WO2021204928A1 - Genetically reprogrammed mycoplasma bacteria and uses thereof - Google Patents

Abstract

The present invention concerns oligonucleotide arrangements that express gene products having an anti- biofilm activity, and bacteria comprising said oligonucleotide arrangements. Also intended is the use of such arrangements and bacteria as a medicament. Further intended are pharmaceutical compositions comprising genetically modified (e.g. attenuated) Mycoplasma bacteria.

Description

GENETICALLY REPROGRAMMED MYCOPLASMA BACTERIA AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to the field of molecular biology, more specifically the field of genome engineering and synthetic biology. Aspects of the invention relate to genetically reprogrammed Mycoplasma bacteria. Further aspects of the invention relate to the use of these reprogrammed Mycoplasma bacteria for use as a medicament.

BACKGROUND OF THE INVENTION

Biofilms are complex and dynamic structures formed by different pathogens that cause chronic persistent and recurrent infections. In biofilms, the adherent cells become embedded within a slimy extracellular matrix that is composed of extracellular polymer substances. It is estimated that approximately 65-80% of human infections are associated with biofilm formation (Jamal etal, Bacterial biofilm and associated infections, Journal of the Chinese Medical Association, 2018). Pathogenic biofilms are especially frequent in pulmonary infectious diseases, like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), bronchiectasis and ventilator-associated pneumonia (VAP) (Boisvert et al. , Microbial biofilms in pulmonary and critical rare diseases, Annals of the American Thoracic Society, 2016).

One of the major challenges in treating biofilm infection is the increased resistance of the bacteria within the biofilm to antimicrobial agents and host defense mechanisms (Hoiby etal., Antibiotic resistance of bacterial biofilms, International Journal of Antimicrobial Agents, 2010). Metabolic activity of bacterial cells is low, resulting in many slow-growing cells. Thus, cell division occurs at radically down-regulated rates. Therefore, antibiotics such as b-lactams which are only active against dividing cells are not efficient at eradicating biofilm infections (Amanatidou et al, Biofilms facilitate cheating and social exploitation of b-lactam resistance in Escherichia coli, Biofilms and Microbiomes, 2019). These diseases are very difficult to manage therapeutically, as the effective antibiotic minimum bactericidal concentration for biofilm eradication in vivo are impossible to reach without causing adverse effects and renal and/or hepatic injury. Moreover, many of the pathogenic bacterial strains are antibiotic resistant.

Bacterial viruses or bacteriophages are currently being re-evaluated as alternatives to antibiotics given the dramatic resistance increase observed in numerous bacterial infections (Sulakvelidze, Phage therapy: an attractive option for dealing with antibiotic-resistant bacterial infections, Drug discovery today, 2005). Phage therapy was used in the first half of the twentieth century, but was largely abandoned when antibiotics were discovered. Also, the development of molecular biology techniques has enabled the evolution of “traditional” phage therapy towards new phage-inspired antibacterial, which includes the use of phage-encoded proteins responsible for bacterial envelopes. Engineering bacteria provides several advantages as a therapy delivery vehicle compared with simple drugs, nanoparticles or phages: i) they contain all biological machinery needed to synthesize complex therapeutics; ii) complex regulatory circuits can be integrated into bacteria to sense and to respond specifically to diseased tissue; iii) there is a low risk of bacterial DNA integration into the host genome; iv) in most cases, bacteria proliferation can be effectively controlled by using antibiotics as contingency strategy; and v) killing circuits or auxotrophic dependence modules can be engineered to control their growth for biocontainment and biosafety.

Bacteria can be considered to be natural factories able to produce recombinant proteins. Over time, Escherichia coli became a model bacteria for biotechnological applications, in particular to produce proteins with therapeutical applications in a cheap manner. Furthermore, bacteria have been proposed as new therapeutic anti-cancer tools, for the delivery of RNA therapeutics to treat colon diseases, to prevent HIV infection in women and to avoid dental caries. More recently, the human microbiome project has revealed that in the human body, bacteria like Lactobacillus play important roles in health by modulating the immune system and by helping said immune system to fend off infectious diseases. Engineered L. lactis strains to treat ulcerative mucositis and bowel disease (http://www.ilyapharma.se/) are currently in clinical trials. Taken together, these observations support the notion that engineered bacteria suited to deliver therapeutic agents to treat diseases is gaining momentum. Nevertheless, a major bottleneck towards utilizing bacteria for (human) therapy is the difficulty to predict the behavior of engineered bacteria in host organisms to which said bacteria are introduced. In addition, several bacterial strains that prima facie appear interesting have been historically difficult to engineer due to a lack of genetic tools. Furthermore, the niche or site of action is one of the multiple factors to consider for bacterial therapeutics. Ideally, a bacterium should be used that is naturally present in the organ to be treated, to ensure the survival of the bacterium and to limit its spreading to other organs. For example, although a previous study reported on the engineering of a E. coli Nissle 1917 strain to treat P. aeruginosa infections in the gut, it cannot be used to treat respiratory infections as the respiratory tract is not its natural niche (De Smet et al, Pseudomonas predators: understanding and exploiting phage- host interactions, Nat Rev Microbiol, 2017). In view of the paucity of efficient therapy strategies to counter biofilm formation, especially in the respiratory tract, a bacterium capable of replicating in the host organism that is also able to locally produce and disperse antimicrobial agents against biofilms associated with bacterial infections would be of considerable value.

SUMMARY OF THE INVENTION

The inventors provide a new and innovative approach to treat biofilms formed by bacterial pathogens. In particular, the inventors have discovered that genetically reprogrammed bacteria such as but not limited to Mycoplasma bacteria are suited for avoiding biofilm formation and/or dissolving biofilms once formed, in a host organism. Generation of such bacteria have now become feasible due to advances in the field of bacterial genome engineering, to which the Applicant has made considerable contributions. In particular, M. pneumoniae strains have been developed that are effective against Pseudomonas aeruginosa and/or Staphylococcus aureus biofilms. The invention further concerns pharmaceutical compositions comprising a reprogrammed (i.e. genetically modified) Mycoplasma bacterium as described herein. Hence, in response to the clear need that is formulated in the state of the art, the invention provides a new strategy to treat pulmonary infections associated with biofilm formation.

The invention therefore provides the following numbered aspects:

Aspect 1. A genetically modified Mycoplasma bacterium comprising in its genome an oligonucleotide arrangement, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium.

Aspect 2. The genetically modified Mycoplasma bacterium according to aspect 1, further comprising in its genome an inactivating mutation, deletion, and/or substitution in at least one gene selected from the group consisting of: MPN051, MPN133, MPN142, MPN257, MPN294, MPN372, MPN400, MPN415, MPN453, MPN483, MPN491, MPN592, and MPN626.

Aspect 3. The genetically modified Mycoplasma bacterium according to aspect 2, comprising in its genome at least an inactivating mutation, deletion, and/or substitution in at least the MPN372 gene encoding CARDs toxin and the MPN133 gene encoding nuclease.

Aspect 4. The genetically modified Mycoplasma bacterium according to aspect 2 or 3, further comprising in its genome at least an inactivating mutation, deletion, and/or substitution in at least the MPN051 encoding glycerol-3 -phospate dehydrogenase.

Aspect 5. The genetically modified Mycoplasma bacterium according to any of aspects 2 to 4, which is attenuated.

Aspect 6. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 5, wherein said oligonucleotide arrangement comprises at least one further nucleotide sequence encoding one or more heterologous proteins, preferably one or more DNA degrading enzymes and/or heterologous proteinases under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacteria. Aspect 7. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 6, wherein the one or more nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes and the one or more heterologous nucleotide encoded antimicrobial proteins are each under the control of the same or different promoters or a functional variant of said promoter(s) or fragment thereof.

Aspect 8. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 7, wherein the one or more nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes, the one or more nucleotide encoded heterologous antimicrobial proteins and/or the one or more nucleotide encoded heterologous DNA degrading enzymes are each under the control of the same or a different promoter or a functional variant of said promoter(s) or fragment thereof.

Aspect 9. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 8, wherein at least one of the nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes and at least one of the nucleotide encoded heterologous antimicrobial proteins are operably linked under the control of a single promoter or a functional variant of said promoter(s) or fragment thereof.

Aspect 10. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 9, wherein at least one oligonucleotide sequence comprises a constitutive promoter, preferably a promoter with a sequence selected from the group of sequences comprising of: P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11).

Aspect 11. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 10, wherein at least one nucleotide sequence comprises a synthetic promoter with a nucleotide sequence of at least 65% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 8.

Aspect 12. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 11, wherein the nucleotide sequence encoding the exopolysaccharide hydrolyzing enzyme and/or the nucleotide sequence encoding the antimicrobial protein further comprises an exposure signal sequence or a secretion signal sequence.

Aspect 13. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 12, wherein the nucleotide sequence encoding the exopolysaccharide hydrolyzing enzyme, and/or the nucleotide sequence encoding the antimicrobial protein, and/or the nucleotide sequence encoding the DNA degrading enzyme further comprises a nucleotide sequence encoding an exposure signal sequence or a secretion signal sequence, preferably the optimized secretion signal from MPN142 (SEQ ID NO 2). Aspect 14. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 13, wherein at least one of the oligonucleotide sequences further comprises a regulatory sequence capable of modulating transcription, preferably wherein the regulatory sequence is a riboswitch.

Aspect 15. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 14, wherein the exopolysaccharide hydrolyzing enzyme is selected from the group comprising: LysK (CHAP-AMID)-Lyso(PEP), endolysin LysH5, HydH5 (HydH5SH3b and HydH5Lyso), Cpl-1 lysozyme, Cpl-7 lysozyme, Pal amidase, PL3 Amidase, MV-L lysin, PlySs2, Major autolysin (Atl) of Staphylococcus aureus, CF-301, N-Rephasin, P128, Art-175, gp49, LysK (CHAP1-AMID-SH3), LysAB-SH3, SAP-1 SAL-1, P128, LysGH15/GH15, CF-301, ClyF, PaVDpl, Cpi-l/CP-1, LytA, Cpi- 7/Cp-7, Cpi-7S, Cpl-711, PL3, PlyPy, PlyC/Ct, Lys8/Bxz2, LysA/BTCU-1, LysBIBTCU-1, Lysl521/8. amyloliquefaciens phage, E1188/EL, KZ144, OBPgp279, LysPA26, LysAB2, LysABP-01, PlyABl, PlyF307, LysAB3, LysAB4, Lysep3, Lysep3, Colicin-lysep3, EndoT51T5, PlyE146, Kl lgp3.5, KP32gpl5, KP27 lysin, CfPl lysin, P28, AP3gpl5, lysB4, LysBPS13, Plyl2, Ply21, PlyBa, PlyG, PlyB, Phage APSOc lysine, PlyBT33, PlyPH, Plyl, AmiBA2446, Alginase Al-II, Alginase Al-IG, Alginase Al-III, Alginase ProtA, a-amylase, Dispersin B, a-mannosidase, b-mannosidase, cellulase, hyaluronidase, PelAh, and PslGh..

Aspect 15. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 14, wherein the antimicrobial protein is selected from the group comprising defensins, pyrrhocoricin, GramicidinA, IL37, Magainin, SMA2P9, CAP18, bacteriocinE50-5, Peptide LL-37, 1018, 1037, 17BIPHE2, Bac8c, Battacin, BMAP-27, BMAP-28, CAMA, DJK-5, DJK-6, GF-17, LL-31, LL7-31, LL7-37, Melittin, P10, P60.4Ac, SMAP-29, Lysostaphin, pyocin Sn, pyocin SI, pyocin S2, pyocin S3, pyocin AP41, pyocin S5, pyocin S2, pyocin S3C, pyocin S6, pyocin S8, pyocin SD1, pyocin S13, pyocin SD2, pyocin SD3, pyocin SA189, pyocin LI, pyocin L2, pyocin L3, pyocin Ml, pyocin M4, pyocin PAEM4, pyocin PAEM, pyocin LI, putidacin LI, pyocin Rl, pyocin H108 (8-type), pyocin 1577, colicin R, colicin N, colicin M, colicin D, colicin El, colicin E3, and colicin E9, haemocin A, CLB pesticin, tailocin chimeras, S 1 chimeras, S2 chimeras, E2 chimeras, E3 chimeras, pyocin R, pyocin F, Enterocoliticin, AvR2-V10.3, and lactoferrin.

Aspect 16. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 15, wherein the exopolysaccharide hydrolyzing enzyme is Dispersin B, or PelAh, PslGh and Alginate lyase A1-IG.

Aspect 17. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 15, wherein the exopolysaccharide hydrolyzing enzymes are PelAh, PslGh and Alginate lyase A1-IG and wherein the antimicrobial protein is pyocin LI. Aspect 18. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 17, wherein the exopolysaccharide hydrolyzing enzyme is Dispersin B and wherein the antimicrobial protein is Lysostaphin.

Aspect 19. A method for altering het genome of a Mycoplasma bacterium comprising introducing an oligonucleotide arrangement into the genome of a Mycoplasma bacterium, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium.

Aspect 20. The method according to aspect 19, wherein the Mycoplasma bacterium is a. Mycoplasma pneumonia bacterium.

Aspect 21. Use of an oligonucleotide arrangement for altering the genomic sequence of a Mycoplasma bacterium, wherein the oligonucleotide arrangement comprises: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant or fragment thereof which is active in said Mycoplasma bacterium.

Aspect 22. The use according to aspect 21, wherein the first and/or second nucleotide sequences encode a gene product able to reduce biofilm formation.

Aspect 23. The use according to aspect 22, wherein the biofilm is a microbial biofilm.

Aspect 24. The use according to aspect 22 or 23, wherein said biofilm is formed in the respiratory system of said subject.

Aspect 25. The use according to aspects 22 to 24, wherein said biofilm comprises hexosamine- containing polymers (PI A).

Aspect 26. The use according to aspects 22 to 25, wherein said biofdm comprises Pel and/or Psl and/or alginate exopolysaccharides. Aspect 27. The use according to aspects 22 to 26, wherein said biofilm is produced by a group of bacteria comprising Pseudomonas aeruginosa, preferably wherein said biofdm is produced by Pseudomonas aeruginosa.

Aspect 28. The use according to aspects 22 to 27, wherein said biofilm is produced by a group of bacteria comprising Staphylococcus aureus, preferably wherein said biofdm is produced by Staphylococcus aureus.

Aspect 298. The use according to aspects 22 to 28, wherein the Mycoplasma bacterium is an attenuated Mycoplasma bacterium.

Aspect 30. A pharmaceutical composition comprising the genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20.

Aspect 31. The pharmaceutical composition of any of aspect 30, wherein said pharmaceutical composition further comprises an antibiotic, preferably wherein said antibiotic is selected from the group comprising: Piperacillin, Tazobactam, Ciprofloxacin, Levofloxacin, Meropenem, Imipenem/, Cilastatin, Amikacin, Ceftazidime, Avibactam, Ceftolozane, Tazobactam, Ceftriaxone, Vancomycin, and Linezolid or any combination thereof, preferably wherein the antibiotic is selected from the group comprising Piperacillin, Meropenem, Imipenem, Cilastatin, Vactomycin, or any combination thereof, more preferably wherein the antibiotic is Meropenem.

Aspect 32. A genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 30 or 31, for use as a medicament.

Aspect 33. A genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 30 or 31, for use in treating pneumonia.

Aspect 34. A genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 29 or

30, for use in treating ventilator-associated pneumonia (VAP).

Aspect 35. A genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 30 or

31, for use in dissolving microbial biofilms produced by a group of bacteria comprising Pseudomonas aeruginosa and/or Staphylococcus aureus. Aspect 36. Use of a genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 30 or 31, for avoiding biofilm formation.

Aspect 37. Use of an genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspects 19 or 20, or a pharmaceutical composition according to aspect 30 or 31, for dissolving biofilms.

Aspect 38. Use of a genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or obtained by the method of aspect 19 or 20, or a pharmaceutical compositions according to aspect 30 or 31, for the manufacture of a medicament for the prevention or treatment of (a) pathogenic biofilm (formation).

Aspect 39. The use according to aspect 38, wherein the genetically modified Mycoplasma bacterium is a live genetically modified Mycoplasma bacteria, more preferably a live genetically modified Mycoplasma pneumoniae bacteria.

Aspect 40. A method of treating a subject diagnosed with, or suspected to have a pathogenic biofilm formation, wherein the method comprises a step of contacting the subject with a genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, or a pharmaceutical composition according to aspect 30 or 31.

Aspect 41. A synthetic promoter with a nucleotide sequence of at least 65% sequence identity to the nucleotide sequence of SEQ ID NO: 8.

Aspect 42 The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, wherein said Mycoplasma bacterium comprises a functional modification such as a deletion, insertion, and/or substitution in MPN372 and/or MPN133, and further comprising in its genome one or more oligonucleotide arrangements encoding for PelAh, PslGh, Alginate lyase Al-IT, and pyocin, preferably pyocin LI.

Aspect 43. The genetically modified Mycoplasma bacterium according to aspect 42, further comprising a functional modification such as a deletion, insertion, and/or substitution in MPN051.

Aspect 44. A genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18, comprising a functional modification such as a deletion, insertion, or substitution in MPN372 and/or MPN133, and further comprising in its genome one or more oligonucleotide arrangements encoding for Dispersin B and lysostaphin.

Aspect 45. The genetically modified Mycoplasma bacterium according to aspect 44, further comprising a functional modification such as a deletion, insertion, and/or substitution in MPN051. Aspect 46. The genetically modified Mycoplasma bacterium according to any one of aspects 1 to 18 or 42 to 45, further comprising a functional modification such as a deletion, substitution, and/or insertion in one or more genes or operons encoding a protein capable of eliciting Guillain-Barre in a host organism, preferably in MPN257 and/or MPN483.

The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Biofilm degradation activity. A) Capacity of different enzymes to degrade biofilms formed P. aeruginosa PAOl wt (solid bars) and PAOlAmucA (striped bars). The concentration of enzyme was identical in each sample (7.09 pmol/mΐ), corresponding to 0.32 mg/ml for Alg2A; 0.14 mg/ml for Al-II; 0.22 mg/ml for Al-IT; and 0.23 mg/ml for Al-III. The values are normalized against the control sample (non-treated biofilm) that has 100% biomass. The decrease in the percentage of biomass reflects the effect of the treatment. The asterisk (*) indicates a significant difference (p <0.05) when the sample is compared with the control. The (&) indicates a significant difference (p <0.05) when the PAOl wt strain is compared with PAOlAmucA. B) Synergism between antibiotics and the AlyAl, Al-III and Alg2A alginate lyase proteins. The asterisk (*) indicates a significant difference (p <0.05) when the sample is compared with the control. The ampersand (&) indicates a significant difference (p <0.05) when alginase is compared with alginase + ciprofloxacin. The infinity symbol (¥) indicates a significant difference (p <0.05) when a sample is compared with ciprofloxacin.

Figure 2. Quantification of alginate lyase activity from supernatant of engineered Mycoplasma strain. Degradation of brown seaweed alginate as the substrate by AI-IT protein (0.0001 mg/ml alginase in Hayflick medium (HF)) added in the HF medium (striped line). WT-1 IF SN is the supernatant from Mycoplasma cells producing AI_IT grown in HF at 37°C for 72 hours (200 pi of 25 ml culture; dotted line). Myco_Al_IF Cells sample (stripe and dot line) represents ~6xl0⁷ cells of Mycoplasma pneumoniae expressing the Al-IF alginase added to the alginate. Measurements were done at three different time points (0.5 and 24h) by triplicates.

Figure 3. Biofilm degradation activity of media samples obtained from Mycoplasma strains. Media obtained after 3 days of Mycoplasma growth in Hayflick medium (HF no amp, negative control) was used to test activity in degradation of biofilms formed by the P. aeruginosa PAOl strain. WT indicates wild type strain. WT AI-IF is the strain obtained after transformation of WT with pTnMCSlox66Cm71_EfTu_MPN142_AI-IF vector. WT_AI-IT expresses and secretes the alginate lyase protein AI-IF. Figure 4. DNA degradation activity in the CV2 strain. A) Study of degradation of plasmid DNA after 15 min and 60 minutes of incubation times. Lane labelled with indicates negative control (sample of DNA treated with Hayflick medium). Lane CV2 corresponds with the sample of DNA incubated with the medium obtained after growing the CV2 strain for 90h. B) Study of degradation of PCR amplified DNA after addition of EDTA and EGTA in the sample of the medium obtained from the CV2 strain (right panel, CV2 SN) or sample from Hayflick medium (left panel, INPUT in HF).

Figure 5. DNA degradation assay in WT and CV4. The CV4 lacks the nucleases encoded by MPN133 and MPN491 genes and is therefore not able to degrade DNA amplified by PCR.

Figure 6. Biofilm degradation by DNAsel. Left: Haflick medium negative control, middle WT Mycoplasma pneumoniae strain, right: recombinant hDNAsel (50 pg/ml).

Figure 7. Western Blot showing expression of heterologous proteins form at total cell extract of different engineered strains. Primary antibody used: anti-Flag.

Figure 8. Biofilm degradation activity of media samples obtained from different Mycoplasma strains. Media obtained after 3 days of Mycoplasma growth was used to test activity in degradation of biofilms formed by PAOl strain. WT indicates wild type strain and CV2 is the attenuated chassis strain. Panel A: antibiofilm activity of different genetically modified Mycoplasma strains on the P. aeruginosa PAOl strain. PelAh is the WT strain transformed with the minitransposon vector pTnlox66Tetra71- EfTu-MPN142(OPT)-PelAh that secretes PelAh protein. PslGh is the strain obtained after transformation of WT with pTnMCSlox66Cm71-EfTu-MPN142(OPT)-PslGh vector. This strain secretes PslGh protein. PelAh+PslGh is the strain derived from transformation of WT with pTnMCSlox66Cm71-EfTu-MPN 142(OPT)-PelAh-EfTu-MPN 142(OPT)-PslGh vector. WT H strain expresses and secretes both the PelAh and the PslGh protein. PelAh+PslGh+AI-IT is the corresponding transformed WT strain that secretes the PelAh protein, the PslGh protein, and the alginate lyase protein AI-IT. Panel B: antibiofilm activity of the CV2 M pneumoniae chassis versus antibiofilm activity of CV2 wherein PelAh, PslGh, and alginate lyase AI-IT are expressed and secreted (i.e. CV2-HA). SAT290, PAOl, Boston 41501, and NCTC13437 indicate distinct P. aeruginosa strains. . Panel C: Lung infection dynamics of M. pneumoniae WT and CV2 chassis strains. In mice Higher bacterial counts were obtained at 2 dpi than at 4 dpi or 14 dpi for both strains, showing a similar CFU decay over time.

Figure 9. Growth curve of P. aeruginosa PAOl strain. Absorbance of the culture at 600 nm was measured every 3 minutes by using TECAN for 24 hours. Triplicates were performed for every condition that represents the mixture of P. aeruginosa PAO 1 inoculum with medium of different strains obtained after 72h growth. The top line depicts the PAOl with WT medium and PAOl with CV2 medium (both conditions displayed a near identical growth curve) and the lower line depicts PAOl with CV2 H+A medium. This evidences that the proteins expressed by the CV2 chassis could, aside from dissolving biofdms, have a killing activity on P. aeruginosa.

Figure 10. Growth curves of CV2 H+A and different strains of P. aeruginosa in presence of Piperacillin-Tazobactam antibiotic. In black is represented the growth of the strains (labelled in each graph) not treated with antibiotic. From dark to clear grey, growth curves of cells treated with the doses of 1000 pg/ml; 500 pg/ml and 100 pg/ml.

Figure 11. Biofilm assays with P. aeruginosa SAT290 (top) and PAOI GFP (bottom) strains. Outer left condition is the positive control (medium of CV2 H+A strain). From left to right for each group of samples: increasing concentrations (in pg/ml) of different antibiotics.

Figure 12. SAT290 biofilm degradation by combining different antibiotics with medium or cells of the CV2 H+A strain. From left to right for each group of samples: increasing concentrations (pg/ml) of different antibiotics. Left sample: Hayflick medium control condition, second sample: CV2 H+A cells positive control, third sample: CV2 H+A medium positive control.

Figure 13. Validation of expression by Western blot. Lane 1: Negative control: WT; lane 2: Positive control: CV2 PyoPAEM4Flag (45 kDa); lane 3: WT H+A (35+50 kDa); lane 4: CV2 H+A (35+50 kDa); Lane 5: WT H+A+PyoLlFlag (30+35+50 kDa); Lane 6: CV2 H+A+PyoLlFlag (30+35+50 kDa). RL7 was used as loading control.

Figure 14. Capacity of different strains to dissolve P. aeruginosa biofilms (crystal violet). Top full line and top dotted line:PA01 strain, bottom full line and bottom dotted line: NCTC 13437 strain. In both strains, CV2 Mycoplasma is indicated by the full line, and CV2 H+A+PyoLl is indicated by the dotted line. It can be observed that the further genetically engineered Mycoplasma CV strain displayed increased antibiofilm activity.

Figure 15. PAOl tissue burden in mice lung tissue 8 hours post infection. Animals were infected with PAOl alone or in combination with WT H+A+PyoLl (=WT VAP2) or CV8 H+A+PyoLl (=CV8 VAP2). Values between brackets indicate CFUs infected. After 8 hours, PAOl CFUs were quantified.

Figure 16. Antibiofilm and antimicrobial activities of M. pneumoniae strain CV8_H+A+PyoS5.

(A) Crystal violet assay showing that the supernatant of CV8_H+A+PyoS5 degrades the biofilm of the indicated strains of Pseudomonas aeruginosa. (B) Quantification of the crystal violet assay of panel A. Left samples: CV8; right samples: CV8_H+A+PyoS5. (C) Growth curve showing that the supernatant of CV8_H+A+PyoS5 inhibits the growing of . aeruginosa strain SAT290.

Figure 17. In vivo preventive treatment against PAOl infection. A: Schematic representation of the experimental design. Two amounts (1x105 or 1x107 CFUs) of different M pneumoniae strains were inoculated in the animals with or without P. aeruginosa PAO 1 cells. After 8 h, animals were sacrificed, and the lungs were homogenized and seeded on Hayflick agar plates to quantify the CFUs of P. aeruginosa PAOl (in B) and the CFUs for M. pneumoniae (in C). p values of Kruskal-Wallis H test compared to the control at 8h are indicated (* = <0.05; *** = <0.001). hpi=hours post injection. D: The mean values of the clinical scores of different animal groups, treated with PBS or with CV2_HA_P1 strain at different time points, are shown. Outer left sample corresponds to the pre-treatment sample (e.g., the group infected with P. aeruginosa, sacrificed at 2 hpi and not treated). E: Progression of body weight of animals at different experimental timepoints. F: Number of CFUs of different strains of M. pneumoniae recovered from the lung at 8 hpi and 24 hpi.

Figure 18. Degradation of a 12 h S. aureus biofilm by WT EfTuD. Crystal violet assay for measuring S. aureus biofilm degradation by quantifying the biofilm measuring absorbance at OD595. The number of cells is CFUs (colony forming units). The legend (from top to bottom) corresponds to the order of the samples (left to right for every time point).

Figure 19. Degradation of a 24 h S. aureus biofilm by WT EfTuD. Crystal violet assay for measuring S. aureus biofilm degradation by quantifying the biofilm measuring absorbance at OD595. The number of cells is CFUs (colony forming units). The legend (from top to bottom) corresponds to the order of the samples (left to right for every time point).

Figure 20. Degradation of a 12h S. aureus biofilm by WT EfTuD and CV2_EfTuD strains.

Figure 21. Study of degradation of a S. aureus biofilm formed in vitro in a catheter. (A) Schematic representation of in vitro and ex vivo assays performed with different Mycoplasma strains. S. aureus biofilms generated in vitro or in vivo on the surface of sealed catheters (n=5) were treated (37°C, 4h) in vitro with 10⁸ CFU of either WT-DispB or with CV2-DispB. (B) Representative pictures of the catheters from the ex vivo experiment after crystal violet staining. Catheter treated with WT-DispB shows reduction in the bacterial biofilm (stained in violet) (C) Plots showing the efficacy of different treatments to dissolve S. aureus biofilms in vitro expressed as the OD595 (crystal violet staining) found in the different treated catheters. (D) Plots showing the efficacy of different treatments to dissolve S. aureus biofilms ex vivo expressed as the OD595 (crystal violet staining) found in the different treated catheters. Catheters treated with WT or CV2 were used as controls. Data are represented individually (symbols) and as the mean ±SD (n>4). Statistical comparison of means (p values) by one-way ANOVA test: * p<0.05; ** p<0.005; *** p<0.0005.

Figure 22. In vivo assay to study S. aureus biofilm degradation by WT EfTuD and CV2_EfTuD strains.

Figure 23. Growth curve of S. aureus in presence of increasing amounts of LysAB2_SH3b protein.

Effect of adding increasing amounts of LysAB2_SH3b in the inoculum of S. aureus (from clear grey to dark grey). Inhibition of growth was observed when increasing the amount of protein corroboration that this protein could have an effect as antimicrobial agent.

Figure 24. Characterization of strength of different synthetic promoters. Decrease in absorbance indicates the reduction of .S' aureus cells as consequence of the Lysostaphin activity present in the medium of different Mycoplasma strains. Samples are supernatant of cells grown for 3 days inoculated in a 6h culture of S. aureus. The data is normalized by the total number of CFUs obtained or derived for each culture.

Figure 25. In vivo assay to study S. aureus biofilm degradation by WT DispB and CV2_DispB strains. M. pneumoniae platforms efficacy against & aureus catheter-associated biofilm in mice by [18F]-FDG-MicroPET. S. aureus pre-colonized sealed catheters were implanted subcutaneously in CD1 mice between the shoulder blades, and 24h later mice were treated by a single subcutaneous injection of 108 CFU of the correspondent M. pneumoniae strain (abscissas axis). [18FJ-FDG- MicroPET uptake images were taken at days 1 (Dl) and 4 (D4) post-treatment. (A) Schematic representation of in vivo assays performed with different Mycoplasma strains. (B) Representative longitudinal slices of [18F]-FDG-MicroPET uptake in mice carrying implanted catheters (red arrows). Micro-PET images have been co-registered with CT-3D images used as anatomical reference. Brain (b) and spinal cord (sp) show physiological uptake of [18F]-FGD. (C) Increase or decrease from Dl to D4 of the [18F]-FDG-MicroPET uptake SUV60 (%) values on the implanted catheters. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Data are represented individually (symbols) and as the mean ±SD (n>4). Statistical comparison of means by ANOVA and post-hoc PLSD tests: * p<0.05; ** p<0.005; *** p<0.0005.

Figure 26. Biofilm and antimicrobial activities of different strains expressing different combinations of Dispersin B and Lysostaphin. Samples from left to right for each group: Myco WT, Myco EfTu-DispB, Myco EfTu-Lyso, Myco EfTu-DispB-Lyso, Myco EfTu-DispB-EfTu-Lyso.

Figure 27. S. aureus biofilm degradation assay. Crystal violet assay performed with recombinant proteins and different M pneumoniae strains. TBS-glu and HF correspond to samples treated with two different medium TBS-glu and Hayflick respectively. We observed that there is not impact in biofilm growth by using HF medium when compared with the TBS-glu. In the section with recombinant proteins, L is lysostaphin (0.25 pg/pl), D is Dispersin B (0.25 pg/pl) and, L+D (mixture of lysostaphin and Dispersin B; 0.25 pg/pl) and protA (negative control, Alg2A alginate lyase protein that is not active in S. aureus biofilms also used at 0.25 pg/pl). In the samples of WT and CV2, are the Mycoplasma strains that do not express any heterologous protein M(-), expressing only Lysostaphin protein (ML), expressing Dispersin B protein (MD) and expressing both, Dispersin B and Lysostaphin (ML-D), and strains expressing negative control protein (M-ProtA). The inoculum of different Mycoplasma strains was normalized and used at 10⁸ cells per well to be able to compare the different strains. Figure 28. In vivo degradation assay of a biofilm formed in a subcutaneous catheter model in mice.

Increase or decrease from D1 to D4 of the [18F]-FDG-MicroPET uptake SUV60 (%) values on the implanted catheters. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post treatment. Data are represented individually (symbols) and as the mean ±SD (n>4). Statistical comparison of means by ANOVA and post-hoc PLSD tests: * p<0.05; ** p<0.005; *** p<0.0005.

Figure 29. Nucleotide and amino acid sequences described throughout the specification.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from... to... ” or the expression “between... and... ” or another expression.

The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/- 10% or less, preferably +/- 5% or less, more preferably +/- 1% or less, and still more preferably +/- 0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

The term “subject”, “patient”, and “subject in need” may be used interchangeably herein and refer to animals, preferably warm-blooded animals, more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals. The term “mammals”, or “mammalian subjects” refers to any animal classified as such and hence include, but are not limited to humans, domestic animals, commercial animals, farm animals, zoo animals, sport animals, pet and experimental animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferred patients are human subjects. Particularly preferred are human subjects, including both genders and all age categories thereof.

Amino acids are referred to herein with their full name, their three-letter abbreviation or their one letter abbreviation. Unless explicitly stated otherwise, reference herein to any peptide, polypeptide, protein, or nucleic acid, or fragment thereof may generally also encompass modified forms of said peptide, polypeptide, protein, or nucleic acid, or fragment thereof, such as bearing post-expression modifications including the following non-limiting examples: phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, ghitathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, combinations thereof.

A first aspect of the invention is related to a genetically modified bacterium comprising: i) a first oligonucleotide arrangement comprising a nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium, and ii) a second nucleotide comprising a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium. In certain embodiments, the genetically modified bacterium is a live genetically modified bacterium. In certain embodiments, the genetically modified bacterium is a Mycoplasma bacterium. In certain embodiments, the promoter is a naturally occurring promoter. In alternative embodiments, the promoter is an artificially optimized non-naturally occurring promoter having a sequence identity to a naturally occurring promoter of at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85% to a naturally occurring promoter. In certain embodiments, the first and second oligonucleotide arrangement are physically linked with each other. In further embodiments, the first and second oligonucleotide arrangement are directly physically linked with each other by a phosphodiester bond, separated by an optional nucleotide sequence. In certain embodiments, the exopolysaccharide hydrolyzing enzyme(s) and antimicrobial protein does not occur in the genome of the unmodified (wild type) bacterium.

The term “active” as described herein indicates the capacity of the nucleotide encoded gene product to fulfill its commonly accepted function in the bacterium. Hence, for a gene product such as an enzyme to be active, it is understood that expression of the gene product is required in the bacterium. A skilled person understands that a heterologous gene product expressed by a bacterium does not equal that activity of a gene product can be observed upon expression of said heterologous gene product due to differences in for example post translational machinery present in different organisms. Assays to determine the activity of gene products, in particular to measure the enzymatic activity when said gene product is an enzyme, have been described in detail in the art (Bums et al. , Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts, Biol Proced Online, 1998). A skilled person further understands that the “degree” or “level” of activity depends on numerous parameters, including but not limited to substrate availability, competing enzymes, and half-life of both substrate and enzyme. Hence, “active” as used herein can be interpreted as a quantitative reduction of substrate amount after incubation with an enzyme.

Alternatively, the aspect can be described as an oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant or fragment thereof which is active in bacteria, viruses, (bacterio)phages, or Archaea , and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant or fragment thereof which is active in bacteria, viruses, (bacterio)phages, or Archaea. Non-limiting examples of bacteria envisaged herein are gram-positive bacteria, Chlamydiae, Green nonsulfur bacteria, Actinobacteria, Planctomycetes, Spirochaetes, Fusobacteria, Cyanobacteria (blue-green algae), Thermophilic sulfate- reducers, Acidobacteria, and Protobacteria. It is intended that any embodiments described herein citing the oligonucleotide arrangement also apply to a genetically modified bacteria, viruses, (bacterio)phages, or Archaea comprising said oligonucleotide arrangement and vice versa. The term “(bacterio)phage” is well defined in the art is therefore clear to a skilled person. Non-limiting examples of (bacterio)phages include Podoviridae, Siphoviridae, Ackermannviridae, Myoviridae, Rudiviridae, Lipothrixviridae, Spiraviridae, Clavaviridae, Guttaviridae, Bicaudaviridae, Fuselloviridae, Corticoviridae, Leviviridae, Microviridae, Tectiviridae, Inoviridae, Cystoviridae, Tristromaviridae, Plasmaviridae, Pleolipoviridae, Globuloviridae, Portogloboviridae, Sphaerolipoviridae, Turriviridae, Ampullaviridae. In certain embodiments, the oligonucleotide arrangement is introduced in a (bacterio)phage selected from the group comprising: 186 phage, l phage, F6 phage, F29 phage, FC174, G4 phage, M13 phage, MS2 phage, N4 phage, PI phage, P2 phage, P4 phage, R17 phage, T2 phage, T4 phage, T7 phage, and T12 phage.

Methods and protocols to introduce oligonucleotide arrangements into bacteria, i.e. methods of bacterial transformation, are known to a person skilled in the art (Johnston et al, Bacterial transformation: distribution, shared mechanisms and divergent control, Nature reviews Microbiology, 2014). The term “transformation” is indicative for a genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material. Transformation is a horizontal gene transfer process and is commonly used in context of introducing foreign DNA to a bacterial, yeast, plant, animal, or human cell. Cells capable of taking up foreign DNA are named competent cells. In other embodiments, transformation may be indicative for the insertion of new genetic material into animal and human cells, albeit the term “transfection” is more common for these cells. Non-limiting examples of suitable transformation methods that can be applied to bacteria include heat- shock transformation and electroporation. In heat shock transformation, artificial competence is typically induced by making the cell permeable to DNA by subjecting them to non-physiological conditions. In such atypical transformation experiment, the cells are incubated in a solution containing divalent cations often in cold conditions, before the cells are exposed to a heat shock. It is theorized that exposure of the cells to divalent cations are responsible for a weakening of the cell surface structure, rendering it (more) permeable to DNA. The heat shock generates a thermal imbalance across the membrane, forcing entry of DNA through cell pores (i.e. adhesion zones or Bayer junctions) or through the damaged cell wall. An alternative method to induce transformation is by means of electroporation, which is hypothesized to create pores in the cellular membrane. In electroporation the bacterial cells are briefly exposed to an electric field of 10-20kV/cm. After the shock, cellular membrane repair mechanisms remove the pores.

The term “oligonucleotide arrangements” as used herein, or synonymously “nucleotide sequences”, “polynucleotide arrangements”, “polynucleotide sequences”, refers to a sequence of a multitude of nucleotides physically connected to form a nucleotide sequence. Unless the contrary is mentioned, the oligonucleotide arrangements are not presented in their naturally occurring genome. Means and methods to obtain, generate and modify isolated polynucleotide sequences are well known to a person skilled in the art (Alberts et al, Molecular Biology of the Cell. 4th edition, 2002). In certain embodiments, the oligonucleotide arrangement is one or more double stranded DNA sequences. In alternative embodiments, the oligonucleotide arrangement is one or more single stranded DNA sequences. In yet alternative embodiments, the oligonucleotide arrangement is one or more single stranded RNA sequences. In yet alternative embodiments, the oligonucleotide arrangement is one or more double stranded RNA sequences.

In certain embodiments, the oligonucleotide arrangements described herein may be multiple DNA sequences. In certain embodiments, the oligonucleotide arrangements described herein may be multiple RNA sequences. In alternative further embodiments, the oligonucleotide arrangements as described herein may comprise both DNA nucleotide sequences and RNA nucleotide sequences.

By means of guidance and not limitation, any oligonucleotide arrangement described herein can be part of an expression vector such as a plasmid optionally a non-replicative plasmid, a phagemid, a bacteriophage, a bacteriophage-derived vector, an artificial chromosome, a minicircle, a lentiviral vector, a retroviral vector, an adenoviral or adeno-associated viral vector, a piggyback vector, or a tol2 vector. A skilled person is aware of these different types of constructs and their generation and manipulation has been detailed at numerous instances (Sambrook et al, Molecular cloning: a laboratory manual, 4^th edition, Cold Spring Harbor Laboratory press, 2012). Furthermore, it is evident to a skilled person that plasmid DNA, or (circular) recombinant DNA is commonly referred to in the art as copy DNA, complement DNA, or even referred to by the abbreviation “cDNA”. In certain embodiments, an oligonucleotide arrangement as described herein is part of a bicistronic expression construct. In further embodiments, an oligonucleotide arrangement as described herein is incorporated in a cellular (e.g. bacterial) genome. In yet further embodiments, one or more oligonucleotide arrangements as described herein are part of a cellular genome. In further embodiments, a oligonucleotide arrangement as described herein is comprised in a bacterial artificial chromosome or a yeast artificial chromosome. In certain embodiments, the 5’ and/or 3’ end of one or more nucleotide sequences part of an oligonucleotide arrangement as described herein is modified to improve the stability of the sequence in order to actively avoid degradation. In certain embodiments, the oligonucleotide sequence is comprised in a bacteriophage. The term “bacteriophage” as described herein is indicative for a virus that infects and optionally is able to replicate within bacteria and archaea.

The term “promoter” as defined herein is a region of DNA that initiates transcription of a particular gene and hence enables a gene to be transcribed. A promoter is recognized by RNA polymerase, which then initiates transcription. Thus, a promoter contains a DNA sequence that is either bound directly by, or is involved in the recruitment, of RNA polymerase. A promoter sequence can also include “enhancer regions”, which are one or more regions of DNA that can be bound with proteins (namely the trans acting factors) to enhance transcription levels of genes in a gene-cluster. The enhancer, while typically at the 5 ’ end of a coding region, can also be separate from a promoter sequence, e.g., can be within an intronic region of a gene or 3’ to the coding region of the gene. Promoters may be located in close proximity of the start codon of genes, in preferred embodiments on the same strand and typically upstream (5’) of the gene. Promoters may vary in size, and are preferably from about 100 to 1000 nucleotides long. In certain embodiments, the promoter may be a constitutive promoter. A constitutive promoter is understood by a skilled person to be a promoter whose expression is constant under the standard culturing conditions, i.e. a promoter which expresses a gene product at a constant expression level. In alternative embodiments, the promoter may be an inducible (conditional) promoter. It is understood that inducible promoters are promoters which are responsive at least one induction cue. Inducible promoters, and more specifically bacterial inducible promoter systems have been described in great detail in the art ( inter alia in Brautaset et al, Positively regulated bacterial expression systems, Microbial biotechnology, 2009). In certain embodiments, the inducible promoter is chemically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a chemical inducing agent such as an alcohol, tetracycline, a steroid, a metal, or other small molecule) or physically regulated (e.g., a promoter whose transcriptional activity is regulated by the presence or absence of a physical inducer such as light or high or low temperatures). An inducible promoter can also be regulated by other transcription factors that are constitutive or are themselves directly regulated by chemical or physical cues. In certain embodiments, the promoter is a TetR promoter part of a Tet-On or Tet-off system (Krueger et al, Tetracycline derivatives: alternative effectors for Tet transregulators, Biotechniques, 2004, and, Loew etal, Improved Tet-responsive promoters with minimized background expression, BioMedCentral Biotechnology, 2010). In further embodiments, the concatenation of different sequence elements may be considered as an operon. “Operon” as used herein refers to a functional unit of DNA containing a cluster of genes in which all genes are controlled by a single promotor. It is evident to a skilled person that genes from an operon are co-transcribed. Transcribed genes from an operon are transcribed to a single mRNA strand and may be either translated together in the cytoplasm or spliced to generate monocistronic mRNAs that may be translated separately. In certain embodiments, one or more oligonucleotide arrangements as described herein may comprise a regulatory sequence.

“Control sequences” or “regulatory sequences” as used interchangeably herein refer to any nucleotide sequence which capable of increasing or decreasing the expression of specific genes. This regulation may be imposed by either influencing transcription rates, translation rates, or by modification of the stability of the sequence. In further embodiments, the polynucleotide sequence comprises regulatory elements such as but not limited to the following: enhancers, selection markers, origins of replication, linker sequences, polyA sequences, terminator sequence, and degradation sequences. In certain embodiments, at least one oligonucleotide arrangement comprises one or more suitable control sequences. In certain embodiments, the control sequences are identical for all oligonucleotide arrangements. In alternative embodiments, different control sequences are used for or within different oligonucleotide arrangements. In certain embodiments, the control sequences are control sequences naturally occurring in Mycoplasma bacteria. In other embodiments, the control sequences are adapted to perform their intended function in Mycoplasma bacteria. It is evident to the skilled person that any component of the oligonucleotide arrangement as described herein may further comprise tag sequences that ameliorate purification or localization of either the nucleotide sequence, or one or more gene products encoded in the nucleotide sequences of the oligonucleotide arrangement. Both oligonucleotide motifs and sequences that bind to other oligonucleotides or proteins and amino acid motifs or sequences are envisaged.

From the various examples provided herein, it can be observed that optionally either the naturally occurring (amino acid sequence: SEQ ID NO: 1) or optimized MPN142 secretion sequence (nucleotide sequence: SEQ ID NO: 2 and amino acid sequence: SEQ ID NO: 3) may be incorporated (i.e. attached, fused) to any heterologous gene products (including exopolysaccharide hydrolyzing enzymes, DNAses, proteases, antimicrobial proteins, DNA binding proteins, DNA cleaving proteins, etc.) disclosed herein. Unexpectedly, the (optimized) MPN142 sequence was observed to be a potent and universally applicable secretion signal for the gene products of the oligonucleotide arrangements described herein that is able to promote secretion of said product(s). The term “exopolysaccharide hydrolyzing enzyme” as used herein, is indicative for any enzyme that is capable of performing exopolysaccharide hydrolysis. As envisaged herein, the term enzyme includes biologically active analogs, (natural and synthetic) variants, fragments and chemically modified derivatives of the enzyme, which are capable of degrading exopolysaccharides. According to the present invention, the primary, secondary and/or tertiary structure of the enzyme can be modified as long as its biological activity is retained. “Exopolysaccharides” are a major component of the Extracellular polymeric substance that establishes the functional and structural integrity of biofilms. are high- molecular-weight polymers that are composed of sugar residues and are secreted by a microorganism into the surrounding environment. Exopolysaccharides generally comprise monosaccharides and some non-carbohydrate substituents (including as acetate, pyruvate, succinate, and phosphate). Functions of exopolysaccharides have been described in detail in the art (Harimawan and Ting, Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion, Colloids and Surfaces B: Biointerfaces, 2016). Non-limiting examples of suitable exopolysacchararide hydrolyzing enzymes are further discussed below. The term “biofilm” as used herein is a term that indicates any syntrophic consortium of microorganisms such as bacteria in which cells adhere to one another and/or to a surface. Biofilms are characterized by a viscous extracellular matrix composed of extracellular polymeric substances produced by the bacteria (Lopez, et al. , Biofilms, 2010). Biofilms can form in natural, medical, and industrial settings. In medical settings, biofilm is a characteristic of several difficult to treat diseases including but not limited to cystic fibrosis and chronic obstructive pulmonary disease. Furthermore, biofilm formation on medical devices such as catheters and/or implants is responsible for an increasing incidence of chronic infections that are hard to effectively treat. Biofilms hamper treatment of such infection by different mechanisms, such as an increased amount of persister cells that are present in the biofilm which are non-dividing cells with a high antibiotic resistance (Lewis, Persister cells and the riddle of biofilm survival, Biochemistry, 2005). Besides persister cells, the biofilms also achieve increased protection from antibiotics by the extracellular matrix, which acts as a physical barrier. A skilled person is aware that specific molecular mechanisms and biofilm constituents vary between biofilms formed by different microorganisms. However, the extracellular matrix described above (comprising exopolysaccharides, proteins, and/or DNA) is a general feature of biofilms (Monds and O’Toole, The developmental model of microbial biofilms: ten years of a paradigm up for review, Trends Microbiol, 2009) The nature of the matrix containing exopolysaccharides is dependent on numerous parameters such as but not limited to the involved microorganisms, growth conditions, medium, and substrates. These parameters have been described in the art and are therefore known to a skilled person (Branda et al. , Biofilms: the matrix revisited, Trends Microbiol. 2005).

“Antimicrobial proteins” as described herein, and interchangeably annotated in the art as “antimicrobial peptides” indicate proteins or peptides that demonstrate a toxic effect to (one or more classes of) bacteria. Antimicrobial peptides have been demonstrated to kill Gram negative and Gram positive bacteria. Unlike the majority of conventional antibiotics antimicrobial peptides frequently destabilize biological membranes, can form transmembrane channels, and may also have the ability to enhance immunity by functioning as immunomodulators. Both naturally occurring antimicrobial proteins (peptides) and synthetic antimicrobial peptides are envisaged herein. A large number of antimicrobial peptides have been described in the art, and are therefore known to a skilled person, as are their (potential) application(s) (for example in Reddy et al, Antimicrobial peptides: premises and promises, International Journal of Antimicrobial Agents, 2004). Non-limiting examples of suitable antimicrobial proteins are further discussed below.

In certain embodiments, the genetically modified bacterium or oligonucleotide arrangement comprises a third oligonucleotide arrangement comprising a third nucleotide sequence encoding one or more heterologous DNA degrading enzyme and/or heterologous proteinases under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in (the) bacteria. “DNA degrading enzyme”, “deoxyribonuclease”, or “DNAse” as used herein is indicative for an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Numerous DNA degrading enzymes have been described in the art (for example in Yang, Nucleases: diversity of structure, function and mechanism, Q Rev Biophys, 2011) and are further discussed below. In alternative embodiments, the genetically modified bacterium or oligonucleotide arrangement comprises a third nucleotide sequence encoding one or more RNA degrading enzymes. A non-limiting example of an RNA degrading enzyme is the RNAse A superfamily member hRNAse 7 as described in Rademacher et al, RNAse 7 in cutaneous defense, Int J Mol Sci, 2016). “Proteinases”, also known in the art as “proteases” and “peptidases” are enzymes responsible for the breakdown of proteins into smaller polypeptides or even single amino acids. Proteinases act through peptide bond cleavage by hydrolysis. In certain embodiments, the one or more DNA degrading enzyme and/or proteinases do not occur in the genome of the unmodified (wild type) bacterium. In certain embodiments wherein the third oligonucleotide arrangement comprises a nucleotide sequence encoding one or more heterologous proteases, the heterologous protease is selected from the group of protease comprising: Ficin (described in Baidamshina et al., Sci Rep, 2017), Aureolysin, LapG protease, Proteinase K, Spl proteases, Staphopain A (ScpA), Staphopain B (SspB), Staphylococcal cysteine protease (SpeB), Surface-protein releasing enzyme (SPRE), Trypsin, and V8 serine protease (SspA).

In certain embodiments, the one or more nucleotide encoded exopolysaccharide hydrolyzing enzymes and the one or more nucleotide encoded heterologous antimicrobial proteins are each under the control of the same (i.e. identical) promoter or a functional variant of said promoter(s) or fragment thereof. In alternative embodiments, the one or more nucleotide encoded exopolysaccharide hydrolyzing enzymes and the one or more heterologous antimicrobial proteins are each under the control of a distinct (i.e. different) promoter or a functional variant of said promoter(s) or fragment thereof. In certain embodiments where the third oligonucleotide arrangement encoding a heterologous DNA degrading enzyme and/or heterologous proteinase is present, the one or more nucleotide encoded exopolysaccharide hydrolyzing enzymes, the one or more nucleotide encoded antimicrobial proteins and/or the one or more nucleotide encoded DNA degrading enzymes are each under the control of the same (i.e. identical) promoter or a functional variant of said promoter or fragment thereof. In alternative embodiments where the third oligonucleotide arrangement encoding a DNA degrading enzyme and/or proteinase is present, the one or more nucleotide encoded exopolysaccharide hydrolyzing enzymes, the one or more antimicrobial proteins and/or the one or more DNA degrading enzymes are each under the control of a distinct (i.e. different) promoter or a functional variant of said promoters or fragment thereof.

In certain embodiments, at least one of the nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes and at least one of the nucleotide encoded heterologous antimicrobial proteins are operably linked under the control of a single promoter or a functional variant of said promoter or fragment thereof. In certain embodiments, a least one of the nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes and at least one of the nucleotide encoded heterologous antimicrobial proteins, and/or at least one of the nucleotide encoded DNA degrading enzymes are operably linked under the control of a single promoter or a functional variant of said promoter or fragment thereof. The wording “operably linked” refers to a multitude of genetic elements that are joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is under transcriptional initiation regulation of the promoter or in functional combination therewith. In certain embodiments, the at least one of the nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes, at least one of the nucleotide encoded heterologous antimicrobial proteins, and/or at least one of the nucleotide encoded heterologous DNA degrading enzyme are comprised in a polycistronic construct, preferably bicistronic or tricistronic. The terms “polycistronic”, “bicistronic”, and “tricistronic” as used herein indicate that respectively multiple, two, or three separate proteins are encoded in a single messenger RNA. In certain embodiments, the polycistronic construct comprises one or more 2A peptides as described in the art (Liu et al. , Systematic comparison of 2A peptides for cloning multi -genes in a polycistronic vector, Scientific Reports, 2017) that separates the at least one of the nucleotide encoded exopolysaccharide hydrolyzing enzymes, at least one of the nucleotide encoded antimicrobial proteins, and/or at least one of the nucleotide encoded DNA degrading enzyme. In further embodiments, the 2A peptide encoded in the polycistronic construct is selected from the group of 2A peptides consisting of T2A, P2A, E2A or F2A. In alternative embodiments, the at least one of the nucleotide encoded exopolysaccharide hydrolyzing enzymes, at least one of the nucleotide encoded antimicrobial proteins, and/or at least one of the nucleotide encoded DNA degrading enzyme are separated by one or more Internal Ribosomal Entry Site (IRES) sequences. IRES sequences and their use in bacterial systems have been described in the art and are therefore known to a skilled artisan (Colussi el al. , Initiation of translation in bacteria by a structured eukaryotic IRES RNA, Nature, 2015).

In certain embodiments, at least one oligonucleotide sequence as described herein comprises a constitutive promoter. In further embodiments, the constitutive promoter is a promoter having a sequence identity of at least 65%, preferably at least 75%, at least 80%, at least 85%, at least 90% to a promoter selected from the group consisting of P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11). In yet further embodiments, the constitutive promoter is selected from the group consisting of P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11). In certain embodiments, at least one oligonucleotide arrangement as described herein comprises a synthetic promoter with a nucleotide sequence of at least 65% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: (P3).

Methods and tools to verify sequence homology or sequence identity between different sequences of amino acids or nucleic acids are well known to a person skilled in the art and include non-limiting tools such as Protein BLAST, ClustalW2, SIM alignment tool, TranslatorX and T-COFFEE. The percentage of identity between two sequences may show minor variability depending on the algorithm choice and parameters. The term “sequence identity” refers to the relationship between sequences at the nucleotide (or amino acid) level. The expression “% identical” is determined by comparing optimally aligned sequences, e.g. two or more, over a comparison window wherein the portion of the sequence in the comparison window may comprise insertions or deletions as compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not comprise insertions or deletions. A reference window is chosen and the “% identity” is then calculated by determining the number of nucleotides (or amino acids) that are identical between the sequences in the window, dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) in the window and multiplying by 100. Unless indicated otherwise, the sequence identity is calculated over the whole length of the reference sequence.

In certain embodiments, any of the herein described nucleotide encoded gene products may comprise an exposure signal and/or a secretion signal. By the term nucleotide encoded gene product is intended any protein encoded by a oligonucleotide arrangement as described herein. In certain embodiments, the nucleotide sequence encoding the heterologous exopolysaccharide hydrolyzing enzyme and/or the nucleotide sequence encoding the heterologous antimicrobial protein further comprises an exposure signal sequence or a secretion signal sequence. In alternative embodiments, the nucleotide sequence encoding the heterologous DNA degrading enzyme and/or proteinase comprises an exposure signal sequence or a secretion signal sequence. The term “exposure signal sequence” and is indicative for sequences encoding exposure signal peptides that targets the linked protein for exposure on the cell membrane. “Secretion signal sequence” as used herein refers to a sequence provoking or mediating secretion of a protein. As commonly accepted in the art, it is evident that when the term “secretion” is used, the secreted protein containing the secretion signal is no longer physically attached to the cell wherein said protein was produced, and it is intended that the protein is secreted into an extracellular space. Alternatively, when terms such as “exposed” or “displayed” are employed it is meant that the protein is still physically attached to the cell wherein the protein is produced, preferably to the outer cell surface of said cell. In certain embodiments, after the protein has been exposed on the cell surface (i.e. displayed), or after the protein has been secreted, the signal sequence may be removed from the linked protein by proteolytic cleavage. In certain embodiments, the exposure or secretion signal sequence is located at the N-terminus of the nucleotide-encoded gene product, here a protein.

In certain embodiments, the secretion signal sequence is a naturally occurring sequence in Mycoplasma, preferably M. pneumoniae. In yet further embodiments, the secretion signal sequence is a. Mycoplasma, preferably M. pneumoniae secretion signal sequence. In alternative embodiments, the secretion signal sequence is a not-naturally occurring Mycoplasma sequence. Mycoplasma secretion signals have been described in International patent application WO2016/135281 and are therefore known to a person skilled in the art. A skilled person furthermore understands that (mutagenized) exposure or secretion signals may be further mutagenized to improve exposure or secretion respectively of one or more nucleotide-encoded heterologous gene products described herein. In certain embodiments, concatenated secretion signals are comprised in one or more nucleotide-encoded gene products comprised herein. In certain embodiments, a plurality of distinct secretion signals is comprised in one or more nucleotide- encoded gene products described herein. In further embodiments, different secretion signals are comprised at different locations of nucleotide-encoded gene products described herein. In embodiments wherein the heterologous nucleotide-encoded gene product is polycistronic, the polycistronic sequence may contain both at least one secretion signal sequence and at least one exposure signal sequence.

In certain embodiments, the nucleotide sequence encoding the heterologous exopolysaccharide hydrolyzing enzyme, and/or the nucleotide sequence encoding the heterologous antimicrobial protein, and/or the nucleotide sequence encoding the heterologous DNA degrading enzyme further comprises a nucleotide sequence encoding an exposure signal sequence or a secretion signal sequence. In further embodiments, the nucleotide sequence encoding the heterologous exopolysaccharide hydrolyzing enzyme, and/or the nucleotide sequence encoding the heterologous antimicrobial protein, and/or the nucleotide sequence encoding the heterologous DNA degrading enzyme further comprises a nucleotide sequence encoding a secretion signal sequence from MPN036 (SEQ ID NO: 12), MPN142 (SEQ ID NO: 1), MPN645 (SEQ ID NO: 13), MPN400 (SEQ ID NO: 14), MPN200 (SEQ ID NO: 15), MPN213 (SEQ ID NO: 16), MPN489 (SEQ ID NO: 17). In yet further embodiments, the nucleotide sequence encoding the heterologous exopolysaccharide hydrolyzing enzyme, and/or the nucleotide sequence encoding the heterologous antimicrobial protein, and/or the nucleotide sequence encoding the heterologous DNA degrading enzyme further comprises a nucleotide sequence encoding the optimized secretion signal from MPN142 (nucleotide sequence: SEQ ID NO: 2; amino acid sequence: SEQ ID NO: 3).

In certain embodiments, at least one of the oligonucleotide arrangements described herein further comprises a regulatory sequence capable of modulating transcription. In certain embodiments, the regulatory sequence capable of modulating transcription is an enhancer sequence. The concept and meaning of “enhancer sequence” is known to a skilled person and are disclosed in the art (Xu and Hoover, Transcriptional regulation at a distance in bacteria, Curr Opin Microbiol, 2001). In further embodiments, the regulatory sequence capable of modulating transcription is a riboswitch. “Riboswitch” as defined herein is a regulatory sequence comprised in messenger RNA that may bind to a small molecule, wherein said binding has as consequence a change in the production of the one or more proteins encoded by the messenger RNA. A riboswitch is commonly divided into two parts: an aptamer and an expression platform. The aptamer directly binds a small molecule, and the expression platform undergoes structural changes in response to the changes in the aptamer. The expression platform is what regulates gene expression. Depending on the type of riboswitch, binding by a small molecule may enable translation, or inhibit translation. Non-limiting examples of riboswitches include cobalamin riboswitches, cyclic AMP-GMP riboswitches, cyclic di-AMP riboswitches, cyclic di-GMP riboswitches, fluoride riboswitches, FMN riboswitches, glmS riboswitches, glutamine riboswitches, glycine riboswitches, lysine riboswitches, manganese riboswitches, NiCo riboswitches, preQl riboswitches, purine riboswitches, SAH riboswitches, SAM riboswitches, SAM-SAH riboswitches, tetrahydrofolate riboswitches, TPP riboswitches, ZMP/ZTP riboswitches and the Moco RNA motif, the latter which is presumed to be a riboswitch. In certain embodiments, each promoter-containing oligonucleotide arrangement comprises a different riboswitch. In alternative embodiments, each promoter-containing oligonucleotide arrangement comprises a different riboswitch. In yet alternative embodiments, at least one oligonucleotide arrangement comprises two different riboswitches.

In certain embodiments, the exopolysaccharide hydrolyzing enzyme is a peptidoglycan hydrolase or a glycoside hydrolase. Both peptidoglycan hydrolases and glycoside hydrolases have been described in the art (Sharma et al , Prediction of peptidoglycan hydrolases-a new class of antibacterial proteins, BMC genomics, 2016, and Bourne et al, Glycoside hydrolases and glycosyltransferases: families and functional modules, Current opinion in structural biology, 2001). In certain embodiments, the exopolysaccharide hydrolyzing enzyme is a peptidoglycan hydrolase selected from the group consisting of: LysK (CHAP-AMID)-Lyso(PEP) (Becker et al, Scientific reports, 2016), endolysin LysH5 (Rodriguez-Rubio etal, PLOS one, 2013), HydH5 (HydH5SH3b and HydH5Lyso) (Garcia etal, Int J of Food Microb, 2010), Cpl-1 lysozyme (Sanz et al, Eur J Biochem, 1990), Cpl-7 lysozyme (Bustamante et at, J Biol Chem, 2010), Pal amidase (Varea et at, J Biol Chem, 2004), PL3 Amidase (Blazquez et at, Front Microbiol, 2016), MV-L lysin (Rashel et at, J Infect Dis, 2007), PlySs2 (Gilmer et at, Antimicrob Agents Chemother, 2013), Major autolysin (Atl) of Staphylococcus aureus (Porayath et at, Int J Biol Macromol, 2018), CF-301, N-Rephasin, P128, Art-175 (all described in Crit Care. 2016;20:397; J Infect Dis. 2014;209: 1469; Antimicrob Agents Chemother. 2016;60:7280; Antimicrob Agents Chemother. 2014;58:3774;Lancet Infect Dis. 2016;16:239;Curr Opin Biotechnol. 2016;37:76;Int J Med Microbiol. 2010;300(6):357;Future Microbiol. 2012;7(10): 1147), gp49 (Rodriguez-Rubio et at, Appl Environ Microbiol, 2013), LysK (CHAP1-AMID-SH3) (Becker et al., FEMS Microbiol Lett, 2009), , LysAB-SH3 (Lai etal. , Appl Microbiol Biotechnol, 2011), SAP-1 SAL- 1, P128, LysGH15/GH15, CF-301, ClyF, PaVDpl, Cpi-l/CP-1, LytA, Cpi-7/Cp-7, Cpi-7S, Cpl-711, PL3, PlyPy from MGAS315 prophage, PlyC/Ct, Lys8/Bxz2, LysA/BTCU-1, LysBIBTCU-1, Lysl521/8. amyloliquefaciens phage, E1188/EL, KZ144, OBPgp279, LysPA26, LysAB2, LysABP-01, PlyABl, PlyF307, LysAB3, LysAB4, Lysep3, Lysep3, Colicin-lysep3, EndoT51T5, PlyE146, K1 lgp3.5, KP32gpl5, KP27 lysin, CfPl lysin, P28, AP3gpl5 (all described in Amarante-Mendes et at, Front Immunol, 2018), lysB4 (Son et at, BMC Microbiol, 2012), LysBPS13 (Park et at, FEMS Microbiol Lett, 2012), Plyl2, Ply21, PlyBa (all described in Loessner, J Bacteriol, 1997), PlyG (Kikkawa et at, Biochem Biophys Res Commun, 2007), PlyB, Phage APSOc lysine (both described in Porter et at, J Mol Biol, 2007), PlyBT33 (Yuan et al, BMC Microbiol, 2012), PlyPH (Y oong et at, J Bacteriol, 2006), Plyl (Low et at, J Biol Chem, 2005), and AmiBA2446 (Mehta et at, 2013). In alternative embodiments, the exopolysaccharide hydrolyzing enzyme is a glycoside hydrolase selected from the group consisting of: Alginase Al-II, Alginase A1-IG, Alginase Al-III, Alg2A form Flavobacterium sp. S20 (Alg2A; AEB69783.1), Alginase-ProtA, a-amylase (Kalpana et at, Appl Biochem Biotechnol, 2012), Dispersin B (Darouiche et at, J Antimicrob Chemother, 2009), a- mannosidase, b-mannosidase (both described in Banar et at, PLoS One 2016), cellulase (Loiselle and Anderson, Biofouling, 2003), hyaluronidase (Pecharki et at, Microbiology, 2008), PelAh, and PslGh (both described in Baker et at, Sci Adv, 2016).

In certain embodiments, the exopolysaccharide hydrolyzing enzyme is selected from the group comprising LysK (CHAP-AMID)-Lyso(PEP), endolysin LysH5, HydH5 (HydH5SH3b and HydH5Lyso), Cpl-1 lysozyme, Cpl-7 lysozyme, Pal amidase, PL3 Amidase, MV-L lysin, PlySs2, Major autolysin (Atl) of Staphylococcus aureus, CF-301, N-Rephasin, P128, Art-175, gp49, LysK (CHAP1- AMID-SH3), , LysAB-SH3, SAP-1 SAL-1, P128, LysGH15/GH15, CF-301, ClyF, PaVDpl, Cpi-l/CP- 1, LytA, Cpi-7/Cp-7, Cpi-7S, Cpl-711, PL3, PlyPy, PlyC/Ct, Lys8/Bxz2, LysA/BTCU-1, LysBIBTCU- 1, Lysl521/8. amyloliquefaciens phage, E1188/EL, KZ144, OBPgp279, LysPA26, LysAB2, LysABP- 01, PlyABl, PlyF307, LysAB3, LysAB4, Lysep3, Lysep3, Colicin-lysep3, EndoT51T5, PlyE146, Kl lgp3.5, KP32gpl5, KP27 lysin, CfPl lysin, P28, AP3gpl5, lysB4, LysBPS13, Ply 12, Ply21, PlyBa, PlyG, PlyB, Phage APSOc lysine, PlyBT33, PlyPH, Plyl, AmiBA2446, Alginase Al-II, Alginase Al- IG, Alginase Al-III, Alginase ProtA, a-amylase, Dispersin B, a-mannosidase, b-mannosidase, cellulase, hyaluronidase, PelAh, and PslGh. In further embodiments, the exopolysaccharide hydrolyzing enzyme is selected from the group consisting of Dispersin B, PelAh, PslGh, alginate lyases, and any fusion proteins combining two or more proteins of said group. In further embodiments, at least two, preferably at least three exopolysaccarde hydrolyzing enzymes as described herein are encoded by a nucleotide sequence in the genetically modified bacterium or oligonucleotide arrangement.

In certain embodiments, the antimicrobial protein is an antibiotic peptide, preferably an antibiotic peptide selected from the group comprising: defensins, pyrrhocoricin, GramicidinA, IL37, Magainin, SMA2P9, CAP18, bacteriocinE50-5, Peptide LL-37, 1018, 1037, 17BIPHE2, Bac8c, Battacin, BMAP- 27, BMAP-28, CAMA, DJK-5, DJK-6, GF-17, LL-31, LL7-31, LL7-37, Melittin, P10, P60.4Ac, SMAP-29. In alternative embodiments, the antimicrobial protein is a bacteriocin, preferably a pyocin, more preferably a pyocin selected from the group comprising: pyocin Sn, pyocin SI, pyocin S2, pyocin S3, pyocin AP41, pyocin S5, pyocin S2, pyocin S3C, pyocin S6, pyocin S8, pyocin SD1, pyocin S13, pyocin SD2, pyocin SD3, pyocin SA189, pyocin LI, pyocin L2, pyocin L3, pyocin Ml, pyocin M4, pyocin PAEM4, pyocin PAEM, pyocin LI, putidacin LI, pyocin Rl, pyocin H108 (8-type), and pyocin 1577. Pyocins and their potential as therapeutics have been described in the art (Behrens et al, The therapeutic potential of bacteriocins as protein antibiotics, Emerging topics in life sciences, 2017). In alternative embodiments, the antimicrobial protein is a colicin, preferably a colicin selected from the group comprising of: colicin R, colicin N, colicin M, colicin D, colicin El, colicin E3, and colicin E9. In alternative embodiments, the antimicrobial protein is a haemocin, preferably haemocin A. In yet alternative embodiments, the antimicrobial protein is an engineered bacteriocin, preferably an engineered bacteriocin selected from the group comprising of: CLB pesticin, tailocin chimeras, SI chimeras, S2 chimeras, E2 chimeras, and E3 chimeras. In yet alternative embodiments, the antimicrobial protein is a taylocin, preferably selected from the group comprising pyocin R, pyocin F, Enterocoliticin, AvR2-V10.3 (all described in Behrens et al, The therapeutic potential of bacteriocins as protein antibiotics, Emerg Top Life Sci, 2017). In yet alternative embodiments, the antimicrobial protein is lactoferrin (van der Kraan et al, Peptides, 2004). In certain embodiments the antimicrobial protein is Lysostaphin. In certain embodiments, the antimicrobial protein is selected from the group comprising defensins, pyrrhocoricin, GramicidinA, IL37, Magainin, SMA2P9, CAP18, bacteriocinE50-5, Peptide LL-37, 1018, 1037, 17BIPHE2, Bac8c, Battacin, BMAP-27, BMAP-28, CAMA, DJK-5, DJK-6, GF- 17, LL-31, LL7-31, LL7-37, Melittin, P10, P60.4Ac, SMAP-29, Lyostpahin, pyocin Sn, pyocin SI, pyocin S2, pyocin S3, pyocin AP41, pyocin S5, pyocin S2, pyocin S3C, Pyocin S6, Pyocin S8, Pyocin SD1, pyocin S13, pyocin SD2, pyocin SD3, pyocin SA189, pyocin LI, pyocin L2, pyocin L3, pyocin Ml, pyocin M4, pyocin PAEM4, pyocin PAEM, pyocin LI, putidacin LI, pyocin Rl, pyocin H108 (8- type), pyocin 1577, colicin R, colicin N, colicin M, colicin D, colicinEl, colicin E3, and colicin E9, haemocin A, CLB pesticin, tailocin chimeras, SI chimeras, S2 chimeras, E2 chimeras, E3 chimeras, pyocin R, pyocin F, Enterocoliticin, AvR2-V10.3, and lactoferrin.

In certain embodiments describing the genetically modified bacterium or the oligonucleotide arrangement, the exopolysaccharide hydrolyzing enzyme is Dispersin B or Lysotaphin. In certain embodiments describing the genetically modified bacterium or the oligonucleotide arrangement, the genetically modified bacterium and the oligonucleotide arrangement, the exopolysaccharide hydrolyzing enzymes are PelAh, PslGh and Alginate lyase Al-IE and wherein the antimicrobial protein is Pyocyn LI.

In certain embodiments, the genetically modified bacterium or oligonucleotide arrangement further comprises a nucleotide encoded DNAse. In certain embodiments, the DNAse does not occur in the genome of the unmodified (wild type) bacterium.

It is evident that any combination of the herein envisaged gene products may be expressed from one or more oligonucleotide arrangements as described herein as a fusion protein. In certain embodiments, the fusion protein comprises at least two gene products independently selected from the group comprising: exopolysaccharide hydrolyzing enzymes, antimicrobial proteins, DNA degrading enzyme, and proteinases. In certain embodiments, the fusion protein further comprises an N-terminal secretion signal sequence as disclosed herein. In further embodiments, the secretion signal is the optimized MPN142 secretion signal sequence (SEQ ID NO: 2). In certain embodiments, expression of the fusion protein is controlled by a synthetic promoter, preferably a synthetic promoter having a sequence identity of at least 65%, preferably at least 75%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably 100% to SEQ ID NO: 8. In certain embodiments, the fusion protein is LysAB2_SH3b. In certain embodiments, the fusion protein is Lysostaphin-Dispersin B.

A further aspect of the invention is directed to a method for altering het genome of a bacterium comprising introducing an oligonucleotide arrangement as described herein into a bacterium. In certain embodiments, the method comprises a selection step for detecting, and optionally isolating, genetically modified bacteria comprising the oligonucleotide arrangements in their genomic sequence. In further embodiments, the selection step is a phenotype selection step. Non-limiting examples phenotypic selection steps are antibiotic resistance of the correct genetically modified bacterium and fluorescence of the correct genetically modified bacterium. In alternative embodiments, the selection step is a genetic detection step. A non-limiting example of a genetic detection step is a polymerase chain reaction (partially) over spanning the targeted region of the genome. In certain embodiments, the selection step is based on a counter selection by a designer nuclease targeting the unmodified genomic sequence where cleavage of this sequence is toxic for said bacterium. In certain embodiments of the methods described herein, the bacterium is a Mycoplasma bacterium, preferably a Mycoplasma pneumonia bacterium. “ Mycoplasma ”, “ Mycoplasma bacteria”, or Mycoplasmas as used interchangeably herein refers to the mollicute genus Mycoplasma which is characterized by lack of a cell wall around their cell membranes. Therefore, the plasma membrane forms the outer boundary of the Mycoplasma bacterial cell. Due to the absence of a cell wall, Mycoplasma has been found to have versatile shapes ranging from round to oblong, and display pleomorphism. “Pleomorphism” as used herein is a term used in histology and cytopathology to describe cells and/or their nuclei that may contain variable sizes, shape and staining. Culturable Mycoplasma species typically form small umbonate colonies on agar. The exact shape of the Mycoplasmas may depend on numerous parameters including osmotic pressure, nutritional quality of the culture medium, and growth phase. Certain Mycoplasma bacteria may be filamentous in their early and exponential growth phases or when attached to surfaces or other cells. The filamentous form may be transitory, and in certain conditions the filaments may branch or fragment into chains of cocci or individual vegetative cells. Alternative species are typically coccoid and do not develop a filamentous phase. Certain species develop specialized attachment tip structures involved in the process of colonization and/or contribute to virulence. Mycoplasma bacteria comprise 16S and 70S type ribosomes and a replicating disc to assist the replication process, and isolation of the genetic material. Mycoplasma bacteria may either live as saprophytes or more commonly as parasites. The term “saprophytes” refers to the chemoheterotrophic extracellular digestion that takes place in the processing of decayed organic matter. Mycoplasma bacteria are commonly described as one of the smallest and simplest self- replicating organisms known to date. Naturally occurring Mycoplasma genomes vary from about 500 kilobases (kb) to 1500 kb and GC contents between 23-41 mole percent (mol%) have been described. Different Mycoplasma species have been described and catalogued in the art (inter alia in Thompson et al. , Towards a genome based taxonomy of Mycoplasmas, 2011). It is evident to a skilled person that the term Mycoplasma additionally includes any Mycoplasma strain or species that is generated by genetic or chemical synthesis, or any sort of rational design and/or the reorganization of a naturally occurring Mycoplasma genomic sequence and that the term therefore also covers those Mycoplasma strains and species that are termed “synthetic Mycoplasma” , alternatively “ Mycoplasma laboratorium” , “ Mycoplasma synthia ”, or even short “Synthia” in the art (Gibson et al. , Creation of a bacterial cell controlled by a chemically synthesized genome, Science, 2010). Hence, in certain embodiments described throughout this specification, the Mycoplasma species subject of the invention have as genomic sequence a sequences comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% global sequence identity to a naturally occurring Mycoplasma bacterium. In certain embodiments, the Mycoplasma bacterium is M. pneumoniae, preferably M. pneumoniae M129(-B7) (ATCC identifier 29342).

Techniques for enrichment and/or isolation of Mycoplasmas from humans, various species of animals, and cell cultures have been extensively described in the art and are well-known to a skilled person (Tully and Razin, Molecular and diagnostic procedures in Mycoplasmology, Vol. 2, 1996). A skilled person is also aware that minimal standards for descriptions of new species have been outlined (Brown ei al , Revise standards for description of new species of the class Mollicutes (division Tenericutes), International Journal of Systematic and Evolutionary Microbiology, 2007).

Another aspect of the invention is directed to the use of an oligonucleotide arrangement described herein for altering the genomic sequence of a Mycoplasma bacterium. In certain embodiments, the genomic sequence of the Mycoplasma bacterium is altered by inserting the oligonucleotide arrangement in said genomic sequence. In alternative embodiments, the genomic sequence of the Mycoplasma bacterium is altered by substituting a wild type (i.e. naturally occurring) genomic sequence of said Mycoplasma bacterium with the oligonucleotide arrangement. In yet alternative embodiments, the genomic sequence of the Mycoplasma bacterium is modified by the oligonucleotide arrangement by a combination of insertion, substitution, and optionally deleting one or more genomic sequences of a Mycoplasma bacterium. In certain embodiments, the first and/or second nucleotide sequences encode a gene product able to reduce biofilm formation. In further embodiments, the biofilm is a microbial biofilm. In certain embodiments, the use of an oligonucleotide arrangement as described herein is intended wherein said biofilm is formed in the respiratory system of said subject. In further embodiments, biofilm is formed in the lower respiratory system (tract). In alternative further embodiments, the biofilm is formed in the upper respiratory system (tract). A skilled person understand the meaning of the term “respiratory system”, which may be annotated in the art as “respiratory apparatus”, or even “ventilatory system” in the art and is aware that the respiratory system comprises organs and structures used for gas exchange in animals, human being a non-limiting example hereof. In mammals such as humans, the upper respiratory tract includes the nose, nasal cavities, sinuses, pharynx and the part of the larynx above the vocal folds. In mammals such as humans, the lower respiratory tract includes the lower part of the larynx, the trachea, bronchi, bronchioles and the alveoli. In certain embodiments, said biofilm is formed in the lungs of said subject. In certain embodiments, said biofilm is formed in the trachea of the subject. In certain embodiment, the biofilm is formed in the bronchi and/or bronchiole of the subject.

In certain embodiments, the use of an oligonucleotide arrangement as described herein for dispersing biofilms comprising hexosamine-containing polymers (PIA) is intended. Hexosamine-containing polymers (PIA) have been described in the art (for example in Kaplan et al, Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms, Journal of Bacteriology, 2004). In certain embodiments, the biofilm comprises between 10% and 90% weight percentage, preferably between 20% and 80% weight percentage hexosamine-containing polymers. In certain embodiments, the biofilm comprises at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60% hexosamine- containing polymers. In certain embodiments, the use of an oligonucleotide arrangement as described herein for dispersing biofilms comprising Pel and/or Psl is envisaged. In further embodiments, the Pel and/or Psl present in the biofilm are P. aeruginosa Pel and/or Psl (Colvin et al, The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofdm matrix, Environ Microbiol, 2012). In certain embodiments, the use of an oligonucleotide arrangement as described herein for dispersing biofdms comprising alginate exopolysaccharides is envisaged. The protective role of alginate exopolysaccharide for P. aeruginosa biofdms is known in the art (Leid el al. , The exopolysaccharide alginate protects Pseudomonas aeruginosa biofdm bacteria from IFN-gamma- mediated macrophage killing, J Immunol, 2005). In certain embodiments, the use of an oligonucleotide arrangement as described herein for dispersing biofdms comprising Pel, Psl, and/or alginate exopolysaccharides is envisaged. In certain embodiments, the use as described herein for dispersing biofdms produced by Pseudomonas aeruginosa (or a group of bacteria comprising or consisting essentially of P. aeruginosa) is intended. In certain embodiments, the use as described herein for killing and/or inactivating P. aeruginosa present in a microbial biofdm is intended. In certain embodiments, the use as described herein for reducing the growth rate of a biofdm comprising P. aeruginosa bacteria is intended. In further embodiments, the growth rate of the biofdm comprising P. aeruginosa bacteria is reduced by at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% when compared to a P. aeruginosa comprising biofdm not contacted with a nucleotide arrangement as described herein or a genetically modified bacterium comprising a nucleotide arrangement as described herein. In certain embodiments, the use as described herein for dispersing biofdms produced by Staphylococcus aureus (or a group of bacteria comprising or consisting essentially of S. aureus) is envisaged. In certain embodiments, the use as described herein for killing and/or inactivating S. aureus present in a microbial biofdm is intended. In certain embodiments, the use as described herein for reducing the growth rate of a biofdm comprising S. aureus bacteria is intended. In further embodiments, the growth rate of the biofdm comprising S. aureus bacteria is reduced by at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% when compared to an S. aureus comprising biofdm not contacted with a nucleotide arrangement as described herein or a genetically modified bacterium comprising a nucleotide arrangement as described herein. It is evident to a skilled person that biofdms commonly comprise a plethora of bacterial species. Hence, as intended herein are biofdms comprising a relative amount of P. aeruginosa and/or S. aureus bacteria of at least 10%, preferably at least 25%, preferably at least 35%, preferably at least 50%, preferably at least 60%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 95% when assessed in view of the total amount of bacterial species present in the biofdm.

In certain embodiments, the use of an oligonucleotide arrangement as described herein for altering the genomic sequence of genetically modified (e.g. attenuated) Mycoplasma bacterium is envisaged. In alternative embodiments, the use of an oligonucleotide arrangement as described herein for altering the genomic sequence of an naturally occurring Mycoplasma bacterium is envisaged. In yet alternative embodiments, the use of an oligonucleotide arrangement as described herein for altering the genomic sequence of a synthetic Mycoplasma bacterium is intended. The term “attenuated” as described herein can be used interchangeably with terms such as "weakened" and "diminished". The wording "attenuated strain" is commonly used in the art and refers to weakened disease agents, i.e. attenuated pathogens. An attenuated bacterium is a weakened, less vigorous, less virulent bacterium when compared to the traditionally occurring counterpart. Multiple vaccines against different diseases are based on inclusion of an attenuated strain of a bacterium or virus that is still capable of inducing an immune response and creating immunity but not causing illness. An attenuated Mycoplasma bacterium according to embodiments of the invention is indicative for a genetically modified Mycoplasma bacterium wherein expression of genes whereof the gene product is responsible for a certain degree of virulence or toxicity have been modified in order to diminish the adverse effect of said gene on an infected subject.

The genetically modified (e.g. attenuated ) Mycoplasma bacterium used to introduce the oligonucleotide arrangement has a genomic sequence comprising a (functional) modification, such as but not limited to an inactivating mutation, deletion, and/or substitution in at least one gene selected from the group consisting of: MPN051 (glycerol-3 -phospate dehydrogenase), MPN133 (Ca2+ dependent cytotoxic nuclease gene), MPN142 (Adhesin PI), MPN257 (UDP-glucose 4-epimerase), MPN294 (chaperone protein YajL), MPN372 (ADP-ribosyltransferase CARDS gene), MPN400 (hypothetical protein MPN_400), MPN415 (high affinity transport system protein p37), MPN453 (adhesin P30), MPN483 (glycosyltransferase enzyme), MPN491 (membrane nuclease A), MPN592 (Uncharacterized lipoprotein MPN_592), and MPN626 (RNA polymerase sigma-D factor). The intended Mycoplasma genes are indicated throughout this specification by their MPN (M pneumoniae) number. A skilled person is aware that the MPN nomenclature is a standard manner of gene annotation in the technical field and that gene and/or protein names are readily derivable from publicly available resources such as the M. pneumoniae database http://mympn.crg.eu/essentiality.php or (academic) publications (including but not limited to Lluch-Senar et al, Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium, Molecular Systems Biology, 2015). It is evident that the MPN numbers are intended to also cover Mycoplasma genes from different strains, and it is thus evident for a skilled person that alternative annotations and classifications may be used to specify the same, or essentially the same genes. For example, a commonly used yet non-limiting system to annotate certain gene product is the IUBMB enzyme nomenclature. Reference works and tools to link certain enzymatic activities to specific IUBMB EC numbers are readily available in the art (e.g. McDonald et al., ExplorEnz: the primary source of the IUBMB enzyme list, Nucleic Acids Research, 2009). Hence, when reference herein is made to a certain MPN number, such references also encompass the corresponding enzymes in orthologue Mycoplasma bacteria categorized under the same IUBMB EC number. In certain embodiments, the attenuated Mycoplasma bacterium used to introduce the oligonucleotide arrangement has a (functional) modification such as but not limited to an inactivating mutation, deletion, and/or substitution in MPN133 and/or MPN372. Example 1 describes how such a genetically modified (e.g. attenuated) Mycoplasma bacteria can be constructed. The resulting genetically modified (e.g. attenuated) Mycoplasma bacterium with a modification in MPN133 and MPN372 is herein referred to as the CV2 chassis. The resulting genetically modified (e.g. attenuated) Mycoplasma bacterium with a further modification in MPN051 is herein referred to as the CV8 chassis.

In a further aspect, the invention is directed to a genetically modified (e.g. an attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use as a medicament. It is understood that a medicament as used in the context herein refers to a substance, or drug, that is used to diagnose, cure, treat, or prevent disease.

In certain embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in treating pneumonia is envisaged. “Pneumonia” as used herein refers to an inflammatory condition of the lung affecting in particular the alveoli of the subject. The diagnosis of pneumonia is usually based on the assessment of physical signs, a chest radiograph, PCR-based methods, lung ultrasound, sputum cultures, or a combination thereof. Typical physical signs include but are not limited to low blood pressure, high heart rate, low oxygen saturation, increased respiratory rate, decreased chest expansion on the side affected by the pneumonia, bronchial breathing, crackling noises during inspiration, altered percussion of an affected lung, and increased vocal resonance. In certain embodiments, a genetically modified (e.g; attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in treating Cystic Fibrosis (CF) is intended. Cystic fibrosis an autosomal recessive genetic disorder caused by a mutated CFTR gene that mainly affects the lung, while also affecting other organs such as the pancreas, liver, kidneys, and intestine. The main symptoms related to lung function are mucus build up, decreased mucociliary clearance and inflammation. These symptoms develop as a consequence of bacterial colonization and infection of the lungs of the patients. Non-limiting examples of bacteria responsible for lung infections in cystic fibrosis patients are P. aeruginosa, S. aureus, and Haemophilus influenzae. Often biofilms are formed in the lungs of cystic fibrosis patients due to presence of one or more of these bacterial species (Johnson et al. , Novel understandings of host cell mechanisms involved in chronic lung infection: Pseudomonas aeruginosa in the cystic fibrotic lung, Journal of Infection and Public Health, 2019). In alternative further embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in treating Chronic Obstructive Pulmonary Disease (COPD) is envisaged. Chronic obstructive pulmonary disease may be alternatively indicated by “chronic bronchitis” in the art and is an obstructive lung disease having a shortness of breath and cough with sputum production as main symptoms (Vogelmeier et al, Global Strategy for the Diagnosis, Management and Prevention of Chronic Obstructive Lung Disease 2017 Report: GOLD Executive Summary". Respirology, 2017). In certain embodiments, the genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein is used to treat subjects diagnosed with, or showing symptoms adequate to be diagnosed with pneumonia, preferably ventilator associated pneumonia, (recurrent) pneumonia as a consequence of cystic fibrosis, or pneumonia as a consequence of chronic obstructive pulmonary disease. In further embodiments, the subject having, or suspected to have pneumonia has a reduced standard lung volume of at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferable between 70% and 90%, wherein the standard lung volume is selected from the group consisting of; tidal volume, inspiratory reserve volume, expiratory reserve volume, residual volume. In certain embodiments, the subject having, or suspected to have pneumonia has a reduced standard lung volume of between 10% and 95%, preferably between 10% and 50%, between 25% and 50%, between 50% and 95%, between 75% and 95%, wherein said standard lung volume is selected from the group consisting of; tidal volume, inspiratory reserve volume, expiratory reserve volume, residual volume. In certain embodiments, the subject having, or suspected to have pneumonia has a reduced standard lung capacity of at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferable between 70% and 90%, wherein said standard lung capacity is selected from the group consisting of; inspiratory capacity, functional residual capacity, vital lung capacity, and total lung capacity. In certain embodiments, the subject having, or suspected to have pneumonia has a reduced standard lung capacity of between 10% and 95%, preferably between 10% and 50%, between 25% and 50%, between 50% and 95%, between 75% and 95%, wherein the standard lung capacity is selected from the group consisting of; inspiratory capacity, functional residual capacity, vital lung capacity, and total lung capacity. Standard lung volumes, standard lung capacities, and means to measure them have been described in detail in the art (Lufnti, The physiological basis and clinical significance of lung volume measurements, Multidiscip Respir Med, 2017). A non-limiting method to assess the lung volumes and lung capacities described above is by spirometry. In further embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in treating ventilator associated pneumonia is intended. It is understood that ventilator associated pneumonia is a type of lung infection occurring in patients subjected to mechanical ventilation breathing machines in hospitals (Michetti et al, Ventilator- associated pneumonia rates at major trauma centers compared with a national benchmark: a multi- institutional study of the AAST, J Trauma Acute Care Surg, 2012).

In certain embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in treating mastitis is intended. The term “mastitis as used herein covers any inflammation of the breast and/or udder of a subject. Commonly observed symptoms in subject considered to have mastitis include a local redness (inflammation) and pain of the breast area (Berens, Breast Pain: Engorgement, Nipple Pain, and Mastitis, Clinical Obstetrics and Gynecology, 2015). A subject is considered to have mastitis when a condition has been diagnosed as mastitis, or when a subject is suspected to have mastitis. Especially intended herein is mastitis caused by a Staphylococcus infection, such as for example S. aureus. However, other bacterial sources of mastitis may also benefit from treatment with a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein. In certain embodiments, the mastitis is pregnancy-related mastitis. In alternative embodiments, the mastitis is not pregnancy related. In certain embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium described herein or obtained by any of the methods described herein is intended for use in treating mastitis, wherein said Mycoplasma bacterium is used in a combination therapy further comprising the use of at least one antibiotic. In further embodiments, the antibiotic is selected from the group comprising: Dicloxacillin, Cephalexin, and Vancomycin.

In certain embodiments, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein for use in dissolving microbial biofilms produced by Pseudomonas aeruginosa or Staphylococcus aureus is intended. In certain embodiments, the microbial biofilm is dissolved by at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 100% when compared to the dissolvement rate of a biofilm not treated by the genetically modified (e.g. attenuated) Mycoplasma bacterium. Methods to quantitatively assess biofilm growth are known to a skilled person (Haney el al. , Critical assessment of methods to quantify biofilm growth and evaluate antibiofilm activity of host defence peptides, Biomolecules, 2018).

A further aspect of the invention is thus a genetically modified Mycoplasma bacterium as described herein, wherein said Mycoplasma bacterium comprises a functional modification such as a deletion, insertion, and/or substitution in MPN372 and/or MPN133, and further comprising in its genome one or more oligonucleotide arrangements encoding for PelAh, PslGh, Alginate lyase Al-IG, and pyocin, preferably pyocin LI . In addition, this genetically modified Mycoplasma bacterium further comprising a functional modification such as a deletion, insertion, and/or substitution in MPN051.

A further aspect of the invention is thus a genetically modified Mycoplasma bacterium as described herein, comprising a functional modification such as a deletion, insertion, or substitution in MPN372 and/or MPN133, and further comprising in its genome one or more oligonucleotide arrangements encoding for Dispersin B and lysostaphin. In addition, this genetically modified Mycoplasma bacterium according to aspect 39, further comprising a functional modification such as a deletion, insertion, and/or substitution in MPN051.

A further aspect of the invention is thus the genetically modified Mycoplasma bacterium as described herein, further comprising a functional modification such as a deletion, substitution, and/or insertion in one or more genes or operons encoding a protein capable of eliciting Guillain-Barre in a host organism, preferably in MPN257 and/or MPN483. Also intended by the invention is a pharmaceutical composition comprising the genetically modified Mycoplasma bacterium as described herein or obtained by any of the methods described herein. A person skilled in the art is aware that the terms “pharmaceutical composition”, “pharmaceutical formulation”, and “pharmaceutical preparation” can be used interchangeably herein and are meant to describe compositions containing a genetically modified Mycoplasma bacterium as active pharmaceutical ingredient, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. It is evident that pharmaceutical compositions are indicative for those compositions that comprise a therapeutically effective amount of genetically modified Mycoplasma bacteria, or at least an amount of genetically modified Mycoplasma bacteria that, when introduced into a host organism as live bacteria, can propagate to express or deliver a therapeutically effective amount of a desired gene product and/or bacterial cargo.

The term “therapeutically effective amount” as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include a reduction or complete removal of the symptoms associated with the disease or condition being treated. Methods to determine pharmaceutically effective amounts are known in the art and are therefore known to a skilled person. It is further evident that therapeutic effective amounts are determined in function of the specific subject in need of treatment. Further, a wording such as “a subject in need of treatment” includes any subject or group of subjects that would benefit from treatment of a given condition. Such subjects may include, without limitation, those that have been diagnosed with a condition susceptible to treatment with the genetically modified Mycoplasma bacterium, those prone to develop said condition and/or those in who said condition is to be prevented.

The terms “treat” or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed pulmonary disease, as well as prophylactic or preventive measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of a pulmonary infection. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the object is to bring a subjects body or an element thereof from an undesired physiological change or disorder, including but not limited to pulmonary infections, to a desired state, such as a less severe or unpleasant state (e.g., amelioration or palliation), or back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it (i.e., not worsening) at said undesired physiological change or disorder (e.g., stabilization), or to prevent or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder.

In certain embodiments, the pharmaceutical formulation further comprises one or more further pharmaceutical active ingredients. In certain embodiments, the pharmaceutical formulation further comprises one or more non-active pharmaceutical ingredients or inactive ingredients, commonly referred to in the art as excipients. In further embodiments, the pharmaceutical composition may be a lyophilized pharmaceutical composition.

The term “excipient”, commonly termed “carrier” in the art may be indicative for all solvents, including but by no means limited to: diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubilisers (e.g., Tween 80, Polysorbate 80), colloids, dispersion media, vehicles, fdlers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, sweeteners, colorants, flavorings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives (e.g., benzyl alcohol), antioxidants (such as, e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol) and the like. The use of such media and agents for formulating pharmaceutical compositions is well known in the art.

In certain embodiments, the pharmaceutical composition is a lyophilized composition that may need to be reconstituted prior to administration. In further embodiments, the pharmaceutical composition can be formulated into a unit dosage form, including but not limited to hard capsules, soft capsules, tablets, coated tablets such as lacquered tablets or sugar-coated tablets, granules, aqueous or oily solutions, syrups, emulsions, suspensions, ointments, pastes, lotions, gels, inhalants or suppositories, which may be provided in any suitable packaging means known in the art, non-limiting examples being troches, sachets, pouches, bottles, films, sprays, microcapsules, implants, rods or blister packs.

In certain embodiments, the pharmaceutical composition described herein further comprises at least one antibiotic. In further embodiments, the pharmaceutical composition further comprises at least one antibiotic selected from the group comprising Piperacillin, Tazobactam, Ciprofloxacin, Levofloxacin, Meropenem, Imipenem, Cilastatin, Amikacin, Ceftazidime, Avibactam, Ceftolozane, Tazobactam, Ceftriaxone, Vancomycin, and Linezolid. In further embodiments, the pharmaceutical composition comprises at least one antibiotic selected from the group comprising Piperacillin, Tazobactam, Meropenem, Imipenem, Cilastatin, and Vactomycin. more preferably the antibiotic is Meropenem. In further embodiments, the Meropenem is used at a final concentration of about 100 pg/ml. In certain embodiments, the pharmaceutical composition described herein comprises Piperacillin and Tazobactam, preferably wherein the Piperacillin-Tazobactam is used at a final concentration of about 500 pg/ml. In alternative embodiments, the pharmaceutical composition as described herein comprises Imipenem and Cilastatin, preferably wherein the Imipenem-Cilastatin combination is used at a final concentration of about 300 pg/ml. In alternative embodiments, the pharmaceutical composition comprises Vactomycin, preferably wherein the Vactomycin is used at a final concentration of about 200 pg/ml. In certain embodiments, the pharmaceutical composition is a lyophilized pharmaceutical composition.

Further envisaged is a synthetic promoter with a nucleotide sequence of at least 65% sequence identity to the nucleotide sequence of SEQ ID NO: 8. In certain embodiments, the synthetic promoter comprises a nucleotide sequence of least 75% sequence identity, more preferably at least 85% sequence identity, preferably at least 95% sequence identity, most preferably 100% sequence identity to the nucleotide sequence of SEQ ID NO: 8 able to drive expression of a coding or non-coding bacterial nucleotide sequence such as a nucleotide encoded gene product as described herein. In certain embodiments, the synthetic promoter is the P3 promoter (SEQ ID NO: 8) and the heterologous gene product is lysostaphin.

In a further aspect, the expression of one or more heterologous gene products is controlled by a synthetic promoter. In further embodiments, the synthetic promoter for each heterologous gene product is independently selected from a group of sequences comprising : P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11). In certain embodiments wherein a synthetic promoter is used, the expression of the one or more heterologous gene product is increased with at least 25%, preferably at least 35%, preferably at least 45%, preferably at least 50%, preferably at least 75%, preferably at least 100%, preferably at least 150%, preferably at least 200% compared to the expression level of the one or more heterologous gene product whereof expression is controlled by a naturally occurring promoter, preferably a naturally occurring M. pneumoniae promoter. In certain embodiments, the efficacy of a heterologous gene product as described herein part of a oligonucleotide arrangement as described herein is increased by at least 25%, preferably at least 35%, preferably at least 45%, preferably at least 50%, preferably at least 75%, preferably at least 100%, preferably at least 150%, preferably at least 200% when compared to said gene product under control of a naturally occurring promoter, preferably a naturally occurring M. pneumoniae promoter. Efficacy as used herein may be indicative of the potency of a heterologous gene product to dissolve and/or prevent formation of (microbial) biofilms, preferably wherein said biofilm is formed by P. aeruginosa, S. aureus, or a combination hereof.

An aspect of the invention is directed to a method of treating a subject diagnosed with, or suspected to have a pathogenic biofilm formation, wherein the method comprises a step of contacting the subject with a genetically modified bacterium as described herein or a pharmaceutical composition as described herein. In certain embodiments, the bacterium is a (live) Mycoplasma bacterium. In certain embodiments, the pathogenic biofilm formation is a respiratory biofilm formation. In further embodiments, the subject is diagnosed with, or suspected to have, ventilator associated pneumonia (VAP), Cystic Fibrosis (CF), or Chronic Obstructive Pulmonary Disease (COPD). In alternative embodiments, the pathogenic biofdm formation is causative for the development of mastitis. In certain embodiments, the bacterium is allowed to propagate after administration. In certain embodiments, said modified bacterium is co-administered with at least one antibiotic. In further embodiments, the at least antibiotic is selected from the group comprising: Piperacillin, Tazobactam, Ciprofloxacin, Levofloxacin, Meropenem, Imipenem, Cilastatin, Amikacin, Ceftazidime, Avibactam, Ceftolozane, Ceftriaxone, Vancomycin, Linezolid, or any combination thereof. A particularly preferred combinations of the described compounds is the combination of Piperacillin and Tazobactam, wherein Tazobactam will prevent Piperacillin degradation. A further particularly preferred combination is the combination of Imipenen and Cilastatin, wherein Cilistatin will prevent Imipenen degradation. Yet a further particularly preferred combination is the combination of Ceftazidime and Avibactam, wherein Avibactam will prevent Ceftazidime degradation. Yet a further particularly preferred combination is the combination of Ceftolozane and Tazobactam, wherein Tazobactam will prevent Ceftozolane degradation. In most preferred embodiments, the antibiotic is selected from the list consisting of: Piperacillin, Ciprofloxacin, Levofloxacin, Meropenem, Imipenem, Amikacin, Ceftazidime, Ceftolozane, Ceftriaxone, or any combination thereof. In certain embodiments, the genetically modified bacterium or pharmaceutical composition comprising said genetically modified bacterium is administered once. In alternative embodiments, the genetically modified bacterium or pharmaceutical composition comprising said genetically modified bacterium is administered periodically, preferably at regularly time intervals. In certain embodiments where a subject is treated for mastitis, a genetically modified (e.g. attenuated) Mycoplasma bacterium as described herein or obtained by any of the methods described herein is used in combination with an invasive procedure. In further embodiments, the invasive procedure is (ultrasound-guided) fine needle aspiration, or surgical incision and drainage.

In certain respiratory biofilm related embodiments, the contacting step comprises inhalation of the bacterium. In certain mastitis related embodiments, the contacting step comprises local injection of the bacterium. A further aspect of the invention relates to the use of a genetically modified bacterium as described herein, for the manufacture of a medicament for the prevention or treatment of (a) pathogenic biofilm (formation). In certain embodiments, the medicament comprises live genetically modified bacteria, preferably live genetically modified Mycoplasma bacteria, more preferably live genetically modified Mycoplasma pneumoniae bacteria. In certain embodiments, the use of a genetically modified bacterium as described herein is intended, for the manufacture of a medicament for the prevention or treatment of pneumonia. In certain embodiments, the use of a genetically modified bacterium as described herein is intended, for the manufacture of a medicament for the prevention or treatment of mastitis.

Additionally, the disclosure provides the following statements: Statement 1. A genetically modified bacterium comprising an oligonucleotide arrangement, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium.

Statement 2. The genetically modified bacterium according to statement 1, wherein said oligonucleotide arrangement further comprises a third nucleotide sequence encoding one or more heterologous DNA degrading enzymes and/or heterologous proteinases under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacteria.

Statement 3. The genetically modified bacterium according to statement 1 or 2, wherein the one or more nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes, the one or more nucleotide encoded heterologous antimicrobial proteins and/or the one or more nucleotide encoded heterologous DNA degrading enzymes are each under the control of the same or a different promoter or a functional variant of said promoter(s) or fragment thereof.

Statement 4. The genetically modified bacterium according to any one of statements 1 to 3, wherein at least one oligonucleotide sequence comprises a constitutive promoter, preferably a promoter with a sequence selected from the group of sequences comprising : P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11).

Statement 5. The genetically modified bacterium according to any one of statements 1 to 4, wherein at least one nucleotide sequence comprises a synthetic promoter with a nucleotide sequence of at least 65% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 8.

Statement 6. The genetically modified bacterium according to any one of statements 1 to 5, wherein the exopolysaccharide hydrolyzing enzyme is selected from the group LysK (CHAP-AMID)- Lyso(PEP), endolysin LysH5, HydH5 (HydH5SH3b and HydH5Lyso), Cpl-1 lysozyme, Cpl-7 lysozyme, Pal amidase, PL3 Amidase, MV-L lysin, PlySs2, Major autolysin (Atl) of Staphylococcus aureus, CF-301, N-Rephasin, P128, Art-175, gp49, LysK (CHAP1-AMID-SH3), Lysostaphin, LysAB- SH3, SAP-1 SAL-1, P128, LysGH15/GH15, CF-301, ClyF, PaVDpl, Cpi-l/CP-1, LytA, Cpi-7/Cp-7, Cpi-7S, Cpl-711, PL3, PlyPy, PlyC/Ct, Lys8/Bxz2, LysA/BTCU-1, LysBIBTCU-1, Lysl521/8. amyloliquefaciens phage, E1188/EL, KZ144, OBPgp279, LysPA26, LysAB2, LysABP-01, PlyABl, PlyF307, LysAB3, LysAB4, Lysep3, Lysep3, Colicin-lysep3, EndoT51T5, PlyE146, Kl lgp3.5, KP32gpl5, KP27 lysin, CfPl lysin, P28, AP3gpl5, lysB4, LysBPS13, Ply 12, Ply21, PlyBa, PlyG, PlyB, Phage APSOc lysine, PlyBT33, PlyPH, Plyl, AmiBA2446, Alginase Al-II, Alginase Al-IG, Alginase Al-III, Alginase ProtA, a-amylase, Dispersin B, a-mannosidase, b-mannosidase, cellulase, hyaluronidase, PelAh, and PslGh..

Statement 7. The genetically modified bacterium according to any one of statements 1 to 6, wherein the antimicrobial protein is selected from the group comprising defensins, pyrrhocoricin, GramicidinA, IL37, Magainin, SMA2P9, CAP 18, bacteriocinE50-5, Peptide LL-37, 1018, 1037, 17BIPHE2, Bac8c, Battacin, BMAP-27, BMAP-28, CAMA, DJK-5, DJK-6, GF-17, LL-31, LL7-31, LL7-37, Melittin, P10, P60.4Ac, SMAP-29, pyocin Sn, pyocin SI, pyocin S2, pyocin S3, pyocin AP41, pyocin S5, pyocin S2, pyocin S3C, Pyocin S6, Pyocin S8, Pyocin SD1, pyocin S13, pyocin SD2, pyocin SD3, pyocin SA189, pyocin LI, pyocin L2, pyocin L3, pyocin Ml, pyocin M4, pyocin PAEM4, pyocin PAEM, pyocin LI, putidacin LI, pyocin Rl, pyocin H108 (8-type), pyocin 1577, colicin R, colicin N, colicin M, colicin D, colicinEl, colicin E3, and colicin E9, haemocin A, CLB pesticin, tailocin chimeras, SI chimeras, S2 chimeras, E2 chimeras, E3 chimeras, pyocin R, pyocin F, Enterocoliticin, AvR2-V10.3, and lactoferrin.

Statement 8. The genetically modified bacterium according to any one of statements 1 to 7, wherein the exopolysaccharide hydrolyzing enzyme is Dispersin B, or wherein the exopolysaccharide hydrolyzing enzymes are PelAh, PslGh and Alginate lyase Al-IT .

Statement 9. A method for altering het genome of a bacterium comprising introducing an oligonucleotide arrangement into a bacterium, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacterium.

Statement 10. Use of an oligonucleotide arrangement for altering the genomic sequence of a Mycoplasma bacterium, wherein the oligonucleotide arrangement comprises: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant or fragment thereof which is active in said bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant or fragment thereof which is active in said bacterium.

Statement 11. The use according to statement 10, wherein the first and/or second nucleotide sequences encode a gene product able to reduce biofilm formation, preferably a microbial biofilm.

Statement 12. An attenuated Mycoplasma bacterium according to any one of statements 1 to 8, or obtained by the method of statement 9, for use as a medicament.

Statement 13. An attenuated Mycoplasma bacterium according to statement 1 to 8, or obtained by the method of claim 9, for use in treating pneumonia, preferably for use in treating ventilator-associated pneumonia (VAP).

Statement 14. Use of an attenuated Mycoplasma bacterium according to any one of statements 1 to 15, or obtained by the method of statement 9 for avoiding biofilm formation and/or for dissolving biofilms.

Statement 15. A pharmaceutical composition comprising the genetically modified Mycoplasma bacterium according to any one of statements 1 to 8, or obtained by the method of statement 9.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects, statements, and embodiments of the invention are further supported by the following non limiting examples.

EXAMPLES

The inventors herein provide substantial experimental evidence for the findings disclosed herein and provide means to a skilled person to reproduce the findings of the current invention. By implementing state of the art techniques situated in the fields of systems biology and synthetic biology, rationally engineered M. pneumoniae strains were generated that are able to target the biofilms formed by P. aeruginosa and S. aureus. In earlier research endeavors, the secretome of M. pneumoniae has been unraveled (WO/2016135281). The putative secretion signals of eleven proteins found enriched in the medium in the secretome study were further characterized and five signals were identified that are able to promote the secretion of heterologous proteins in M. pneumoniae, a preferred example being the secretion signal of MPN142. The MPN142 N-terminal sequence was optimized in order to enhance the expression of heterologous proteins. Example 1. Method for producing modified Mycoplasma strains

The genetically modified Mycoplasma strains were generated as described in co-pending application PCT/EP2021/057122. In brief, 0.5 nmol of editing oligo’s were co-transformed with pUC57PuroSelector plasmid into a M. pneumoniae strain expressing GP35 from a constitutive promoter and Cre recombinase from the inducible Ptet promoter. A mock transformation without oligo served as control condition to monitor non-specific plasmid integration. After transformation, cells were allowed to recover for at least 3 hours in Hayflick medium at 37°C.

Cre recombinase was transiently expressed to mediate the integration of pUC57PuroSelector plasmid which allows for selection of edited clones. Therefore, the complete amount of the oligo+ plasmid co transformations was inoculated into T75 flasks containing 25 ml of Hayflick medium supplemented with 5 ng/ml of anhydrotetracycline and 3 gig/ ml puromycin. Cultures were allowed to grow in the presence of inducer for a period of time of minimum 12 hours and maximum 72 hours. Afterwards, cells were removed from the flasks in 500 mΐ of Hayflick medium, and half of this volume was spread onto Hayflick 0.8% bacto agar plates containing 3 pg/ml puromycin. Plates were incubated at 37°C and 5% C02 for a minimum period of 10 days before screening of the resulting colonies.

Two non-limiting examples of Mycoplasma chassis that were generated according to the above method that are of particular interest in the context of the present findings are the CV2 chassis and the CV8 chassis. The CV2 Mycoplasma chassis comprises functional modifications (i.e. deletions, insertions, and/or substitutions) in the MPN372 and MPN133 genes. The CV8 Mycoplasma chassis is further genetically modified to further include at least one further functional modification (i.e. deletion, insertion, and/or substitution) in MPN051. As was shown in PCT application PCT/EP2021/057122, both these chassis versions are particularly suited to function as a delivery vehicle for exogenous (e.g. therapeutic) proteins. Optionally, each of the chassis described herein may evidently comprise further genetic modifications.

As described before, a skilled person appreciates that preferably these chassis versions may be further genetically modified to provide an even further reduction of the chance for a recipient subject to develop one or more (adverse) side-effects. As an illustrative and non-limiting example, such modifications may comprise modifications to genes that are capable of eliciting immune disorders such as Guillain-Barre, preferably MPN257 and/or MPN483.

Example 2. Platform active against biofilm formation by P. aerusinosa

Dispersal and antimicrobial activities have been thoroughly characterized and combined to rationally engineer the optimal platform (i.e. combination of genes) to be inserted in the genome of a M. pneumoniae M129 wild type strain (WT, ATCC strain #29342) or in the genome of a genetically modified Mycoplasma strain CV2 (comprising at least an inactivating mutation in, or replacement of, gene MPN372 encoding CARDs toxin and gene MPN133 encoding nuclease). DifferentM pneumoniae derivate strains have been obtained and characterized for their use in degrading P. aeruginosa biofilms. An exemplary preferred optimized platform, and thus M. pneumoniae strain, has been tested in vivo in a mice model to further corroborate the activity.

The biofdm matrix that is formed by P. aeruginosa comprises 5 major components, namely: extracellular DNA, proteins and three exopolysaccharides (alginate, Pel and Psl) (Mann and Wozniak, Pseudomonas biofdm matrix composition and niche biology, FEMS Microbiology Reviews, 2012). Any activity targeting these components thus has a reasonable chance to be active against biofdms. In the following section, the targeting of each of these components has been systematically assessed.

2.1 Characterization of a preferred genetic platform and bacterial strain to degrade biofdms formed by P. aerusinosa.

2.1.1. Characterization of key enzymes for alginate degradation.

As mentioned above, a main component of biofdms is alginate; a homo- and hetero-polysaccharide that consists of b-D-mannuronate (M) and a-L-guluronate (G) units. Alginate lyases degrade this polysaccharide and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofdms. However, there are contradictory reports in the art regarding the efficacy of alginate lyases against biofdms and any synergistic effect they might have with antibiotics. From a literature study it could be concluded that several positive reports used a commercial crude extract from Flavobacterium multivorum as alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, two distinct enzymes in this extract were identified by the inventors. The first enzyme contains both polyM and polyG (polyM/G) degradation activity and has a sequence that is similar to a broad- spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The second enzyme only shows polyG degradation activity and it is similar in sequence to AlyAl from Zobellia galactanivorans . By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), it could be deducted that only enzymes with polyM/G activity such as Alg2A and Al- IG (alginate lyase from Sphingomonas sp.) are effective in dissolving biofdms (Figure 1A). Furthermore, both activities appear to be required to have a synergistic effect with antibiotics (Figure IB). Hence, as a proof of concept Al-IF was selected to be expressed in a M. pneumoniae WT strain to target alginate in P. aeruginosa biofdm (see below). It was validated that the Al-IF protein could be expressed and secreted by M. pneumoniae . A first platform was therefore generated that comprises the Al-IF gene fused to the MPN142(OPT) signal (secretion patent) in a M. pneumoniae WT strain by transposon mutagenesis, hereby obtaining the strain Myco-Alginase (WT_A). The pTnMCSlox66Cm71_EfTu_MPN142_AI-IF vector was used to insert the exogenous gene construct, which is a minitransposon vector that has the MPN142_A1-IT gene under the EFTu promoter between the inverted repeated sequences. After transforming M. pneumoniae the expression and activity of the protein was measured by UV and BSA turbidity assays (Figure 2). Additionally, the activity of the M. pneumoniae strain in dissolving P. aeruginosa POl strain biofdms was assessed by using crystal violet assay. It could be observed that the generated WT_AI-IF strain was able to degrade 20% of a 24h biofdm formed by PAOl strain after 4h of treatment (Figure 3).

2.1.2. Characterization of components suitable for degradation ofDNA present in the biofilm.

Degradation of DNA by DNase-coated nanoparticles enhances antibiotic delivery in P. aeruginosa biofilm (Baelo el al. , Disassembling bacterial extracellular matrix with DNase-coated nanoparticles to enhance antibiotic delivery in biofilm infections. J Control Release, 2015). This suggests that DNA could be an important target to promote biofilm degradation. In M. pneumoniae, two proteins with nuclease activity have been reported (Somarajan et al, Mycoplasma pneumoniae MPN133 is a cytotoxic nuclease with a glutamic acid-, lysine- and serine-rich region essential for binding and internalization but not enzymatic activity. Cell Microbiol. 2010, and Yamamoto et al, Mpn491, a secreted nuclease of M. pneumoniae, plays a critical role in evading killing by neutrophil extracellular traps. Cell Microbiol. 2017). We have performed an experiment ofDNA degradation to evaluate the intrinsic capacity ofM pneumoniae to degrade exogenous DNA (Figure 4). We show that the CV2 strain (mutant strain defective in MPN133 nuclease and the MPN372 CARDs toxin) digests DNA (Figure 4) and thus still has nuclease activity that can be inhibited by chelation of divalent cations by addition of EDTA or EGTA (Figure 4). These experiments confirm that Mycoplasma contains solvent exposed nucleases other than MPN133. The Mycoplasma strain CV4 (defective in MPN133 and MPN491 nucleases) cannot degrade DNA (Figure 5). Thus, we have characterized the two principal solvent exposed DNAses of M. pneumoniae . Mass spectroscopy analysis of WT cell supernatants in two experiments revealed that MPN491 protein is not only exposed in the membrane but also secreted to the medium. The sequences labelled in bold show the peptides of the proteins that have been detected in the supernatant sample by MS (Figure 29). Despite this DNAse activity we found that medium samples from the WT M. pneumoniae did not degrade P. aeruginosa biofilms in vitro (Figures 3 and 6). Theoretically, it could be that in vitro DNA is not the main component of the biofilm or that MPN491 released to the medium is unable to penetrate the biofilm or it is not produced in enough amounts to dissolve the biofilms.

2.1.3. Degradation of Pel and P si exopolysaccharides.

Pel and Psl play an important role in the biofilm formation and stability. Mutant strains overproducing Pel and Psl exopolysaccharide showed highly increased antibiotic tolerance (Colvin et al. , The Pel and Psl polysaccharides provide P. aeruginosa structural redundancy within the biofilm matrix. Environ Microbiol, 2012, and Goltermann and Tolker-Nielsen et al, Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates. Antimicrob Agents Chemother, 2017). Glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, have been shown to target specifically P. aeruginosa biofilms. We have assembled different platforms expressing the A1 IF alginase and/or the PelAh and PslGh glycoside hydrolases in minitransposon vectors in E. coli (Table 1).

Table 1. Vectors used to obtain different strains expressing heterologous proteins for biofilm degradation.

These vectors have been transformed in different M. pneumoniae strains to study the expression, secretion (by Western Blot and Mass Spectrometry) and activity (on biofdms and growth curves) of strains expressing: PelAh; PslGh;, PleAh_ PslGh (platform H), and PleAh_ PslGh_AI-IF. The obtained strains are depicted in Table 2. Table 2: Obtained M. pneumoniae strains.

Expression was validated by Western Blot, using an antibody that recognizes the Flag-tag added to PslGh and PslAh proteins. Both proteins were expressed in both WT and CV2 transformed strains (Figure 7). Next, relative levels of expression of heterologous proteins were determined by MS (Table 3). All recombinant proteins showed values in the supernatant higher than 10⁷.

Table 3. Values of areas for heterologous proteins expressed in different CV2 and WT strains. CL samples correspond to cell extracts and the SN samples to medium supernatant. Sample SNCV23 did not work properly in the MS due to technical issues. However, expression could be confirmed for the heterologous proteins by Western blotting. They are labeled at the beginning with CV2 and additional names (separated by scripts) indicate the heterologous proteins that are expressed in the strain. In brackets I indicate the abbreviated name and CL indicates total cell extract and SN medium of the cells that should comprise the secreted proteins. For example CV2-AiIF-PelAh-PslGh (CV25;CL) is the CV2 derived strain that expresses AI-IG, PelAh and PslGh strain (named CV25). CL is indicative for a sample derived from total cell extract.

Additionally, it could be observed that when P3 synthetic promoter is used instead than EFTU (strains expressing Lysosthapin, see below) higher expression levels were achieved, suggesting that the synthetic promoter P3 may be more efficient than EFTU. Strains combining the activities of PelAh, PslGh and alginate lyase (annotated as the “H+A” strains) showed the most outspoken biofilm degradation effect in both WT and CV2 (Figure 8). In Figure 8 A, a beneficial effect from each of the exogenous proteins can be observed. This antibiofilm activity was observed for different P. aeruginosa strains (Figure 8B). Both WT and CV showed a near identical lung infection dynamics profile in the lungs of infected mice.

Subsequently, the antimicrobial activity of the CV2 H+A engineered strain by plotting the growth curve of P. aeruginosa PAO 1 in presence or absence of the Mycoplasma strain. For this, P. aeruginosa PAO 1 strain was co-seeded with medium obtained from cell culture of WT, CV2 or CV2 H+A. The effect of the addition of the medium on P. aeruginosa growth was measured by optical density at different time points. The growth curve of P. aeruginosa PAOl incubated with the medium from CV2 H+A shows a different profile than that treated with WT or CV2 medium (Figure 9). Thus, we could conclude that the platform comprising the PelAh, PelGh and AI-IG proteins is a preferred genetic combination to be included in different Mycoplasma strains to implement the biofilm degradation activity against P. aeruginosa PAOl strain. It is evident that the platform can readily be transported to other bacterial frameworks different from that of Mycoplasma to implement functions related with activities that imply biofilm degradation of Pseudomonas or other pathogens that form biofilms that have as main components alginate, Pel and Psl exopolysaccharides.

22. Characterization of antimicrobial activity of a preferred genetic platform a d Mycoplasma strain.

A preferred bacterial framework for the genetic platform is Mycoplasma (pneumoniae). Due to the lack of a cell wall, it can be used as an efficient delivery system since it can produce antimicrobial agents that can target the cell wall of pathogenic bacteria. In addition, it can be combined with antibiotics that target the cell wall and thus, reducing their dose or rescue those that had been discarded because they cannot target pathogenic bacteria that from biofilms. We have evaluated the effect in growth curves of different strains of . aeruginosa, as well as the effect in the CV2 H+A strain in presence of antibiotics. Different doses of antibiotics commonly used in the clinic have been tested (Table 4; Figure 10). All antibiotics that affect cell wall do not kill CV2H+A strain and they would act against most . aeruginosa strains and other lung pathogenic bacteria. We have identified as optimal antibiotics and doses to be combined withM pneumoniae chassis: Piperacillin-Tazobactam (500 pg/ml); Meroprem (100 pg/ml); Imipenem-Cilastatin (300 pg/ml); Vactomycin (200 pg/ml). Some antibiotics also targeting the cell wall are not good because they do not effectively kill each P. aeruginosa strain (not broad spectra) or the effective dose may impact the growth ofM pneumoniae. Table 4. Description of different antibiotics tested in growth curves. Effect of different doses of antibiotics (in pg/ml) are represented in standard font when the strain does not grow; in bold font when the strain is growing; in underlined font when there is a delay of more than 24h in the growth. Italics: results that were not available for PA SAT2 90 strain.

Further, the hypothesis was tested whether the dose of some antibiotics used in clinics or to rescue some antibiotics that are not effective because the biofdm does not allow their action could be lowered when administered in combination with the genetic platform (e.g. CV2 H+A). First, the minimal dose that resulted in an inhibitory effect on the growth curve of P. aeruginosa was determined. For most of the antibiotics we did not observe an effect in biofdm degradation, reflecting the current problem in treatment of biofdm associated diseases that many antibiotics cannot effectively act on biofdms (Figure

11). To assess if combination of dispersal activity of CV2 H+A strain with antibiotics could enhance killing of antibiotics, we tested the combination of CV2 H+A (medium and cells) with Piperacillin- Tazobactam and Imipenem-Cilastatin in biofdm formed by the P. aeruginosa SAT290 strain (Figure

12). We tested the doses which were previously found to not be effective in biofdm degradation (Figure 11). The combination of the engineered platform (both medium and cells) had a synergistic effect with the selected antibiotics (Figure 12).

2 3 Production of antimicrobial agents bvM pneumoniae to kill P. aerusinosa.

In order to further reinforce antimicrobial activity, we tested the capacity of Mycoplasma to produce proteins that could have antimicrobial properties against P. aeruginosa. As non-limiting examples, the expression, secretion and activity of Pyocin LI (UniProtKB ID T2LG16, Genomic DNA translation CDG56231.1), Pyocin S5 (UniProtKB ID Q9I4Y4-1), LKAlgp49, and PAEM4 (NCBI Reference Sequences ERY59288.1) was assessed.

Table 5 indicates the minitransposon vectors used to obtain differentM pneumoniae strains that express candidate antimicrobial proteins with and without Flag tag. The tag is used to validate their expression by Western Blot. Version without tag is obtained to ensure that there is not any interference by the tag in the protein activity. The sequences of the genes were optimized by considering the codon usage of M. pneumoniae and using a translation model disclosed in the art (Yus etal, A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants, Nat Commun, 2017). For Pyocin_PAEM4 specifically the second Met was mutated to an lie, ensuring that there was no production of protein without secretion signal. Secretion signal of MPNG142(OPT) was used to promote protein secretion. The DNA sequence of the signal was also optimized.

Table 5. Description of vectors used to express different antimicrobial proteins.

After transformation of WT, CV2, WTH+A and CV2 H+A M. pneumoniae strains by electroporation with the different vectors described in Table 5, the expression of different proteins was validated by Western Blot (example of Pyocin LI in Figure 13). All the proteins could be expressed by M. pneumoniae. By transforming the WT H+A and CV2 H+A strains with the platform comprising PyocinLl gene we show that strains containing the dispersal platform can also express the antimicrobial agents. Thus, the genetic platform with antimicrobial activity can be combined with the genetic platform containing biofilm dispersal activity. We validated the bactericidal activity of chassis strains that express different antimicrobial proteins by doing growth curves with different strains of P. aeruginosa. We found that Pyocin LI and Pyocin S5 were killing specifically some P. aeruginosa strains (Table 6).

Table 6: Study of antimicrobial effect of different chassis strains on growth curves of different strains of P. aeruginosa.

Thus, strains comprising CV2 H+A PyocinLl are preferred strains that could act against the different strains of P. aeruginosa we have tested since a killing effect was observed in the growth curves. We also tested if such a strain was maintaining its capacity to dissolve P. aeruginosa biofdms after adding the antimicrobial activity. In addition, we measured when the strain started to produce the proteins dissolving PAOl and 13437 P. aeruginosa biofilms by treating said biofilms with medium of cells obtained at different times of the growth curve. We observed that these strains can efficiently dissolve biofilm and the amount of protein secreted into de medium after lh was able to dissolve the biofilm of strain 13437 (Figure 14). For the PAOl strain a higher amount of secreted protein was required (8h onwards, Figure 14).

24 In vivo characterization of P. aerusinosa biofilm degradation bvM pneumoniae CV2 comprising the genetic platform.

The in vivo capacity of CV2 H+A Pyocin LI to dissolve biofilms formed by P. aeruginosa PAOl strain in vivo in mice lung models was assessed. In a first experiment, male CD1 mice (n= 30, 5 mice per group) were infected intranasally with 40 pi inoculum administered into the nares under parental anaesthesia. Animals were preconditioned with Cyclophosphamide 150mg/kg SC (Day -4) & lOOmg/kg SC (Day -1). Infecting organisms were P. aeruginosa PAOl and M. pneumoniae WT VAP2 (M129 strain transformed to express the AI-IF-PelAh- PslGh and PyocinLl) and CV2 VAP2 (CV2 chassis strain modified to express AI-IF-PelAh-PslGh and PyocinLl). WT VAP2 and CV VAP2 strains ofM pneumoniae were used at intended inocula of 10⁵ and 10⁷ CFU/mouse. P. aeruginosa PAOl was infected at inocula of ~lxl0³ CFU/mouse. Animals survived to 8 hours post infection, at which point all animals had decreased in body weight but no other signs of clinical deterioration were observed in any study group. Upon euthanasia and dissection, no obvious macroscopic changes were observed in the lung tissue. WT VAP2 inoculated at both 10⁵ and 10⁷ CFU/mouse significantly reduced PAOl lung tissue burden by 0.99 Logio and 2.07 Logio respectively from PAOl controls at 8 hours. WT VAP2 inoculated at 10⁷ CFU/mouse also significantly reduced PAOl burden by 1.62 Logio from PAOl controls at 2 hours. CV2 VAP2 inoculated at 10⁷ CFU/mouse only, significantly reduced PAOl lung tissue burden by 1.83 Logio from PAOl controls at 8 hours and by 1.39 Logio from PAOl controls at 2 hours. There was no statistically significant difference between the WT and CV2 strains in their ability to reduce PAOl tissue burden in the lung (Results depicted in Table 7 and Figure 15).

Table 7. Results experiment 1.

2 5 In vitro characterization of P. aeruginosa biofilm degradation by M. pneumoniae CV8 comprising the genetic platform. We studied the antibiofilm and antimicrobial activities of M. pneumoniae strain CV8_H+A+PyoS5, by crystal violet. Briefly, biofilms from different strains of Pseudomonas aeruginosa were formed in 96-well plates, and after 24h the biofilms were treated with the supernatant from strain CV8 (as control) or CV8-H+A+PyoS5 (Figure 16A and B). After staining with crystal violet, we observed that the supernatant of CV8_H+A+PyoS5 degrades the biofilm of the all Pseudomonas aeruginosa strains tested. We also tested the capability of CV8-H+A+PyoS5 to inhibit the growth of P. aeruginosa ; we performed a growth curve of . aeruginosa strain SAT290 in presence of the supernatant of CV8_H+A+PyoS5 and found that the supernatant inhibits the strain SAT290 (Figure 16C). 2.6. Use of M. pneumoniae comprising the genetic platform in prophylactic treatment.

We also studied the efficacy of CV2_HA_P1 as a prophylactic treatment. We inoculated mice with a mix of M. pneumoniae (WT HA Pl or CV2 HA P1; doses 10⁵ and 10⁷) and PAOl, and we studied the progression of PAOl infection (Figure 17a). We observed a significant reduction of PAOl burden in the lungs of mice treated with 1 x 10⁷ CFUs of WT HA Pl or CV2 HA P1 strains at 8 hours post infection (hpi), but no significant differences in animals treated with 1 x 10⁵ CFUs, as compared to non- treated control animals (Figure 17b). SimilarM pneumoniae CFUs in the lung were recovered from all groups (Figure 17c). We monitored the clinical score of the mice inoculated with P. aeruginosa and treated or not with the CV2 HA P1 strain. In the control groups treated with PBS at 2 hpi, the clinical score increased over time from 1 that is the basal level to value of 2, whilst mice treated with CV2_HA_P1 showed the same score as pre-treatment mice (score 1; Figure 17d). All the other groups survived to 26 hpi, at which point mice showed decreased body weight but no other signs of clinical deterioration (Figure 17e). After sacrifice, macroscopic changes in the lungs were evaluated. Hemorrhagic lesions were observed exclusively in the PBS group. CV2 HA P1 CFU were not detected in the lungs of mice (except for the condition of a dose of 1 x 10⁸ at 8hpi) in presence of a PAOl- established infection (Figure 17f).

Taken together, these results demonstrate that the engineered M. pneumoniae strain CV2_HA_P1 is efficient in treating acute PAOl infections in a mouse model.

2 7 Methodology of the assays mentioned in Example 2

2.7.1. Measurements of activity by UV assay.

Activity of the alginate lyase was determined by the increase in absorbance at 235 nm due to the formation of a carbon-carbon double bond at the end of the product generated from lyase-mediated cleavage of alginate. Three different substrates were tested: brown seaweed alginate (Sigma# W201502 A straight-chain, hydrophilic, colloidal, polyuronic acid composed of glucuronic and mannuronic acid residues), EUICITYU # DP25-DP45 Guluronate oligosaccharides (polyG) and EUICITYU # DP20- DP35 Mannuronate oligosaccharides (polyM). The substrates were dissolved in a solution of 20% glycerol and 20mM Tris pH 7.4 so as to reach a final alginate concentration of 0.2%. Then, 50 pL of these substrates were added to the wells containing 7.1 pmol of each enzyme. As positive control we used 10 pmols of Sigma A1603 alginase. Absorbance was measured at 235 nm every 2 minutes for 78 minutes using UV-star microplates, 96wells, (Greiner #655801).

2. 7.2. Turbidity assay to measure the activity

To measure alginate lyase activity we used the assay developed by Kitamikado (et al., 1990). Briefly, 0.1% of brown seaweed substrate is added to the sample containing the alginate lyase. At various time points 0.2 ml of media supernatant is added in a test tube and 2.0 ml of an acidic albumin solution (3.26 g sodium acetate, 4.56 ml of glacial acetic acid, 1.0 g of bovine albumin fraction V were filled up to 11 with water and pH adjusted to 3.75 with HC1). In the presence of polymeric alginate, a white precipitate is formed. A small aliquot of the mixture is then transferred to a plate and the absorbance is measured. We tested different wavelengths for the signal to noise ratio and found 300 nm to be the most sensitive, while everything up to 660 nm gave good reliable readings.

2. 7.3. Determination of activity of alginase expressed by Mycoplasma strains

Grow T75 flask for 3 days with 25mL of HF media w/o antibiotics. Supernatant media was frozen and cells scrapped in 3mL of HF. Test 4 samples (each in triplicate A,B,C): HF only, SN, + control (0.000 lmg/ml alginase in HF), and Myco cells. 52.6ul of 2% alginate (make in water and mixed at 60oC) is added to 1ml of each sample to have a final alginate concentration of 0.1%. Samples are mixed and at each time point (T=0, 5, and 24h) 200ul are taken for each tube and added to 1.8mL acidic BSA solution ( For 1L: 3.26g sodium acetate, 4.56mL acetic acid, 1 g BSA , pH to 3.75 with HC1), mixed and lOOul taken to measure OD at 300nm.

2. 7.4. Crystal violet assay to characterize biofilm degradation

Three days before starting the experiment, M. pneumoniae strains are grown in a T25cm2 flaks at 37°C, 5% C02 with 5 ml of Hiflick medium without ampicillin. Medium of Mycoplasma cells is recovered after 3 days and used for the measurements to study the activity of secreted protein. Also, cells are recovered by scraping them in 1ml of Hiflick medium without ampicillin.

First day of experiment to inoculate different P. aeruginosa strains in erlenmeyer flasks (20 pi stock in 20 ml TSB). Incubate at 37°C shaking overnight. Pseudomonas strains tested in those experiments were PAOl, Pseudomonas aeruginosa (Schroeter) Migula (ATCC® 27853™) Boston 41501, Pseudomonas aeruginosa NCTC 13437, Pseudomonas aeruginosa (Schroeter) Migula (ATCC® BAA-2113™); PA SAT290 (Hospital Clinic), PAO GFP (Hospital Clinic), PA Cl 17 (Hospital Clinic), PAOl Pel, PAOl PsI, PA CHA, PDO300.

After 24h, the cultures were diluted to an OD600 of 0.1 in TSB (aprox. dilute in 1:40 in TSB). Then, 100 mΐ of diluted culture were added to sterile 96-well polystyrene microtiter plates . Cells were incubated statically overnight at 25 °C to allow for biofilm formation. The next day, the biofilms were washed with PBS to remove non-adherent cells and TSB media. Then, treatments were added to the wells (50-100 mΐ of medium). Also, A1-IG (218 pg/ml) or PelAh (250 pg/ml) +PslGh (70 pg/ml) recombinant proteins were added as controls.

After incubation of 5 hr at 37°C, the plates were washed with PBS and the wells were stained with 150 mΐ of 0. l%(w/v) crystal violet. After 10 min of incubation, the wells were washed with PBS and the dye was solubilized by addition of 100 mΐ of 95%(v/v) ethanol and incubated for 10 min. Then, we measured absorbance at 595 nm using a TECAN. If the values get saturated, dilute the samples 1 to 10 in 95% EtOH in a separate plate, and read again.

2. 7.5. DNA degradation assay

To check if M. pneumoniae has endonuclease activity we incubated the medium of CV2 cells with plasmid or PCR amplified DNAs. After growing CV2 cells for 90h at 37°C, medium of cells was recovered and passed through a 0,22 pm filter. Then, 5 mΐ of medium was incubated with 800ng of plasmid DNA (pMTnCmLox vector) or 800ng of a DNA amplified PCR fragments at 37°C at two different time points (15min and 1 h). Degradation of DNA was evaluated by running in 1% agarose gel.

Example 3. Platform active against biofilm formation by Staphylococcus aureus.

A second genetic platform was engineered to express and secrete different enzymes that target the matrix of Staphylococcus aureus biofilms. The activity of different combinations of secreting enzymes was compared by using purified recombinant proteins in biofilms formed in vitro, ex vivo and in vivo. Herein, two different applications of this delivery system to dissolve S. aureus biofilms were explored: 1) elimination of biofilms in catheters, and 2) treatment of mastitis. We provide the first proof of concept that an engineered lung bacteria can be used to dissolve biofilms in a more efficient manner when compared to recombinant proteins. This genetic platform, and by extension the resulting bacterial framework could be used as a delivery system to treat amongst others human lung diseases, hereby opening new perspectives in synthetic biology applications.

3 1 Mycoplasma pneumoniae derived strains that express proteins to dissolve S. aureus biofilm.

There are different biomolecules described that attack or prevent biofilm formation. One of these is Dispersin B, which is a glycosyl-hydrolase that is able to break linear polymers of N-acetyl-D- glucosamine present in most common S. aureus biofilm matrices. Weakening the biofilm offers the opportunity for using bacteriolytic agents like antibiotics, or antimicrobial enzymes that attack the cell wall (e.g. Lysostaphin). Hence, a bacterium devoid of cell wall like M. pneumoniae that could express biofilm dispersing agents and bacteriolytic enzymes could be seen as a perfect therapeutic agent against S. aureus biofilms. Therefore, Dispersin B can be regarded as an interesting exogenous protein to express in genetically modified Mycoplasma bacteria and test for antibiofilm activity.

Mycoplasma pneumoniae wild type (WT) and chassis (CV2) strains were transformed by electroporation with different vectors to obtain the different strains described in Table 8. Table 8. Vectors used to transform Mycoplasma and annotation of the resulting strains.

3 2 Implementation of biofilm degradation activity: Dispersion of biofilm by WT EfTuD and CV2 EfTuD.

Both WT_EfTuD and CV2_EfTuD strains are obtained after transformation of M. pneumoniae M129 and CV2 strains with the vector Tnlox66CmLox71-Eftu-142(OPT)-DispersinB, respectively. First we studied the capacity of WT and WT EFTuD strains to dissolve biofilms (12 h; Figure 18) and mature (24 h; Figure 19) biofilms formed by Staphylococcus aureus. Degradation of biofilm by was studied at different time points (15 min, 4 h, 8 h, 12 h and 24 h) after treatment with different samples. The samples were: the medium of the culture recovered after growing Mycoplasma for 3 days (SN) and cells (Cells; two different doses 10⁸ and 10⁴ CFUs). It could be observed that Dispersin B protein secreted by WT EfTuD cells in the medium (SN WT EfTuD sample) is able to dissolve biofilms after 15 minutes treatment. However, since there are not Mycoplasma cells producing continuously the Dispersin B protein at 4 h we observe that the biofilm starts to form again. When WT EfTuD cells are used the biofilm also starts to be dissolved after 15 min treatment, but less efficiently than in the SN sample. This is probably due because in this case after 15 min the amount of protein secreted by the cells is lower than then amount of Dispersin B present in the medium of WT EfTuD cells grown for 3 days. However, we found that after 4 hours the sample with the Mycoplasma cells showed more degradation of biofilm than the SN sample and the biofilm is not formed. The same effect was observed for both, young and mature biofilms. These results suggest that continuous production of Dispersin B protein by WT EfTuD strain could be more efficient than using Dispersin B recombinant protein (present in the SN sample) to dissolve biofilms formed by S. aureus. We further showed that the M. pneumoniae chassis CV2_EfTuD strain (missing the MPN372 and MPN133 pathogenic genes and expressing constitutively the Dispersin B protein) is also able to degrade biofilms formed in vitro in similar levels than the WT EfTuD strain (Figure 20 and 21 C). 3.3. Evaluation of degradation of a S. aureus biofilm formed in a catheter (in vitro . ex vivo . and in vivo) by a genetically Mycoplasma strain expressing EfTuD.

In ventilator associated pneumonia (VAP), biofilms formed by S. aureus and P. aeruginosa are the main reason for a lack of responsiveness to antibiotics. To asses if we can target biofdms formed in a catheter, first, we inoculated a catheter with S. aureus and after biofilm formation (12 h) and we studied the degradation by the two strains (Figure 21A and B). We showed that both strains are able to degrade a biofilm formed in vitro in a catheter. Models of subcutaneous implantation of catheters in mice are known in the art (e.g. Garrido etal., In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose-MicroPET in a mouse model, Antimicrob Agents Chemother, 2014). In this model, the S. aureus biofilms can be formed in vivo in the animal then extracted and exposed to the treatment (ex vivo model) or do the treatment in the regions where the catheter is implanted (in vivo assay). By using these ex vivo and in vivo model, we studied the capacity of WT_EfTuD and CV2_EfTuD strains to dissolve S. aureus biofilms formed in the catheter (Figure 2 ID). Once the biofilm was formed in vivo in the catheter implanted in the animals, the catheter was extracted and exposed to the different treatments. In these experiments, WT EfTuD and CV2_EfTuD strains could degrade the biofilm formed in the catheter ex vivo (Figure 2 ID, Figure 22).

Finally, we studied the degradation of biofilm formed in vivo in the mice by treating directly the animals by [¹⁸F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node (Figures 22 and 25). Whilst a significant decrease in SUV60 signal was observed in mice treated with WT EfTuD strain, no decrease was observed in animals treated with CV2_EfTuD strain. This suggests that one of the virulence factors depleted in the CV2 could be necessary to activate or attract the immune response that could be together with Dispersin B help to eliminate the S. aureus biofilm. To validate this hypothesis we did a mixture of CV2_EfTuD and WT strains and we use it as treatment. WT cells alone could not degrade the biofilms, and neither could the CV2_EfTuD cells used in isolation. However, the combination of both strains was decreasing significantly the SUV60 signal. Thus, confirming the hypothesis that one of the factors removed in the CV2 was triggering a response that was necessary to see the effect in vivo.

3.4 Addition of additional antimicrobial activity by further engineering CV2-efTuD.

Despite the observation that the WT EfTuD strain was effective, it is not a preferred framework for a therapeutic product since this strain may cause lesions in the lung and provoke an inflammatory response. Therefore, it was decided to add other functions or activity to the dispersal platform (CV2- efTuD). By further engineering the CV2_EfTuD strain and including a gene with antimicrobial properties we could enhance or complement its capacity to dissolve S. aureus biofilm in vivo. First, we characterized the capacity of Mycoplasma to produce different antimicrobial agents and studied the efficacy of each one. Suitable proteins that may be expressed and are active against S. aureus include but are not limited to Defensins (described in detail in Jarczak ei al, Defensins: natural component of human innate immunity, Hum Immunol, 2013), LysK (described in Fujita et al, Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage, Pharmaceuticals (Basel), 2018), LysAB (described in Peng et al, Highly potent antimicrobial modified peptides derived from the Acinetobacter baumannii phage endolysin LysAB2, Sci Rep, 2017), LysAB2_SH3, CHAPKl, CHAPK2, and CHAPK3 (described in Hosseini et al, Purification of Antibacterial CHAPK Protein Using a Self-Cleaving Fusion Tag and Its Activity Against Methicillin-Resistant Staphylococcus aureus, Probiotics Antimicrob Proteins. 2016), and Lysostaphin (Bastos et al, Lysostaphin: A Staphylococcal Bacteriolysin with Potential Clinical Applications, Pharmaceuticals (Basel), 2010). As a non-limiting example, representative experiments for LysAB2_SH3 and Lysostaphin are shown in the next paragraphs.

3.4.1. LysAB 2 SH 3

Preliminary generated data showed that LyasAB2 is not optimal to treat S. aureus infections. However, we thought to engineer the LysAB2 as chimeric protein (LysAB2_SH3b). This chimeric protein was a fusion of LysAB2 gene with SH3b domain to promote activity of LysAB2 protein by enhancing interaction or binding of the protein to S. aureus. First, we expressed recombinant the protein cloned in pETM14 in E. coli. We purified it and tests its activity on S. aureus growth curves (Figure 23). We observed that when adding the LysAB2_SH3b protein to S. aureus cells there was an inhibition of cell growth. Thus, this protein is deemed to be a good candidate to be expressed and secreted by a bacterial framework obtained by introducing the genetic platform such as the CV2_EfTuD strain.

3.4.2. Lysostaphin

We designed and obtained seven different strains of M. pneumoniae WT transformed with different minitransposon vectors where the lysostaphin gene was under the expression of different engineered synthetic promoters based on knowledge of promoters from the work of Jae Song et al. (A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants, Nat Commun, 2017). Vectors are described in Table 8. Levels of expression and secretion by different strains was estimated by measuring the effect in the growth curve of S. aureus (Figure 24). The promoter P3 was selected as the one that was producing higher levels of Lysostaphin protein.

3 5 Generation of a strain combining both dispersal activity and antimicrobial activity.

In order to assess whether combining dispersin and lysostaphin in a single bacterial framework would further reduce S. aureus biofilm formation, strains were generated expressing both genes: EfTu-DispB- EFtu lyso (obtained by transformation of a minitransposon that expresses both genes), and EfTu-DispB- EfTu-Lyso (Obtained by transforming a strain with a minitransposon TnCmLox-Eftu-MPN142- Dispersin(no ATG)-GG-TEV-GG-Lysostaphin in Table 8 that allows expression of a fusion chimeric protein (Dispersin B and Lysostaphin genes fused and separated by a linker sequence that includes de TEV protease cleavage site with two GG flanking amino acid residues). We have measured dispersal and antimicrobial activities in the different strains. We have found that having the two genes expressed independently is better for degradation of biofdm and antimicrobial activities (Figure 26 and 27). The strain transformed with the two genes Dispersin B and Lysostaphin under EfTU promoter (Myco EfTu- DispB-EfTu-Lyso strain in Figure 26) is the one that works better to dissolve .S' aureus biofdms. Also when measuring antimicrobial activity on growth curves we found that this strain was avoiding the growth of S. aureus for the 26 h of the experiment. In the case of strain expressing the chimeric protein after 12 h treatment S. aureus starts to grow, suggesting that this protein is not so active or the amount is not enough to have the same effect than in strain expressing Lysostaphin and Dispersin B as independent proteins. However it cannot discarded that by adding two copies of a chimeric molecule we could increase the levels of expression and maybe the activity of the enzyme is better (each molecule of the chimera has the two functions integrated).

Finally, we obtained a platform that was expressing the Dispersin B under the EfTu promoter expression and the Lysostaphin under the P3 promoter. We transformed the WT (WT_EfTuD_P3L ) and CV2 strains (CV2_EfTuD_P3L) with this platform. After validation biofilm degradation activity and antimicrobial activities of the strains WT_EfTuD_P3L and CV2_EfTuD_P3L we studied the effectiveness of the CV2_EfTUD_P3L strain in the in vivo biofilm degradation assay (Figure 28). We found that addition the complementation of CV2DB with Lysostaphin activity was promoting the degradation of the biofilm in vivo in the attenuated background of the chassis. Thus, we could obtain a chassis strain without main virulence factors that is able to degrade a biofilm formed in vivo in the catheter (Figure 28).

3 6 Methodology of the assays mentioned in Example 3

3.6.1 Biofilm degradation assay

Staphylococcus aureus cells were spread on afar plate incubated over night at 37°C. The next day a colony was recovered and grown over night in TSB-glu medium at 37°C on agitation. After 24h growth, a 1:40 dilution in TSB-glu medium was performed and 100 pi of cells were added into 96-well plate wells. To obtain a mature biofilm, the plate was incubated for 24h at 37°C. In the morning remove the TBS-glu from wells wash with Hayflick medium and add the different treatments ( Mycoplasma strains or recombinant proteins, see below). After incubation for 6h, remove the supernatants and wash with water. Then, the wells were stained with crystal violet for 15 minutes. After three washes with water, wells were dried and the stain was solubilize by adding 200 mΐ 80/20 ethanol/acetone. Then 100 mΐ of each sample were transferred to a new plate and the absorbance was measured at OD595. To prepare samples of different Mycoplasma strains. After growing Mycoplasma cells in T75 cm² flasks with 25 ml of Hayflick medium at 37°C with 5% CO2 for 3 days, attached cells were recovered in 1 ml of medium and stock were kept at -80°C in aliquots of 100 pi. Then, the day before the biofilm degradation assay, Mycoplasma strains were grown overnight in T75 flasks with 25 ml of Hiflick medium without ampicillin, at 37°C with 5% CO2. In this condition, cells are not expected to divide but they become metabolically active. Then, cells were scraped in 1 ml of medium, desegregated by passing them ten times through a an insulin syringe, and 10 mΐ were spread on agar plates to count the amount of cells present in the sample that is used for the assy. In the experiment we used 100 mΐ of cells approximately between 10⁸- 10⁹ CFU/ml which is a total of 10⁷-10⁸ cells per well. When recombinant protein was used in the assay, the final concentration of protein was 0.25 pg/pl.

3.6.2. Induction of defensin expression in Myco strains with tet system

Inoculate all strains of Myco-defmsins: 10 mΐ into 5 ml HF with AMP +PURO +TET (2 Ox more diluted than the usually lOOOx dilution). Inoculate one flask with tetracycline and another one the same but without tetracycline. Also as a control inoculated WT Mycoplasma with and without tetracycline. Cells were grown at 37°C over the weekend. Since, induction was not affecting growth, protein extraction with urea was performed to study protein expression by MS. The positive control was a sample resulting of mixing 10 pg of different recombinant proteins (DEFB103A, DEFA2, DEFA5, DEFB1, and DEFB4A). To assess expression by different Mycoplasma strains we mixed the protein extracts of different strains in pairs (30 pg of each total protein extract). The protocol for protein extraction from cells was the following: 1) After 49-50 h of growth (exponential phase), wash twice with PBS IX; 2) Scrape with 200 pi ofPBS; 3) Centrifuge for 10 min at 14100 g; 4) aspirate PBS 5) Resuspend the pellet with 75 pi 7 M Urea + 0.2 M NH4HCO3 (it should be prepared freshly and kept on ice). 6) Sonicate twice for 5 min with an interval ON/OFF of 30 sec. Add enough ice before sonicating and in between sonications to avoid heating the samples. Always keep the samples on ice; 7) Centrifuge in a cooled desktop at 4C, at 16000 g for 5 min; 8) Take the supernatant for further processes in a new 1.5 ml tube; 9) Measure protein concentration with BCA (compatible with 3M urea); 10) Dilute the samples for BCA 10 fold in 0.2 M NH4HCO3 so that the urea concentration is 0,7M. Standards of BSA are also diluted in 0.7 M urea buffer.

3.6.3. Bacterial strains and culture conditions

S. aureus 15981, a human clinical otitis isolate forms a highly adherent hyperbiofilm with an ica- dependent PIA/PNAG polysaccharidic matrix (Valle etal, SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus, Mol Microbiol, 2003), as previously characterized by PET studies (Garrido el al. , In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [18F]fluoro-deoxyglucose-MicroPET in a mouse model, Antimicrob Agents Chemother, 2014). S. aureus bacteria were cultured (37°C, 18 h) in tryptone soy agar (TSA; Laboratories Conda, Spain) or tryptone soy broth (TSB; Laboratories Conda, Spain) supplemented with glucose (0.25%, wt/vol) (TSA- glc and TSB-glc, respectively). The bacterial concentration was spectrophotometrically adjusted, and by dilution in TSB-glc, to lx lO⁶ bacteria/ml for catheter infection. Exact doses (CFU/ml) were retrospectively assessed by serial 10-fold dilutions in phosphate buffer saline (PBS; pH 7.4), by plating (37°C, 18 h) 100 mΐ in triplicate (limit of detection, 3.3 CFU/ml) in TSA and incubation (37°C, 18 h).

3.6.4 In vitro biofilms

Overnight culture of S. aureus in TSB was grown at 37°C 300 rpm, then diluted 1 :40 in fresh TSB and 100 pF was used for coating the wells of polystyrene well plates (Thermofisher) and then incubated at static conditions at 37°C 24h. Then, cells were washed with Hayflick-Amp and treated with 100 pi of fresh cultures of Mycoplasma (lxlO⁹ CFU/ml; lx 10⁸/well) at different point times (15 min, 4h, 8h, 12h, 24h) at 37°C. Then, wells were stained with crystal violet 15 min at room temperature, washed and air dried. The crystal violet biofilm was quantified solubilizing the dye with 100 mΐ ethanol-acetone (80:20, vol/vol) and determining the absorbance at 595 nm (OD 595).

3.6.5. Mouse model ofS. aureus sealed-catheter infection

Female CD lmice (Charles River International) of 20- to 22-g body weight were accommodated in the animal facilities of the Universidad Phblica de Navarra (UPNA; registration code ES/31-2016-000002- CR-SU-US), with water and food ad libitum. Mouse handling and procedures were performed in compliance with the current European and national regulations, following the FEFASA and ARRIVE welfare guidelines, and with the supervision of the UPNA’s Comite de Erica, experimentacion Animal y Bioseguridad (CEEAB) and approval by the competent authority (Gobiemo de Navarra).

Implants were prepared as previously reported (Garrido el al. , In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [¹⁸F]fluoro-deoxyglucose-MicroPET in a mouse model, Antimicrob Agents Chemother, 2014), commercial Vialon 18G 1.3- by 30-mm catheters (Becton-Dickinson) were cut into 20-mm segments and sealed under sterile conditions with petrolatum and tissular glue (Vetbond; 3M Espana S.A.). Cleaning and disinfection were achieved thereafter by immersion in DD445 (A&B Laboratories de Biotecnologia) and ethanol (15 min in each solvent). Sterility was checked by incubation (37°C, 24 h) in TSB. Then, reliable S. aureus catheter precolonization was successfully achieved by incubation (37°C, 4 h) in 1 ml TSB-glc containing 1 x 106 CFU, as previously reported (Kadurugamuwa el al, Direct continuous method for monitoring biofilm infection in a mouse model, Infect Immun, 2003). The number of bacteria adhered to implants prior to infection was systematically assessed. Finally, catheters were rinsed with fresh TSB-glc and immediately implanted subcutaneously through a minimal surgical incision in the interscapular area of mice, previously anesthetized by intraperitoneal administration of ketamine (100 mg/kg of body weight; Imalgene; Merial Laboratories, S.A.) and xylacine (10 mg/kg; Rompun; Bayer Health Care).

3.6.6. In vivo biofilm formation and ex-vivo treatment

After 18 h of in vivo biofilm formation (detailed above), animals were euthanized and catheters obtained. Rinsed in PBS and treated with suspensions of 1 ml of lxl 0⁸ CFU of the Mycoplasma treatment (WT, WTDB, CV2, CV2DB or a Mix WT+CV2DB) and incubated at 37°C for 4h. Bacterial adhesion was quantified by crystal violet staining, rinsed with water and air dried. Crystal violet stained biofilm was quantified solubilizing the dye with 500 pi ethanol-acetone (80:20, vol/vol) and determining the absorbance at 595 nm (OD 595).

3.6.7. In vivo treatment of mice carrying catheters

Suspensions of Mycoplasma spp. strains (100 mΐ) were cultured in T75 flask containing 25 ml of Hayflick-Amp (100 mg/ml) and grown at 37°C, 5% CO2. When the broth turned orange (3 or 4 days) the supernatant was decanted, and 10 ml of PBS lx was added to the flask and discarded. A cell scraper was used to harvest the adherent Mycoplasmas for the bottom of the flask with 10 ml of PBS lx. Cells were disaggregated passing by syringe (25G, Novico) 5 times. Then, ten-fold dilutions were carried to a 109 CFU/ml and retrospectively assessed by plating in Hayflick-Amp (12 days, 37°C, 5% CO2). A volume of 100 mΐ of a suspension that contains 10⁹ CFU/ml were inoculated (dose 10⁸ /mouse) subcutaneously in the surrounding area of the catheter. The treatments tested were WT, WTDB, WTDBLys, CV2, CV2DB, a mix that contained WT plus CV2DB (n= 4 to 5 animals per group). A group non-treated group (PBS) was included (n= 7).

3.6.8. [¹⁸F]FDG-MicroPET in vivo monitoring ofS. aureus biofilm detachment

Sealed catheters precolonized with S. aureus 15981 strain was implanted subcutaneously in a total of 32 mice, as described above. Following catheter implantation and treatment, the infection was longitudinally evaluated in vivo by [¹⁸F]FDG-MicroPET on days 1 post-treatment and 4 post-treatment. The uptake of radiotracer and PET images were performed as previously reported (Garrido el al. , In vivo monitoring of Staphylococcus aureus biofilm infections and antimicrobial therapy by [¹⁸F]fluoro- deoxyglucose-MicroPET in a mouse model, Antimicrob Agents Chemother, 2014). Briefly, fasted mice were anesthetized with 2% isoflurane in O2 gas and intravenously injected with 18.8 1.9 MBq of [¹⁸F]FDG. Following 1 h of radiotracer uptake under continuous anaesthesia inhalation, PET imaging was performed in a small-animal tomograph (MicroPET; Mosaic; Philips, USA) by laying mice in prone position and capturing images for 15 min. Images were reconstructed using the three-dimensional (3D) Ramla algorithm (a true 3D reconstruction) with 2 iterations and a relaxation parameter of 0.024 into a 128 by 128 matrix with a 1-mm voxel size, applying dead time, decay, random, and scattering corrections. For [¹⁸F]FDG uptake assessment, MicroPET images were analysed using the PMOD software (PMOD Technologies Ltd., Adliswil, Switzerland), and semiquantitative results were expressed as the standardized uptake value (SUV) index, obtained by normalization with the formula SUV = [(RTA/cm3)/RID] ^c BW, where RTA is the radiotracer tissue activity (in becquerels), RID is the radiotracer injected dose (in Bq), and BW is the mouse body weight (in grams). After qualitative inspection of the images, volumes of interest (VOI) were manually drawn on coronal 1 -mm -thick consecutive slices including the entire catheter area. For catheter image quantification, to avoid manual bias of surrounding areas, a new VOI was generated semiautomatically using the threshold of 60% of maximum pixel for SUV mean calculation (SUV60 index). The results were expressed as the SUV60 index increase, calculated as follows: ((SUV60 at day 4 * 100)/SUV60 at day 1)-100. The herein disclosed sequences are available in accompanying Figure 29 and the sequence listing.

4. Funding

The projects leading to this application have received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013), the European Union's Horizon 2020 research and innovation programme, and the European Research Council (ERC) under grant agreements No 232913 (CellDoctor), No 335010 (Mico pLung), No 670216 (MycoChassis), and No 825566 (MycoVAP).

Claims

1. A genetically modified Mycoplasma bacterium comprising in its genome an oligonucleotide arrangement, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium.

2. The genetically modified Mycoplasma bacterium according to claim 1, further comprising in its genome an inactivating mutation, deletion, and/or substitution in at least one gene selected from the group consisting of: MPN051, MPN133, MPN142, MPN257, MPN294, MPN372, MPN400, MPN415, MPN453, MPN483, MPN491, MPN592, and MPN626.

3. The genetically modified Mycoplasma bacterium according to claim 2, comprising in its genome at least an inactivating mutation, deletion, and/or substitution in at least the MPN372 gene encoding CARDs toxin and the MPN133 gene encoding nuclease.

4. The genetically modified Mycoplasma bacterium according to claim 2 or 3, comprising in its genome at least an inactivating mutation, deletion, and/or substitution in at least the MPN051 gene encoding glycerol-3 -phospate dehydrogenase.

5. The genetically modified Mycoplasma bacterium according to any of claims 2 to 4, which is attenuated.

6. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 5, wherein said oligonucleotide arrangement comprises at least one further nucleotide sequence encoding one or more heterologous proteins, preferably one or more DNA degrading enzymes and/or heterologous proteinases under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said bacteria.

7. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 6, wherein the one or more nucleotide encoded heterologous exopolysaccharide hydrolyzing enzymes, the one or more nucleotide encoded heterologous antimicrobial proteins and/or the one or more nucleotide encoded heterologous DNA degrading enzymes are each under the control of the same or a different promoter or a functional variant of said promoter(s) or fragment thereof.

8. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 7, wherein at least one oligonucleotide sequence comprises a constitutive promoter, preferably a promoter with a sequence selected from the group of sequences comprising : P438 (SEQ ID NO: 4), EfTu (SEQ ID NO: 5), PI (SEQ ID NO: 6), P2 (SEQ ID NO: 7), P3 (SEQ ID NO: 8), P4 (SEQ ID NO: 9), P5 (SEQ ID NO: 10), and Psyn (SEQ ID NO: 11).

9. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 8, wherein at least one nucleotide sequence comprises a synthetic promoter with a nucleotide sequence of at least 65% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 8.

10. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 9, wherein the exopolysaccharide hydrolyzing enzyme is selected from the group LysK (CHAP-AMID)- Lyso(PEP), endolysin LysH5, HydH5 (HydH5SH3b and HydH5Lyso), Cpl-1 lysozyme, Cpl-7 lysozyme, Pal amidase, PL3 Amidase, MV-L lysin, PlySs2, Major autolysin (Atl) of Staphylococcus aureus, CF-301, N-Rephasin, P128, Art-175, gp49, LysK (CHAP 1 -AMID-SH3), , LysAB-SH3, SAP-1 SAL-1, P128, LysGH15/GH15, CF-301, ClyF, PaVDpl, Cpi-l/CP-1, LytA, Cpi-7/Cp-7, Cpi-7S, Cpl- 711, PL3, PlyPy, PlyC/Ct, Lys8/Bxz2, LysA/BTCU-1, LysBIBTCU-1, Lysl521/8. amyloliquefaciens phage, El 188/EL, KZ144, OBPgp279, LysPA26, LysAB2, LysABP-01, PlyABl, PlyF307, LysAB3, LysAB4, Lysep3, Lysep3, Colicin-lysep3, EndoT51T5, PlyE146, Kl lgp3.5, KP32gpl5, KP27 lysin, CfPl lysin, P28, AP3gpl5, lysB4, LysBPS13, Plyl2, Ply21, PlyBa, PlyG, PlyB, Phage APSOc lysine, PlyBT33, Ply PH, Plyl, AmiBA2446, Alginase Al-II, Alginase Al-IG, Alginase Al-III, Alginase ProtA, a-amylase, Dispersin B, a-mannosidase, b-mannosidase, cellulase, hyaluronidase, PelAh, and PslGh.

11. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 10, wherein the antimicrobial protein is selected from the group comprising defensins, pyrrhocoricin, GramicidinA, IL37, Magainin, SMA2P9, CAP18, bacteriocinE50-5, Peptide LL-37, 1018, 1037, 17BIPHE2, Bac8c, Battacin, BMAP-27, BMAP-28, CAMA, DJK-5, DJK-6, GF-17, LL-31, LL7-31, LL7-37, Melittin, P10, P60.4Ac, SMAP-29, Lyostaphin, pyocin Sn, pyocin SI, pyocin S2, pyocin S3, pyocin AP41, pyocin S5, pyocin S2, pyocin S3C, pyocin S6, pyocin S8, pyocin SD1, pyocin S13, pyocin SD2, pyocin SD3, pyocin SA189, pyocin LI, pyocin L2, pyocin L3, pyocin Ml, pyocin M4, pyocin PAEM4, pyocin PAEM, pyocin LI, putidacin LI, pyocin Rl, pyocin H108 (8-type), pyocin 1577, colicin R, colicin N, colicin M, colicin D, colicin El, colicin E3, and colicin E9, haemocin A, CLB pesticin, tailocin chimeras, S 1 chimeras, S2 chimeras, E2 chimeras, E3 chimeras, pyocin R, pyocin F, Enterocoliticin, AvR2-V10.3, and lactoferrin.

12. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 11, wherein the exopolysaccharide hydrolyzing enzyme is Dispersin B, or wherein the exopolysaccharide hydrolyzing enzymes are PelAh, PslGh and Alginate lyase A1-IG.

13. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 12, wherein the exopolysaccharide hydrolyzing enzymes are PelAh, PslGh and Alginate lyase A1-IG and wherein the antimicrobial protein is pyocin LI.

14. The genetically modified Mycoplasma bacterium according to any one of claims 1 to 12, wherein the exopolysaccharide hydrolyzing enzyme is Dispersin B and wherein the antimicrobial protein is Lysostaphin.

15. A method for altering het genome of a Mycoplasma bacterium comprising introducing an oligonucleotide arrangement into the genome a Mycoplasma bacterium, said oligonucleotide arrangement comprising: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant of said promoter or fragment thereof which is active in said Mycoplasma bacterium.

16. The method according to claim 15, wherein the bacterium is a Mycoplasma pneumonia bacterium.

17. Use of an oligonucleotide arrangement for altering the genomic sequence of a. Mycoplasma bacterium, wherein the oligonucleotide arrangement comprises: i) a first nucleotide sequence encoding one or more heterologous exopolysaccharide hydrolyzing enzymes under the control of a promoter or a functional variant or fragment thereof which is active in said Mycoplasma bacterium; and ii) a second nucleotide sequence encoding one or more heterologous antimicrobial proteins under the control of a promoter or a functional variant or fragment thereof which is active in said Mycoplasma bacterium.

18. The use according to claim 17, wherein the first and/or second nucleotide sequences encode a gene product able to reduce biofilm formation, preferably a microbial biofilm.

19. The use according to claim 18 wherein said biofilm is formed in the respiratory system of said subject.

20. The use according to any one of claims 17 to 19, wherein said biofilm comprises hexosamine- containing polymers (PI A).

21. The use according to any one of claims 17 to 19, wherein said biofilm comprises Pel and/or Psl and/or alginate exopolysaccharides.

22. The use according to any one of claims 17 to 19 and 21, wherein said biofdm is produced by a group of bacteria comprising Pseudomonas aeruginosa, preferably wherein said biofdm is produced by Pseudomonas aeruginosa.

23. The use according to any one of claims 17 to 20, wherein said biofdm is produced by a group of bacteria comprising Staphylococcus aureus, preferably wherein said biofdm is produced by Staphylococcus aureus.

24. A pharmaceutical composition comprising the genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16.

25. The pharmaceutical composition of claim 24, wherein said pharmaceutical composition further comprises an antibiotic, preferably wherein said antibiotic is selected from the group comprising: Piperacillin, Tazobactam, Ciprofloxacin, Levofloxacin, Meropenem, Imipenem, Cilastatin, Amikacin, Ceftazidime, Avibactam, Ceftolozane, Ceftriaxone, Vancomycin, Linezobd, or any combination thereof.

26. A genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25 for use as a medicament.

27. A genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25, for use in treating pneumonia.

28. A genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25, for use in treating pneumonia, preferably for use in treating ventilator-associated pneumonia (VAP).

29. A genetically modified Mycoplasma bacterium according to claim 13, or obtained by the method of claim 14 or 15, or a pharmaceutical composition according to claim 24 or 25, for use in dissolving microbial biofilms produced by Pseudomonas aeruginosa.

30. A genetically modified Mycoplasma bacterium according to claim 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25, for use in dissolving microbial biofilms produced by Staphylococcus aureus.

31. Use of a genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25, for avoiding biofdm formation and/or for dissolving biofilms.

32. Use of a genetically modified Mycoplasma bacterium according to any one of claims 1 to 14, or obtained by the method of claim 15 or 16, or a pharmaceutical composition according to claim 24 or 25, for the manufacture of a medicament for the prevention or treatment of a pathogenic biofilm.

33. The use according to any one claims 17 to 23 or claims 31 or 21, wherein the genetically modified Mycoplasma bacterium is a live genetically modified Mycoplasma bacterium, more preferably a live genetically modified Mycoplasma pneumoniae bacterium.

34. A method of treating a subject diagnosed with, or suspected to have a pathogenic biofilm formation, wherein the method comprises a step of contacting the subject with a genetically modified Mycoplasma bacterium of any of claims 1 to 14 or a pharmaceutical composition according to claim 24 or 25.

35. A synthetic promoter with a nucleotide sequence of at least 65% sequence identity to the nucleotide sequence of SEQ ID NO: 8.