomic masses of the atoms in the moiety or part of a molecule, even though it may not be a complete molecule.
• alkyl has the broadest meaning generally understood in the art, and may include a moiety composed of carbon and hydrogen containing no double or triple bonds.
• Alkyl may be linear alkyl, branched alkyl, cycloalkyl, or a combination thereof, and in some embodiments, may contain from one to thirty-five carbon atoms.
• alkyl may include CMO linear alkyl, such as methyl (-CH 3 ), ethyl (-CH 2 CH 3 ), n-propyl (-CH 2 CH 2 CH 3 ), n-butyl (- CH2CH2CH2CH3), n-pentyl (-CH2CH2CH2CH2CH3), n-hexyl (- CH2CH2CH2CH2CH2CH3), etc.; C 3- io branched alkyl, such as C 3 H 7 (e.g. iso-propyl), C 4 H 9 (e.g. branched butyl isomers), C 5 Hn (e.g.
• CMO linear alkyl such as methyl (-CH 3 ), ethyl (-CH 2 CH 3 ), n-propyl (-CH 2 CH 2 CH 3 ), n-butyl (- CH2CH2CH2CH3), n-pentyl (-CH2CH2CH2CH2CH3)
• branched pentyl isomers C6H13 (e.g. branched hexyl isomers), C 7 Hi 5 (e.g. heptyl isomers), etc.; C3-io cycloalkyl, such as C3H 5 (e.g. cydopropyl), C 4 H 7 (e.g. cydobutyl isomers such as cydobutyl, methylcyclopropyl, etc.), C 5 H 9 (e.g. cyclopentyl isomers such as cyclopentyl, methylcyclobutyl, dimethylcyclopropyl, etc.) ⁇ (e.g. cyclohexyl isomers), C 7 Hi 3 (e.g. cycloheptyl isomers), etc.; and the like.
• C3-io cycloalkyl such as C3H 5 (e.g. cydopropyl), C 4 H 7
• optionally substituted Ci-12 alkyl refers to a Ci-12 alkyl that may be unsubstituted, or may have 1 or more substituents, and does not limit the number of carbon atoms in any substituent.
• Ci-12 optionally substituted alkyl refers to unsubstituted Ci-12 alkyl, or substituted alkyl wherein both the alkyl parent and all substituents have from 1 -12 carbon atoms. Similar conventions may be applied to other optionally substituted moieties such as aryl and heteroaryl.
• Substituents on alkyl may be the same as those described generally above, except that alkyl may not have an alkyl substituent.
• substituents on alkyl are independently selected from F, CI, Br, I, CN, CO 2 H, -O- alkyl, ester groups, acyl, amine groups, and amide groups, and may have a molecular weight of about 15 to about 100 or about 500.
• aryl has the broadest meaning generally understood in the art, and may include an aromatic ring or aromatic ring system such as phenyl, naphthyl, etc.
• heteroaryl also has the meaning understood by a person of ordinary skill in the art, and in some embodiments, may refer to an "aryl” which has one or more heteroatoms in the ring or ring system.
• heteroaryl may include, but are not limited to, pyridinyl, furyl, thienyl, oxazolyl, thiazolyl, imidazolyl, indolyl, quinolinyl, benzofuranyl, benzothienyl, benzooxazolyl, benzothiazolyl, benzoimidazolyl, etc.
• any reference to a compound herein by structure, name, or any other means includes pharmaceutically acceptable salts, such as sodium, potassium, and ammonium salts; prodrugs, such as ester prodrugs; alternate solid forms, such as polymorphs, solvates, hydrates, etc.; tautomers; or any other chemical species that may rapidly convert to a compound described herein under conditions in which the compounds are used as described herein.
• pharmaceutically acceptable salts such as sodium, potassium, and ammonium salts
• prodrugs such as ester prodrugs
• tautomers or any other chemical species that may rapidly convert to a compound described herein under conditions in which the compounds are used as described herein.
• any structure or name for a compound used herein may refer to any stereoisomer or any mixture of stereoisomers.
• Id may be optionally substituted indolyl. If the indolyl is substituted, it may have 1 , 2, 3, 4, or 5 substituents. Any substituent may be included on the indolyl. In some embodiments, some or all of the substituents on the indolyl may have: from 0 to 10 carbon atoms and from 0 to 10 heteroatoms independently selected from: O, N, S, F, CI, Br, and I (provided that there is at least non-hydrogen atom); and/or a molecular weight of 15 g/mol to 500 g/mol.
• the substituents may be CMO optionally substituted alkyl, such as optionally substituted: CH 3 , C 2 H 5 , C 3 H 7 , cyclic C 3 H 5 , C 4 H 9 , cyclic C 4 H 7 , C 5 Hn , cyclic C 5 H 9 , C 6 Hi 3 , cyclic C6H11 , etc.; CMO optionally substituted alkoxy; halo, such as F, CI, Br, I; OH; CN; NO 2 ; Ci -6 fluoroalkyl, such as CF 3 , CF 2 H, C 2 F 5 , etc.; a CMO ester such as -O 2 CCH 3 , -CO 2 CH 3 , -O 2 CC 2 H 5 , -CO 2 C 2 H 5 , -O 2 C-phenyl, -CO 2 -phenyl, etc.; a CMO ketone such as -COCH 3 , -COC 2
• the indolyl is indol-3-yl. In some embodiments, the indolyl is optionally substituted with 1 or 2 substituents independently selected from Ci-6 alkyl and F. In some embodiments, In is unsubstituted. In some embodiments, In may be:
• Some embodiments may include a compound represented by one or more of Formulas 2-18.
• A may be a bond, CH 2 , CH 2 OH, C 2 H 4 , CO, SO 2 , O, or S In some embodiments, A is CH 2 or CH 2 OH.
• Z may be CO, SO 2 , CHOH, CH 2 , or CNR A . In some embodiments, Z is CO.
• X-Y may be X— Y, wherein X is CO, SO 2 , CHOH, CH 2 , or CNR A and Y is CR 3 R 4 (see, e.g. Formulas 5-8) or OCR 3 R 4 (see, e.g. Formula 14); or X and Y are not directly bonded to one another, and X is R 5 , COR 5 , SO 2 R 5 , CHOH, CH 2 , or CNR A R 5 and Y is R 6 (see, e.g. Formulas 9-13 and 15-18).
• X-Y may be -COCH(OR 15 )- or -COCH 2 -.
• X and Y are not directly bonded to each other, and X is R 5 or COR 5 .
• R 1 may be R A .
• R 1 may be H.
• Each R A may independently be H, or Ci-12 alkyl, including: linear or branched alkyl having a formula C a H a +i, or cycloalkyl having a formula C a H a- i, wherein a is 1 ,
• R A may be H or Ci-6 alkyl. In some embodiments, R A may be H or Ci. 3 alkyl. In some embodiments, R A may be H or CH 3 . In some embodiments, R A may be H.
• Each R B may independently be H, or Ci-12 alkyl, including: linear or branched alkyl having a formula C a H a +i, or cycloalkyl having a formula C a H a , wherein a is 1 , 2,
• R B may be H or Ci -3 alkyl. In some embodiments, R B may be H or CH 3 . In some embodiments, R B may be H.
• R 2 may be R A .
• R 2 may be H.
• R 3 may be R A .
• R 3 may be H.
• R 4 may be R A , F, CI,
• R 4 may be H, Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or d -6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl,
• R 5 may be H, Ci-12 optionally substituted alkyl, or OR A .
• R 5 may be H, d -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O-cyclohe
• R 6 may be H or Ci-12 optionally substituted alkyl.
• R 6 may be H, Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 6 may be H, (CH 2 ) 3 CH 3 , or CH(CH 3 )CH 2 CH 3 .
• R 7 -R 39 may be H or any substituent, such as a substituent having from 0 to 6 carbon atoms and from 0 to 5 heteroatoms independently selected from: O, N, S, F, CI, Br, and I, and/or having a molecular weight of 15 g/mol to 300 g/mol.
• R 7 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 7 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 7 may be H.
• R 8 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 8 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 9 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 9 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• R 10 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 10 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• R 11 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 11 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 12 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 12 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 13 may include R A , OR A , CF 3 , COR A , CO 2 R A , CONR A R B , etc.
• R 13 may be H; OH; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; Ci-6 alkoxy, such as -O-methyl, -O- ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O- cyclobutyl, isomers of -O-pentyl, isomers of -O-
• R 14 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 14 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl
• R 14 may be H.
• R 15 may include R A , COR A , CO 2 R A , CONR A R B , etc.
• R 15 may be H; or d -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 15 may be H.
• R 16 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 16 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci -6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl
• R 17 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 17 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 17 may be H.
• R 18 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 18 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• R 19 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 19 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 19 may be H.
• R 20 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 20 may be H; F; CI; CN; CF 3 ; NH 2 ; or d-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 20 may be H.
• R 21 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 21 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 21 may be H.
• R 22 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 22 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 22 may be H.
• R 23 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 23 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• R 24 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 24 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 24 may be H.
• R 25 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 25 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 26 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 26 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci -6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 26 may be H.
• R 27 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 27 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 28 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 28 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 28 may be H.
• R 29 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 29 may be H; F; CI; CN; CF 3 ; NH 2 ; or d-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 29 may be H.
• R 30 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 30 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 31 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 31 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 31 may be H.
• R 32 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 32 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 32 may be H.
• R 33 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 33 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 33 may be H.
• R 34 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 34 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 34 may be H.
• R 35 may include R A , F, CI, CN, OR A , CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 35 may be H; F; CI; CN; CF 3 ; OH; NH 2 ; Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; or Ci-6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl, isomers of -O- cyclohexyl, etc.
• Ci-6 alkyl such
• R 36 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 36 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 36 may be H.
• R 37 may include R A , F, CI, CN, CF 3 , NO 2 , NR A R B , COR A , CO 2 R A , OCORA, NR A COR B , CONR A R B , etc.
• R 37 may be H; F; CI; CN; CF 3 ; NH 2 ; or Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.
• R 37 may be H.
• R 38 may be H, Ci-i 2 optionally substituted alkyl, or OR A .
• R 38 may be H, Ci-6 alkyl, such as methyl, ethyl, propyl isomers, cyclopropyl, butyl isomers, cyclobutyl isomers, pentyl isomers, cyclopentyl isomers, hexyl isomers, cyclohexyl isomers, etc.; Ci -6 alkoxy, such as -O-methyl, -O-ethyl, isomers of -O-propyl, -O-cyclopropyl, isomers of -O-butyl, isomers of -O-cyclobutyl, isomers of -O-pentyl, isomers of -O-cyclopentyl, isomers of -O-hexyl
• R 30 , R 31 , R 32 , R 33 , R 34 , R 35 , R 36 , and R 37 may each be H;
• R 5 and R 6 may each be independently R A ;
• R 8 may be H or OR A , and
• the composition may be an anti-fouling composition, an antibacterial composition, or a bacteriocidal composition.
• the anti-fouling composition is an anti-fouling coating composition for coating surfaces contacting potable water, sea water or brackish water.
• the antibacterial composition or the bacteriocidal composition may be a coating on a surface of a medical device or may be incorporated into a polymeric matrix of a polymeric surface of a medical device.
• the medical device may be an indwelling medical device selected from an ocular lens, a dental implant, a central venous catheter or needleless connector, an endotracheal tube, an intrauterine device, a mechanical heart valve, a coronary stent, a vascular bypass graft, a pacemaker, a peritoneal dialysis catheter, a prosthetic joint, a central nervous system shunt, a tympanostomy tube, a urinary catheter, or a voice prostheses.
• an indwelling medical device selected from an ocular lens, a dental implant, a central venous catheter or needleless connector, an endotracheal tube, an intrauterine device, a mechanical heart valve, a coronary stent, a vascular bypass graft, a pacemaker, a peritoneal dialysis catheter, a prosthetic joint, a central nervous system shunt, a tympanostomy tube, a urinary catheter, or a
• the compound may be an anti-biofilm signaling substance that inhibits a quorum sensing system selected from a Luxl/LuxR quorum sensing system or an oligopeptide/two-component quorum sensing system.
• the article of manufacture may be an antibacterial soap, an antibacterial detergent, an antibacterial health and skincare product, or an antibacterial household cleaning product.
• the article of manufacture may be an anti- fouling paint or coating composition, such as a marine paint or coating composition.
• the anti-fouling paint or coating composition may further comprise one or more of a binder, a pigment, a solvent, and an additive.
• Example 1 Sampling of Coral Reef Fish.
• GPS Global Positioning System
• Fish were either trapped in a fishpot or shot with a spear gun.
• the fishpot used to trap the fish was made of 0.5 inch galvanized square mesh, 36 inches long, 16 inches wide and high.
• the cornucol hole (horn shaped) on one side of the fishpot was 7 inches in diameter on the outer surface and it tapered inside the fishpot to a 5 inch diameter.
• the fishpot was located approximately 10 feet deep, next to a coral reef, approximately 150 feet away from shore. At the surface after caught, each fish was washed twice with autoclaved artificial seawater to remove any loosely associated microbes and then immediately placed in a sterile plastic bag on ice. The fish was then transported back to the laboratory.
• microorganisms were isolated from six S. ninidae and five microorganisms from three L. purpureus (Table 1 ).
• Fish culture collection was created and catalogued as the following: species name, strain designation, isolation source, medium, name of researcher, physiology, biochemistry, and biotechnologically important properties (for example, producer of unidentified antibiotic or anti-fouling agent).
• the letter P was used to designate the isolates from the Parrotfish, followed by a number representing the fish, a dash and a number representing the isolate.
• the letter S was used to designate the isolates from Snapper (Table 1 ).
• Reference microorganisms were grown on suggested media and cultivation conditions according to the instructions of the vendor or depository, such as the ATCC.
• Psychrobacter immobilis and P. phenylpyruvicus were cultivated on Brain Heart Infusion (BHI) broth (Difco 237500) and BHI agar (Difco 241830) at 26° C.
• Marinobacter hydrocarbonclasticus was cultivated on Marine Broth 2216 (BD 2791 10) and Marine Agar 2216 (BD 212185) at 30° C.
• Aerococcus viridans and Desemzia incerta were cultivated on Brain Heart Infusion broth (Difco 0037) and Trypticase Soy Agar (BBL 1 1043) with 5% defibrinated rabbit blood 37° C. Staphylococcus warneri and Serratia marcescens was cultivated on Nutrient broth (Difco 0003) and Nutrient agar (Difco 0001 ).
• Blood agar (Anon, 1953), Mannitol salts agar (Anon, 1953), Mac Conkey Agar (Anon, 1953), and Cystein tryptic agar (Anon, 1953) were used to test type of hemolysis, acid production from mannitol, resistance to bile salts, and oxidation/fermentation of glucose, respectively.
• Catalase reaction and oxidase reaction were performed using traditional techniques known in the art.
• Isolate P3-1 and P3-2 were Gram-positive cocci, 1 -3 m, halophylic, mesophilic (T 28-37°C), consumed mannitol, and did not contain cytochrome- oxidase. Isolate P3-1 was transparent, catalase-negative, and gamma-hemolytic, while P3-2 produces a white pigment, was catalase-positive, alpha-hemolytic, and its generation time at salinity of 40 g/l for P3-2 was 7.35 hours.
• Isolates P1 -5 and P2-2 were gram positive cocci, 1 -2 m, without colony pigmentation, non-motile, halophilic, mesophilic (grew at 28-37°C), catalase-negative, alpha hemolytic, oxidase-negative, and produced acid from mannitol.
• Isolate P5-2 was Gram-positive cocci, 1 -3 n, orange pigmented, non-motile, halophilic, mesophilic (T 28-37°C), its generation time at salinity of 40 g/l was 1 .38 hours, alpha hemolytic, oxidase- positive, catalase positive and it did not consume mannose,.
• Isolate P4-4 was a Gram-positive motile coccobacilli that grew at 28-37°C. P4-4 did not use glucose, it was catalase-positive, and oxidase-positive.
• the growth curves were obtained at 29°C, pH 7.5-8.0, in ASWA. Growth curves are typical for bacteria. Growth begins at a slow rate with the lag phase, then at a fast exponential rate with the log phase, and finally growth stops with the stationary phase. The latter is when secondary metabolites are produced by the bacteria, thus, extracts were taken from bacteria at this phase.
• OD turbidity measures both live and dead cells thus, OD based growth curves over estimates the number of cells. OD reproduces growth curves accurately, however, and underestimates time line for the stationary phase by several hours (Madigan et al, 2000).
• Generation time for P4-4, P5-2, and P3-2 are 2.53 hrs, 1 .38 hrs, and 7.35 hrs respectively.
• Growth rate constant for P4-4, P5-2, and P3-2 are 0.274, 0.502, and 0.094 respectively. Largest OD was observed with isolate P4-4 and longest generation time was observed with isolate P3-2 (FIG. 1 ). Differences were observed because isolates were not grown at optimum conditions. After 48 hours each bacteria was in stationary phase of growth.
• the isolated organisms were grown in 500 ml flasks containing ASW medium in a shaker bath at 29°C at 180 revolutions per minute (RPM) for 48 hours.
• the cells were separated from the culture medium by centrifugation at 5,000 RPM for 20 minutes in 50 ml centrifuge tubes. The cells were washed twice by additional centrifugation with ASWA to ensure removal of supernatant. Crude extracts were obtained from the supernatant culture medium.
• hydrochloric acid (HCI) was added to supernatant to lower pH to 2.0. Supernatant was then shaken with an equal aliquot of diethyl-ether for 5 minutes. After ten minutes standing, the bottom portion was removed and discarded.
• the ether portion was shaken for 5 minutes with EDTA buffer at pH 8.0 to re-extract the metabolites into the water. After ten minutes of standing, the bottom portion of crude extract was collected in sterile containers and the ether portion was discarded. Also, in some cases, the final ether portion was left open to evaporate the diethyl-ether. Then, 70% alcohol was added to re-suspend extract.
• Reference organism see Example 5
• fish isolate were cross streaked on top of the other on NA plate and incubated at 37°C for 48 hours. Clearance zones of inhibited growth for reference organism were measured and recorded in mm as width of the inhibition streak band. A zone of clearance of 1 .0 mm and larger indicates a positive result of inhibition.
• the gel covered slides were attached to rubber stoppers and exposed to the seawater at a depth of 1 meter below the surface in True Blue Bay, Grenada, at N12°00.040 and W 061 "46.177 for 24 hours.
• Experimental slides were screened for biofilm formation and eukaryotic fouling and compared to controls. Slides were stained with acridine orange and observed under a UV microscope (Carl Zeiss Axioscope equipped with UV lamp with LP 420 excitation filter).
• Isolate P5-2 S. warneri
• Non-photosynthesizing eukaryotic inhibition was observed with extracts from strain P3-2 (45.4%) (FIG. 3, Table 6).
• Photosynthesizing eukaryotic inhibition was observed with extracts from isolates P3-2 (41 .7%) and P4-4 (36.5%) and living cells of isolate P4-4 (78.2%) (FIG. 4, Tables 5 and 6).
• a larger percentage of inhibition was observed against photosynthesizing eukaryotic organisms.
• the isolates were taken from reef fish that inhabit water in the photic zone, exposed to light, therefore, it would be beneficial for the bacteria on their surfaces to be more antagonistic towards photosynthesizing organisms.
• 16S rRNA sequence analysis was performed using Applied Biosystems MicroSeg.TM. microbial analysis software and database to obtain the top ten matches. Matches are presented in a percent genetic distance (low percentage indicates a close match) (Anon, 2003e). Neighbor joining phylogenetic trees were made using the top ten matches.
• P3-1 and P3-2 also showed differences with each other, P3-1 was catalase- negative and gamma-hennolytic, while P3-2 was catalase-positive and alpha- hemolytic, indicating that they may be different strains of Aerococcus viridians or new species.
• a sterile, chemically cleaned glass slide can be painted with a regular paint
• a designated area on the side of a boat can be painted with a regular paint to which the extracts produced by the isolates P3-2, P4-4 and P5-2 are added (see Example 9). Another area painted with a regular paint without the extracts can be used as a control.
• the boat can be deployed into the water for a month and observed for fouling. The results will show that fouling was inhibited on the surface of the boat coated with the paint containing the extracts.
• Example 1 Detachment of Biofilm Formers in Microtiter Wells by Extracts of Isolates.
• Biofilms To develop biofilms, 25 L of stationary growth phase Staphylococcus aureus bacterial culture (requiring about 18 h growth at 37°C. in TSB 2% and containing about 2 x 10 9 cells/mL) can be added under aseptic conditions to a well of a tissue culture-treated polystyrene 96-well plate (cell well tissue culture treated polystyrene plates; Corning, Rochester, N.Y., USA), containing 175 L of growth medium (TSB 2%). Biofilms can be developed (at 37°C) for 6 or 48 h, the growth medium being discarded and freshly added every 12 h.
• Each well can be washed three times with phosphate-buffered saline (PBS) under aseptic conditions to eliminate unbound bacteria, and 200 L of the P5-2 extracts can be added, the mixture being maintained at 37°C. After 3, 6 or 24 hours extract solutions can be removed with a micropipette and wells can be filled (200 L) with undiluted dimethyl sulphoxide (DMSO; Panreac, Barcelona, Spain), which can be used as ATP extractant.
• PBS phosphate-buffered saline
• DMSO dimethyl sulphoxide
• Plates can then be wrapped in plastic and placed in a sonicator bath (P- Selecta, Barcelona, Spain) for 15 min (in the case of 6 h biofilms) or 30 min (in the case of 48 h biofilms) at 40 Hz and 22-24°C to favor the disintegration of bacterial clumps.
• the number of viable bacteria can be estimated by measuring the amount of ATP present in the sample using ATP-bioluminescence (Amorena et al., 1999). The results will show that extracts from P5-2 isolate decreased the S. aureus biofilm cell viability indicating the anti-biofilm activity of the extracts.
• the extract solutions can be used to measure cell turbidity, using a UV spectrophotometer, at an optical density at 595 nm (OD 5 9s)- The average OD of the control wells can be subtracted from the OD of all test wells. The result will show that bacterial cells are present in the extract solution indicating that biofilm forming cells have detached from the surface of the wells after the addition of the microbial extracts.
• Example 12 Microtiter Plate Assay for Assessment of Activity of Microbial Extracts on Biofilm Formation. Strains of Staphylococcus aureus and Psuedomonas auriginosa can be grown overnight in 5 ml test tubes at 32°C in respective media. Overnight cultures can be transferred (0.1 ml) to 10 ml of minimal defined media using glucose as the only carbon source and vortexed. After vortexing, 100 I volumes can be transferred in to microtiter wells in a PVC microtiter plate. 200 I of extracts produced by the isolates P3-2, P4-4 or P5-2 (see Example 5) can be added to each well. Plate can then be rinsed with 70% alcohol and air dried. 8 wells of media without bacteria can be included in each plate and used as control wells. Plates can be incubated and covered at 32°C for 40 hours.
• the medium can be removed with a micropipette and cell turbidity can be measured, using a UV spectrophotometer, at an optical density at 595 nm (OD 595 ) after 40 hours.
• the average OD of the control wells can be subtracted from the OD of all test wells.
• the microtiter wells can then be washed five times with sterile distilled water to remove loosely associated bacteria. Plates can be air dried for 45 min and each well can be stained with 150 I of 1 % crystal violet solution in water for 45 min. After staining, plates can be washed with sterile distilled water five times. At this point, biofilms will be visible as purple rings formed on the side of each well.
• the quantitative analysis of biofilm production can be performed by adding 200 I of 95% ethanol to detain the wells. 100 I from each well can be transferred to a new microtiter plate and the level (OD) of the crystal violet present in the destained solution can be measured at 595 nm to determine the amount of biofilm formed.
• Example 13 Detachment of Biofilm from Hydrophobic and Hvdrophilic Chips.
• Strains of Staphylococcus aureus and Psuedomonas auriginosa can be grown overnight in 5 ml test tubes at 32°C in respective media until OD 6 oo of between 0.6 and 0.7 can be observed. Seven milliliters of the bacterial suspension can be poured into a Petri dish (55-mm diameter) containing a chip (3 x 1 cm) of stainless steel (hydrophilic) and polytetrafluoroethylene (PTFE, hydrophobic) and incubated at 32°C for 2 days. The medium can be replaced after 2 h and 24 h (Chavant et al., 2002).
• each chip/slide can be placed in a Petri dish (90-mnn diameter) and washed twice for 1 min each with 35 ml of sterile tryptone salt (TS) (Bacto-tryptone, 0.1 %; NaCI, 0.85%) to remove nonadherent cells.
• TS sterile tryptone salt
• Sessile cells can be fixed on the support with a solution of 3% glyteraldehyde in 0.2M cacodylate buffer (pH 7.4) and rinsed in the same buffer.
• Samples can be stained for 3 min with a solution of 0.05% acridine orange and then washed twice for 1 min with demineralized water. The chips can then be dried in the air for 1 h and observed with a UV microscope to determine the percentage of surface contaminated (Chavant et al., 2002). The area covered by the biofilm can be converted into a percentage of the total area.
• Example 14 Activity of Extracts in a Flow Cell with Marine and Medical Biofilm Formers.
• Biofilms can be grown in glass capillary tubes under continuous flow conditions (Werner et al. 2004 "Stratified growth in Pseudomonas aeruginosa biofilms.” Appl Environ Microbiol. 70: 6188-6196, see especially FIG. 1 ).
• the glass tubes can be square cross sections, allowing direct microscopic observation of the biofilms growing on the inside of the tubes through the flat tube walls.
• the capillaries can be mounted in a flow cell holder to reduce breakage.
• the capillaries can have a nominal inside dimension of 900 m and a wall thickness of 170 ⁇ 10 m (Friedrich & Dimmock, Millville, N.J.).
• the flow cell apparatus can consist of a vented medium feed carboy (4 L capacity), a flow break, a filtered air entry, a peristaltic pump, the capillary and flow cell holder, an inoculation port, and a waste carboy. These components can be connected by silicone rubber tubing.
• the system can also contain a T connector just upstream of the glass capillary to allow mixing of the air as medium flows to enhance the development of biofilm cell clusters of P. aeruginosa. Medium and system components can be sterilized separately by autoclaving and then connected after cooling in a biological hood.
• the capillary flow system can be inoculated with 2 ml of an overnight culture of P. aeruginosa with an optical density at 600 nm of 0.001 to 0.005.
• the flow can be stopped and the tubing can be clamped downstream of the inoculation port.
• the inoculum can be injected via the port to fill the glass capillary.
• the tubing upstream of the glass tube can be clamped, and the system can be allowed to stand without flow for 24 h.
• the flow of medium (1/10- strength TSB) can be initiated at a flow rate of 20 ml/hr.
• Air can be pumped through the capillary at the same flow rate as the medium by use of a parallel tube in the same peristaltic pump. This can result in slug flow of the medium and air bubbles through the capillary tube.
• Microbial extracts produced by the isolates P3-2, P4-4 and P5-2 can be added to the system and allowed to flow for 24 hours. Another similar system without the extracts can be used as a control. Biofilm formed can be counterstained by injecting a solution of rhodamine B at 50 mg/liter into the capillary.
• Biofilm can be observed by scanning confocal laser microscopy after 24 h of continuous flow at 37°C.
• Confocal scanning laser microscopy can be performed with a Leica TCS NT confocal scanning laser microscope, with excitation at 488 and 568 nm and with emission collected at 500 to 530 nm (green channel) and 585 to 615 nm (red channel).
• Microscope images can be analyzed by use of the line-scan function of MetaMorph image analysis software (Universal Imaging Co., Downingtown, Pa.). The results will indicate the detachment of bacteria from the surface of the capillary glass tube pumped with the medium containing the extracts. No detachment of bacteria will be observed from the controls.
• DNA extraction was performed using modified US Department of Agriculture (USDA) Lazo protocol (Anon, 2006). Cells were grown in 250 ml or 50 ml liquid media or scraped off agar plate. Approximately 1 .0 ml of cells was collected in a 1 .5 ml microcentrifuge tube. Liquid cultures of cell were pelleted to remove remainder of liquid. Pellet was resuspended in 0.5 ml 1 .25 TAE. Suspension was frozen. Once frozen, 0.05 ml of 250 mM Tris and 0.05 ml of 10 mg/ml lysozyme was added and allowed to thaw and placed on ice for 45 minutes.
• USDA US Department of Agriculture
• microcentrifuge tube was heated to 50°C for 60 minutes. Following this, to precipitate the protein, 0.1 vol 3M sodium acetate was added and microcentrifuge tube, mixed gently and to remove unwanted salts from the DNA and precipitate the DNA, 2 vol 95% cold ethanol was added and mixed by inverting. Microcentrifuge tubes were centrifuged at 8000 rpm for 1 minute. The top portion was discarded and 0.5 ml 50 mM tris and 1 mM EDTA, and 40 I of 10 mg/ml RNase A was added and dissolved by rocking overnight at 4°C.
• the purity of DNA was determined for the isolated DNA (Table 8). The ratio of DNA (A 2 6o) to protein (A 2 8o) calculated for each isolate and reference organism to determine purity of DNA. Values from 1 .8 to 2.0 are considered pure in 10 mM Tris- HCI. The purity of the isolated DNA range from 0.97 to 2.13, however, the majority of DNA fell within the range considered to be pure.
• DNA-DNA hybridization was performed as described in De Ley et al. (1970) with the following modifications. The absorbance of the DNA from the marine isolates and closest related species was checked to ensure that they gave approximately the same reading. DNA from the marine isolates (500 I) and closest related species (500 I) were combined in quarts cuvettes. The re-cooling start temperature was 90°C and the rate of temperature decrease was 0.1 °C/minute until the temperature of 15°C. The rate of cooling was chosen because preliminary data revealed that cooling at 0.5°C/minute did not give strands of DNA enough time to re- anneal. Data interval was set to collect at the same rate as increase/decrease. Cary Win UV Themial software was used and converted to Excel. The initial absorbance at 15°C was compared to the final absorbance at 15°C and the ratio was used to determine percent hybridization.
• Percent of DNA-DNA hybridization between the closest related species was determined by the ratio of absorbance at the final temperature to absorbance at start temperature (data not shown) related to parts per hundred. Percent hybridization was adjusted using the rate of re-association (between approximately 24°C to 15°C) for reference organisms with closest related species (data not shown). The adjusted percent hybridized ranged from 100% (P3-1 and A. viridans) to 57.14% (P4-4 and M. hydrocarbonoclasticus) (Table 9).
• P4-4 and M. hydrocarbonoclasticus showed 57.14% hybridization indicating that they are not the same species and distantly related. This agrees with the 16S rRNA gene sequencing published previously (Bruno, 2003) that indicated that P4-4 and M. hydrocarbonoclasticus are not the same genus.
• P4-4 and P. immobilis showed 80% hybridization, indicating that they are the same species. This does not agree with the 16S rRNA gene sequencing data that indicated that P4-4 and P. immobilis are not the same genus. To confirm these results, P4-4 will be sent for 16S rRNA gene sequencing using the entire gene instead of 500 bp and compared to the database.
• P3-2 active agent Crude supernatants from bacterial isolates P3-2, P4-4, P5-2, and P6-6 and purified (95%) P3-2 active agent were screened for antibiofilm activity against human pathogens Staphylococcus aureus (ATCC 25923 and ATCC 12600), S. epidermidis (ATCC 12228), and/or P. aeruginosa (ATCC 27853) using a microplate assay [Merritt et al., 2005]. Biofilm assays were performed in triplicate. Purified (95%) P3-2 active agent reduced S. aureus 48-hour biofilm formation an average of 95.86% (p ⁇ 0.00001 ). In addition, S.
• P3-2 crude supernatant was screened for antibiofilm activity against a mixed culture of oral bacteria Streptococcus mutans and S. gordonii using 24-hour microplate assay in duplicate [Merritt et al., 2005].
• P3-2 crude supernatant reduced S. mutans and S. gordonii biofilm an average of 51 .43% (p ⁇ 0.0001 ).
• This example demonstrates that the active agents from marine isolates P3-2, P4-4, P5-2 and P6-6 have the potential to significantly inhibit oral biofilm formation and infection.
• Ames test [Ames et al., 1973] with three auxotrophic Salmonella enterica strains (previously S. typhimurium) (ATCC 29629, ATCC 29630, ATCC 29631 ). The results were considered positive if the number of revertants were at least twice as high as the negative control. P3-2 crude supernatant reverted the S. enterica mutant strains the same as or less than the negative control (Table 10), therefore, the Ames test results were negative. P3-2 crude supernatant was determined to be free from genotoxins according to the Ames test.
• Brine shrimp (Artemia salina) toxicity assay was performed in duplicate on P3- 2 crude supernatant. Dilutions of P3-2 crude supernatant and incubated in duplicate with brine shrimp larvae in a total volume of 10 ml. Approximately ten brine shrimp larvae were placed in each solution and a mixture of artificial seawater was used as a negative control. The nauplii were examined after 24 and 48 hours of growth and the average number of survived larvae was recorded. The mean percentage mortality was plotted against the logarithm of concentrations. The concentration killing 50% of the larvae (LC50) was determined from the graph. P3-2 crude supernatant is non-toxic to brine shrimp with LC50 values >10,000 g/ml.
• Example 20 Structural Characterization
• the active agent in P3-2 crude supernatant was isolated using activity guided fractionation.
• P3-2 supernatant was fractionated and purified (95%) using High Performance Liquid Chromatography (HPLC) using C-18 Reverse Phase (RP)- CombiFlash and Phenomenex Gemini columns (data not shown).
• HPLC High Performance Liquid Chromatography
• RP Reverse Phase
• Phenomenex Gemini columns data not shown.
• Structural elucidation was performed using a battery of Nuclear Magnetic Resonance (NMR) experiments [Proton NMR ( 1 H-NMR), Carbon NMR ( 13 C-NMR)] and 2D experiments [2D-Correlation Spectroscopy (COSY), Heteronuclear Single Quantum Coherence (HSCQ), Heteronuclear Multiple Bond Correlation (HMBC)], combined with Mass Spectrometry (MS) (data not shown).
• NMR Nuclear Magnetic Resonance
• COSY Nuclear Magnetic Resonance
• HSCQ Heteronuclear Single Quantum Coherence
• HMBC Heteronuclear Multiple Bond Correlation
• MS Mass Spectrometry
• the MS data for the purified P3-2 active agent was entered in the National Institute of Standards and Technology (NIST) standard reference database.
• NIST National Institute of Standards and Technology
• the structure is as depicted above.
• the compound should be construed broadly to include salts, prodrugs, tautomers, alternate solid forms, non- covalent complexes, analogs, derivatives and combinations thereof, of a chemical entity of the depicted structure.
• An acceptable salt is any salt of the depicted structure that is suitable for use in the methods and products disclosed herein.
• a salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counter-ions. Salts can form from or incorporate one or more deprotonated acidic groups (e.g. carboxylic acids), one or more protonated basic groups (e.g. amines), or both (e.g. zwitterions).
• tautomer refers to the migration of protons between adjacent single and double bonds. The tautomerization process is reversible. Tautomers will generally reach an equilibrium state wherein the double bond is resonantly shared between two bond lengths.
• Alternate solid forms are different solid forms than those that may result from practicing the procedures described herein.
• alternate solid forms may be polymorphs, different kinds of amorphous solid forms, glasses, and the like.
• Non-covalent complexes are complexes that may form between the compound and one or more additional chemical species that do not involve a covalent bonding interaction between the compound and the additional chemical species. They may or may not have a specific ratio between the compound and the additional chemical species. Examples might include solvates, hydrates, charge transfer complexes, and the like.
• ⁇ gamma hemolysis
• mucus extract isolated from mucus produced by the organism
• Zones of clearance for streak tests of active isolates are in mm.
• the zones for the extract test are shown as the difference between test and control. Isolates that did not show inhibition are not shown.
• Antibacterial results are represented as diameter of clearance zone in mm. Mean bacterial colony area was used to estimate inhibition of bacterial fouling and counts of eukaryotic cells were used to estimate inhibition of eukaryotic fouling.
• Antibacterial results are represented as diameter of clearance zone in mm. Mean bacterial colony area was used to estimate inhibition of bacterial fouling and counts of eukaryotic cells were used to estimate inhibition of eukaryotic fouling.

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