Targeted antibacterial treatment

Abstract

The present invention relates to engineered bacteriophages encoding and expressing at least one inhibitor of a quorum-sensing molecule. The bacteriophages of the present invention are useful for reducing, minimizing the virulence of, and / or eliminating pathogenic bacteria, such as those causing bacterial infections.

Description

[0001]P476852PC00 1Targeted Antibacterial Treatment Field of the inventionThe present invention relates to engineered bacteriophages encoding andexpressing at least one inhibitor of a quorum-sensing molecule. Thebacteriophage of the present invention are useful for reducing or eliminatingpathogenic bacteria, such as bacterial infections. The present invention furtherrelates to methods of using the bacteriophage in targeted antibacterialtreatment. Background of the invention By 2050, the death toll by previously preventable or easily curable bacterial infections will surpass that of cancer deaths, unless the further spread ofantibiotic resistance is prevented and new therapies are developed. Apromising strategy is phage therapy, exploiting bacteriophage (phage) viruses, which are natural enemies of bacteria. However, bacteria fight back, limiting the success of phage therapy. Notably, bacteria rely on cell-cell communication, known as quorum sensing to orchestrate both virulence programs and phage defenses. The alarming rise of antibiotic resistance thus calls for an urgent need to develop alternative therapies. The increasingly antibiotic resistant pathogenicbacterium Pseudomonas aeruginosa is one of the "priority pathogens" thatpose the greatest threat to human health. P. aeruginosa causes severe diseasein chronic cystic fibrosis lung infections and is a major source of hospitalacquired infections in e.g. burn wound victims and immunocompromisedpatients. Moreover, P. aeruginosa is a versatile pathogen, infecting animalsand plants, underscoring its importance for agriculture and food security. The use of bacteriophage viruses (phages) as an option for killing pathogenicbacteria is reemerging. Phages are viruses that infect, replicate within, and killP476852PC00 2bacteria. During one lytic infectious cycle, a phage may produce 100-150 phage progeny, which are released when the bacterium lyses. Alternatively, temperate phages can execute the lysogenic life cycle to integrate theirgenome into the host chromosome and reside dormant (called a prophage),until conditions in the prophage-containing cell (called a lysogen) favor a switch from lysogenic to lytic replication cycle. Shortly after the discovery of phages over 100 years ago, phage therapy was employed to treat bacterial infections, but poor understanding of phage biology meant therapy had limited success, and it was largely abandoned in the wake of antibiotics, except at key phage therapy institutes such as the George Eliava Institute of Bacteriophages. With the threat of antibiotic resistance, phage therapy is reemerging as apromising alternative. In recent years, phage therapies using natural virulentphages or phages engineered to be exclusively lytic, were approved for compassionate care use. These have saved critically ill patients suffering from multidrug-resistant infections. However, in some cases, the bacteria developed resistance to the phages. Although cocktails of different phages all targeting the same pathogen via different receptors reduce the probability that bacteria evolve resistance to multiple phages simultaneously, multi-phage- resistant bacteria still arise. The evolutionary arms race between bacteria and phages has caused the development of multiple defense and anti-defense mechanisms. These include primary defenses preventing phages from infecting the bacteria in the first place, and should these defenses fail, the next in line are systems that serve to prevent the phage from taking over the bacterial cell and converting it into a viral factory. Intracellular phage defenses are diverse and plentiful and include the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) with its CRISPR-Associated (cas) genes. CRISPR-Cas are adaptive microbial immune systems. Additionally, groups of bacteria can coordinate to form physical defenses or spatial barriers to phage infection. As expressing defense systems inappropriately is costly, bacteria have developed mechanisms to activate the defenses when necessary. For example,P476852PC00 3quorum sensing (QS), which is a cell-cell communication mechanism, enables bacteria to monitor cell-population density and behave as though they were a multicellular organism. QS depends on the production, release, and group-wide detection of diffusible quorum sensing signaling molecules (also knownas autoinducers, AIs). At high-cell-density, the accumulation of quorumsensing molecules induces bacterial populations to synchronously activatehundreds of genes involved in group behavior, such as virulence genes and those encoding phage defenses.P. aeruginosa communicates using QS to coordinate behavior such as biofilmformation and virulence in addition to launching anti-phage defense systems. As described above, anti-phage defenses, some of which are QS-regulated, pose one of the major biological challenges to phage therapy and biocontrol.P. aeruginosa has multiple intertwined QS systems, including two canonicalLuxI / R-type QS systems: LasI / R and RhlI / R. LasI / R is conventionally considered at the top of the QS cascade in P. aeruginosa. LasI synthesizes 3- Oxo-C12-homoserine lactone (3OC12-HSL), which is detected by LasR. 3OC12-HSL-bound LasR activates transcription of the other QS circuit genes rhlI / R and pqsABCDEH / R. RhlI / R, in turn, produces and detects C4- homoserine lactone (C4-HSL). The Pseudomonas quinolone signal (PQS) system produces and detects the AI 2-heptyl-4-quinolone (HHQ) and 2-heptyl- 3,4-dihydroxyquinoline (PQS). Collectively, these QS systems control the production of an array of virulence factors including elastase, rhamnolipids, and pyocyanin. Intriguingly, while the Las QS system generally activates the PQS system, phage infection of LasI deficient mutants bypasses this regulatory link and directly activates PQS QS, which is a major regulator of a physical phage defense. Thus, phage infection directly activates PQS QS, which controls virulence factors and phage-defense systems, theoretically limiting successful outcomes of phage therapy. Summary of the inventionP476852PC00 4There is a need in the art for new solutions to overcome the increasingly antibiotic resistant pathogenic bacteria. The object of the present invention isto provide a solution to the problem described herein.Here, the inventors have combined QS inhibition and phage therapy directlyby engineering phages to deliver enzymes that degrade N-acyl homoserinelactones, PQS, HHQ and HQNO in P. aeruginosa. The dual-function anti-QSphages replicate within and kill the target bacteria, providing local amplification of the phages while releasing AI-degrading enzymes. The potential of using anti-QS phages to disrupt bacterial communication and simultaneously limit virulence and anti-phage defense activation in P.aeruginosa is presented herein. Using a Galleria mellonella wax moth larvaeburn wound infection model, the inventors have quantified the ability ofengineered anti-QS phages to save the larvae from succumbing to P. aeruginosa infection, compared to that of phages that do not interfere withQS. Moreover, the inventors have challenged the robustness of the anti-QSphages to inhibit QS in mixed populations of phage-susceptible and phage- resistant cells, demonstrating the ability of the phages to disarm phage-resistant P. aeruginosa. The data highlights the future potential of using anti-QS phages to cure P. aeruginosa infections through effective killing andsilencing emerging phage-resistant mutants rendering them harmless, thereby improving the effectiveness of phage therapy.In support of the concept, the inventors have engineered anti-quorum sensingphages against the notorious human pathogen Pseudomonas aeruginosa. The engineered phages disclosed herein effectively degrade quorum sensingmolecules, simultaneously rendering P. aeruginosa avirulent and vulnerable tophage attack, saving Galleria mellonella from succumbing to P. aeruginosa infection. Moreover, the anti-quorum sensing phages inhibit quorum sensing in mixed populations of phage-susceptible and phage-resistant cells, demonstrating the ability of the phages to disarm phage-resistant P.aeruginosa. The data highlights the potential of anti-quorum sensing phagesP476852PC00 5to circumvent phage defenses, thus improving the effectiveness of phage therapy. The object of the present invention is to provide bacteriophages targeted to reduce or eliminate bacteria, in particular pathogenic bacteria, which are resistant to antibiotics. The solution provided with the present invention, isengineered bacteriophages encoding and expressing at least one inhibitor of aquorum-sensing molecule. The bacteriophage of the present invention allowsfor rapid expression of the inhibitor of a quorum-sensing molecule upon phageinfection and at the same time ensures a balanced expression that does not inhibit phage replication.In a first aspect, the present invention provides a bacteriophage encoding andexpressing at least one inhibitor of a quorum sensing molecule, wherein the expression of said at least one inhibitor of a quorum sensing molecule is operably linked to a promoter, wherein said promoter is regulated by arepressor. The bacteriophage is genetically modified to encode and expresssaid at least one inhibitor of a quorum-sensing (QS) molecule. In another aspect, the present invention provides a composition comprising the bacteriophage of the present invention. In one aspect, the present invention provides a pharmaceutical compositioncomprising the bacteriophage according to the invention and at least onepharmaceutical acceptable excipient.In a further aspect, the present invention provides the bacteriophage of thepresent invention for use as a medicament. The medicament may be a pharmaceutical composition comprising said bacteriophage and at least one pharmaceutical acceptable excipient. In one aspect, the present invention provides the bacteriophage or a composition such as a pharmaceutical composition for use in the treatment ofP476852PC00 6a bacterial infection. The present invention further provides methods of treating a bacterial infection, said method comprising the administration of said bacteriophage or composition comprising said bacteriophage in an amount sufficient to reduce or eliminate the bacterial infection. In a further aspect, the bacteriophage of the present targets a plant pathogenic bacterium. Accordingly, the present invention further provides methods of using said bacteriophage targeting a plant pathogenic bacterium for the treatment of a plant infected with said bacterium. Likewise, the presentinvention provides the use of said bacteriophage to combat a plant pathogenicbacterium. A further aspect of the present invention provides a bacteriophage encodingand expressing at least one heterologous polypeptide (or heterologouspeptide), wherein the expression of said at least one heterologous polypeptide (or heterologous peptide) is operably linked to an anti-CRISPR promoter, wherein said promoter is regulated by a repressor, wherein said repressor is an anti-CRISPR associated repressor. Brief description of the drawings Figure 1. Anti-QS phages effectively reduce the accumulation of QS molecules 3O-C12-HSL and PQS. Cell densities and relative QS moleculeabundances of phage-infected P. aeruginosa PA14 cultures challenged withDMS3 aqs1_FSDARE vir or anti-QS variants. The phages were mixed in a 1:1 ratio for the dual phage treatment. At OD600~0.14, phage-susceptible PA14 was infected with a 10-fold excess of phages (multiplicity of infection = 10). (A) OD600 was measured to monitor bacterial growth post phage infection. The various phage infections inhibited PA14 growth similarly. (B-D) To determine the relative abundance of key QS molecules of the Las, Rhl and PQS system, sterile supernatants were tested using E. coli bioreporters. The relative levels of 3O-C12-HSL, C4-HSL, and PQS were quantified as relative light units as a response to these QS molecules. The values are reported as RLU (lum / OD600)P476852PC00 7(relative light units). Whereas the anti-QS phages had no apparent effect on C4-HSL abundance, both 3O-C12-HSL, and PQS levels were significantly reduced in response to anti-QS phage infection. (B) The aqdC-carrying phageas well as the mixture of aqdC- and qsdA-carrying phages resulted in a 1.9-fold reduction in 3O-C12-HSL 2 h post infection (p = 0.0110 and p = 0.0104 respectively), whereas all anti-QS phages reduced relative 3O-C12-HSL levels 1.4-1.8-fold 6 h post infection (qsdA: p = 0.0021, aqdC: p = 0.0005, and both phages: p < 0.0001). (D) Anti-QS phages carrying aqdC and phagemixtures of aqdC- and qsdA-carrying phages lowered the accumulation of PQS(p = 0.0189 and p = 0.0200 respectively, 2 h post infection). Error bars showstandard deviation, n = 3. Ordinary one-way ANOVA tests. These datademonstrate that the anti-QS phages effectively suppress accumulation of theQS molecules 3O-C12-HSL and PQS during phage infection.Figure 2. Anti-QS prophage rescues G. mellonella larvae from infectionby P. aeruginosa. G. mellonella larvae were either untreated or inflicted witha burn wound and treated as follows: either the wound was left untreated,infected with 10 µl of an OD600=1 standardized inoculum of P. aeruginosa PA14JBD44 WT lysogen or a PA14 JBD44 anti-QS lysogen expressing aqdC andqsdA. Whereas the JBD44 WT lysogen killed 90% of all larvae, the JBD44 anti-QS lysogen yielded survival rates of 77%, comparable to the burnwoundcontrol survival rates (p < 0.0001, log-rank test based on end-point mortality).N = 30 larvae per condition. These data demonstrate that virulence iseffectively attenuated in the anti-QS phage lysogen, thus protecting G.mellonella larvae from PA14-mediated killing.Figure 3. Virulent anti-QS phages protect G. mellonella larvae from P.aeruginosa killing. G. mellonella larvae were either left untreated or inflictedwith a burn wound and treated as follows: either the wound was untreated,treated with a DMS3 aqs1_FSDARE vir phage, or infected with 10 µl of anOD=1 standardized inoculum of P. aeruginosa PA14 WT. Subsequently, G.mellonella larvae survival was investigated in response to different phagetreatments. One hour after bacterial infection, burn wounds were treated withP476852PC00 8a total of 109PFU (plaque forming units) of either the virulent parental DMS3 aqs1_FSDARE vir phage or a 1:1 mixture of virulent DMS3 aqs1_FSDAREphages expressing qsdA or aqdC, respectively. P. aeruginosa PA14 infectionkilled 60% of the larvae. DMS3 aqs1_FSDARE vir phage treatment did notaffect larvae survival significantly (63% of the larvae died). However, anti-QSphage treatment led to 70% of larvae surviving infection by P. aeruginosaPA14 (p = 0.0079, log-rank test based on end-point mortality). N = 30 larvaeper condition. These data demonstrate that the engineered anti-QS virulentphages significantly reduce P. aeruginosa PA14 virulence and protect G.mellonella larvae from killing.Figure 4. P. aeruginosa JBD44 Δgp1-39 is resistant to JBD44 phagekilling. Bacterial growth of P. aeruginosa PA14 and PA14 lysogen JBD44 Δgp1-39 was tested in the presence of a SM buffer solvent control or with anmultiplicity of infection (MOI) of 10 of WT JBD44 phage. OD600 was measuredat 37 °C in a plate reader over a 24 h period. Whereas PA14 growth was noticeably affected in the presence of the WT JBD44 phage, WT JBD44 phage infection did not affect growth of lysogen JBD44 Δgp1-39. Error bars show standard deviation, n = 2. These data show that the JBD44 ∆gp1-39 prophageprovides resistance to JBD44 phage infection. PA14 JBD44 Δgp1-39 lysogenwas utilized as a phage-resistant mutant in the experiments involving JBD44 phage infection of phage-susceptible and phage-resistant co-cultures (Fig. 7).Figure 5. P. aeruginosa DMS3 aqs1_FSDARE Δgp26-48 lysogen isresistant to DMS3 vir killing. PA14 WT and the PA14 DMS3 aqs1_FSDARE Δgp26-48 lysogen were tested in a phage cross-streak assay to determine sensitivity toward the DMS3 vir phage. A drop of DMS3 vir phage solution was added to the center of the agar plate to cover the dotted vertical line. To test phage sensitivity, PA14 WT (top streak) and PA14 DMS3 aqs1_FSDARE Δgp26- 48 lysogen (bottom streak) were streaked left to right, resulting in phage exposure on the right side of the plate. Whereas PA14 WT was killed by theDMS3 vir phage, PA14 DMS3 aqs1_FSDARE Δgp26-48 lysogen growth wasunaffected. These data illustrate that the DMS3 aqs1_FSDARE ∆gp26-48P476852PC00 9prophage provides resistance to DMS3 phage killing. PA14 DMS3aqs1_FSDARE Δgp26-48 lysogen was utilized as a phage-resistant mutant inthe experiments involving DMS3 phage infection of phage-susceptible and phage-resistant co-cultures (Fig. 6). Figure 6. Anti-QS phages effectively limit QS molecule accumulation in a mixed population of phage-susceptible and phage-resistant P.aeruginosa cells. The effect of DMS3 phage infections on bacterial growthand relative QS molecule abundance of a PA14 co-culture consisting of 90% phage-susceptible (PA14 WT) and 10% phage-resistant cells PA14 DMS3aqs1_FSDARE Δgp26-48 lysogen was tested. Phage treatments include thecontrol phage, a parental DMS3 aqs1_FSDARE vir phage, as well as phagesexpressing either of the anti-QS enzymes, or a 1:1 mixture of aqdC- and qsdA-expressing phages. At OD600~0.14 PA14 co-cultures were infected with a 10- fold excess of phages (multiplicity of infection = 10). (A) OD600 was measured to monitor bacterial growth post phage infection. All the phage variants inhibited the co-culture growth similarly. (B-D) The relative abundance of 3O- C12-HSL, C4-HSL, and PQS were quantified by E. coli bioreporters producing relative light units as a response to high concentrations of the QS molecules. The values are reported as RLU (lum / OD600) (relative light units). Anti-QS phages had no direct effect on C4-HSL abundance whereas 3O-C12-HSL and PQS levels were significantly reduced in response to anti-QS phage infection.(B) Anti-QS phages carrying aqdC as well as phage mixtures of aqdC- andqsdA-carrying phages lowered the accumulation of 3O-C12-HSL 1.4-fold (p = 0.0233 and p = 0.0103 respectively, 4 h post infection). (D) All anti-QS phages lowered accumulation of PQS (all anti-QS phages p < 0.0001, 2 h post infection). Ordinary one-way ANOVA tests. Error bars show standard deviation, n = 3. These data highlight that even in populations enriched with phage-resistant cells, the anti-QS phages efficiently inhibit the accumulation of 3O-C12-HSL and PQS QS molecules. Figure 7. Anti-QS phages significantly reduce pyocyanin virulence factor production and show enhanced lytic activity in a mixedP476852PC00 10population of phage-susceptible and phage-resistant P. aeruginosacells. A mixed population of 90% phage-susceptible (WT PA14) and10 %phage-resistant (PA14 JBD44 Δgp1-39 lysogen) cells at OD600 = 0.1 waschallenged with a 5-fold excess (multiplicity of infection of 5) of WT JBD44 oranti-QS JBD44 phage expressing qsdA and aqdC. 5.5 hours post infection supernatants were sampled and tested for pyocyanin abundance and cell density. (A) As compared to the WT JBD44, the anti-QS JBD44 phage significantly reduced pyocyanin production by 7.8-fold. [Welch’s t test; p =0.0003. Error bars show standard deviation, n = 3.] (B) Compared to the WTphage, OD600 was significantly reduced in response to anti-QS phage treatment. [Welch’s t test; p = 0.0004. Error bars show standard deviation, n = 3.]. Overall, these data demonstrate that the JBD44 anti-QS phage effectively impairs virulence factor production while inhibiting bacterial growth of mixed PA14 populations. Figure 8. Virulent anti-QS-phages are effective against P. aeruginosainfections containing phage-resistant cells. G. mellonella larvae wereinflicted with a burn wound and treated as follows: the wound was treated witha DMS3 aqs1_FSDARE vir phage as a control or infected with 10 µl of an OD=1standardized inoculum consisting of a mixture of P. aeruginosa PA14 WT andphage-resistant PA14 JBD44 Δgp1-39 in a 90:10% ratio based on OD600. Onehour after bacterial infection, wounds were treated with a total of 109PFU of either the parental DMS3 aqs1_FSDARE vir phage or a 1:1 mixture of virulentDMS3 aqs1_FSDARE phages expressing qsdA or aqdC, respectively. 65% ofthe larvae were killed by the bacteria. DMS3 aqs1_FSDARE vir phagetreatment did not improve survival (73% died) Anti-QS phage treatment ledto 53% of larvae surviving infection by the mixture of P. aeruginosa PA14 WTand phage-resistant cells (p = 0.0366, log-rank test based on end-point mortality). N = 30 larvae per condition. These data demonstrate that theengineered anti-QS phages limit P. aeruginosa PA14 virulence even in co-cultures of phage-susceptible and phage-resistant cells. Figure 9. QsdA can broadly inactivate acyl homoserine lactones.P476852PC00 11The ability of QsdA to inhibit the bioactivity of diverse acylhomoserine lactoneswas tested. The homoserine lactone-deficient strain PA14 ∆lasI ∆rhlI carryingeither pHERD30T empty vector control or pHERD30T-qsdA expressing qsdAunder an arabinose inducible promoter, were cultured at 37 °C for 4 h in LBcontaining gentamicin (50 µg mL-1), 0.1% arabinose, MOPS buffer (50 mM,pH=7), ZnSO4·7H2O (10 µM), 1 µM N-acylhomoserine lactones: C4-HSL, C6-HSL, 3O-C6-HSL, C8-HSL, 3O-C8-HSL, C10-HSL, and 3O-C12-HSL. Therelative abundance of N-acylhomoserine lactones was quantified by an E. colibioreporter strain that produces light as a response to various homoserine lactones. Measurements are reported as RLU normalized to optical density(lum / OD600). QsdA significantly reduced the bioactivity of all the tested acylhomoserine lactones C4-HSL, C6-HSL, 3O-C6-HSL, C8-HSL, 3O-C8-HSL, C10- HSL, and 3O-C12-HSL with acyl chains ranging from 4 to 12 carbon atoms andwith different side groups (p = 0.0268, 0.0017, 0.0138, 0.0043, 0.0105,<0.0001, and 0.0207, two-sample t-test assuming equal variance). Error barsrepresent standard deviation (n = 3). These data highlight that QsdA caninactivate a broad spectrum of acylhomoserine lactones produced by Gram-negative bacteria, including those not typically produced by Pseudomonas species. Table 2. Anti-QS phages block phage-infection mediated stressresponse. Swarm assays were performed with P. aeruginosa PA14 inoculatedat the center of the swarm and PA14 infected with either DMS3 aqs1_FSDAREvir, DMS3 aqs1_FSDARE vir qsdA or a 1:1 mixture of aqdC- and qsdA-expressing phages. Phage infection causes a PQS stress response, resulting in repulsion of uninfected tendrils from the center swarm. This is evident inresponse to DMS3 aqs1_FSDARE vir infection, where 0 / 4 infected satellitecolonies were invaded by tendrils. However, in response to infection by all three phages expressing either qsdA, aqdC, or both, tendrils from the center swarm collide with all infected colonies, demonstrating that the anti-QS phages block the PQS induced tendril repulsion, which allows for more efficient phage transmission. In one of the four replicates, the tendrils approaching the DMS3 aqs1_FSDARE vir qsdA-infected colony swarmed insufficiently, so itP476852PC00 12could not be determined whether the tendrils would have collided with the colony. These data demonstrate that the anti-QS phages effectively inhibit the phage-infection induced physical phage defense. Detailed description of the invention In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that eachspecific term includes all technical equivalents, which operate in a similarmanner to accomplish a similar purpose. When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited inmutually different dependent claims or described in different embodimentsdoes not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments. As presented herein, the inventors discovered that QS inhibition and phage targeting a pathogenic bacterium is a solution to the problem relating tocombating pathogenic bacteria, which are resistant to conventional treatmentsuch as antibiotics. The solution provided herein is a genetically modified bacteriophage expressing a heterologous inhibitor of QS sensing molecules. The anti-quorum sensing phage is modified to ensure rapid expression upon phage infection and at the same time ensures a balanced expression that does not inhibit phage replication. Thus, the design of the phage allows for high expression of the QS inhibitor shortly after infection of the bacterium without impairing the replication of the phage in the infected bacterium. This dual-arm strategy thus includes (i) targeting the pathogenic bacteria by means of causing lysis and in the same time (ii) targeting pathogenic bacteria that is or has become resistant to infection by the bacteriophage by inhibiting QS and thus rendering them avirulent.P476852PC00 13As mentioned, the anti-quorum sensing phage is modified to ensure rapid expression upon phage infection, but the expression does not violate phage replication. In order to achieve this, any promoter may be used that ensures rapid temporarily expression upon phage infection and decreasing expression such that phage replication is not impaired. Examples of such promoter is apromoter with a negative feedback mechanism such as a promoter / repressorsystem, where the activity of the promoter induces the expression of a repressor of the promoter (e.g. encoded in an operon comprising the promoter), which down regulate the activity of the promoter. A bacteriophage modified to include an expression cassette comprising a promoter regulating the expression of a sequence encoding the QS molecule inhibitor and a repressor of the promoter may thus allow for rapid onset of expression of the QS molecule inhibitor, which is subsequently downregulated by the repressor to maintain capability of phage replication. Anti-QS bacteriophageThe present invention provides a bacteriophage encoding and expressing atleast one inhibitor of a quorum sensing molecule, wherein the expression of said at least one inhibitor of a quorum sensing molecule is operably linked to a promoter, wherein said promoter is regulated by a repressor. In one embodiment, said promoter regulates transcription of said at least oneinhibitor of a quorum sensing molecule and said repressor. Typically, the QSinhibitor and the repressor is encoded by the same transcript whereby the expression of the repressor generates a negative feedback on the promoter activity. The inventors have demonstrated that the anti-QS bacteriophage of thepresent invention is more efficient than its counterpart, which does not haveanti-QS capabilities in treating a P. aeruginosa infection (see Examples andFig. 2-3).P476852PC00 14In one embodiment, the at least one inhibitor of a quorum sensing moleculeand said repressor are encoded by a bi-cistronic or poly-cistronic transcript. The repressor may be any suitable repressor in the sense that repressor is capable of downregulating the activity of the promoter operably linked to and regulating the expression of the QS inhibitor. An example of a suitable promoter is an anti-CRISPR promoter (Pacr), whichinitiates transcription immediately after phage infection. The anti-CRISPRpromoter (Pacr) is one of the strongest expressed and most rapidly expressedpromoters known. Transcripts are detectable a few minutes after phageinfection and accumulates 100-fold within the first 20 min of infection followedby stabilization of transcript levels.Thus, in one embodiment, the promoter directs expression of the transcriptencoding the QS inhibitor within 5 minutes after infection and stabilizes about100-fold higher about 20 minutes after the infection.In one particular embodiment, the promoter is an anti-CRISPR promoter (Pacr). In another embodiment, the promoter comprises the sequence setforth in SEQ ID NO: 1 or a functional variant of said sequence. In the contextof the present invention, the variant of the promoter is a promoter thatdisplays the same activity or essentially the same activity as the anti-CRISPRpromoter (Pacr) and is subject to the same regulation including subject toregulation by a repressor. In one embodiment, the variant promoter comprises a sequence having a sequence identity of at least 80% to the sequence of SEQ ID NO: 1, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the sequence of SEQ ID NO: 1. In order to regulate the activity of the anti-CRISPR promoter, the bacteriophage may advantageously encode and express an anti-CRISPRP476852PC00 15associated repressor (Aca protein). The Aca protein binds and represses thePacr promoter and maintains the ability of the bacteriophage to replicate inthe host cell. Accordingly, in one embodiment, the promoter is an anti-CRISPR promoter and said repressor is an anti-CRISPR associated repressor. In one embodiment, the anti-CRISPR repressor is encoded by a polynucleotidesequence comprising the sequence set forth in SEQ ID NO:2 or a variant ofsaid sequence. In the context of the present invention, the variant of therepressor is a repressor that displays the same activity or essentially the sameactivity as said anti-CRISPR associated repressor. In one embodiment, thevariant sequence encoding the anti-CRISPR associated repressor comprises asequence having a sequence identity of at least 80% to the polynucleotide sequence of SEQ ID NO: 2, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the polynucleotide sequence of SEQ ID NO: 2.In one embodiment, the bacteriophage comprises a promoter comprising thesequence set forth in SEQ ID NO: 1 and encodes an anti-CRISPR repressor,wherein the sequence encoding the anti-CRISPR repressor comprises thesequence set forth in SEQ ID NO:2.In another embodiment, the anti-CRISPR repressor comprises the amino acidsequence set forth in SEQ ID NO: 3 or a functional variant of said sequence.The variant of the repressor is a repressor that displays the same activity oressentially the same activity as said anti-CRISPR associated repressor. In oneembodiment, the variant anti-CRISPR associated repressor comprises anamino acid sequence having a sequence identity of at least 80% to the sequence of SEQ ID NO: 3, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the sequence of SEQ ID NO: 3.The bacteriophage of the present invention may comprise an operoncomprising the promoter operably linked to the sequences encoding the QSP476852PC00 16inhibitor and the sequences encoding the repressor and optionally also asequence encoding an anti-CRISPR (acr) protein.In one embodiment, the bacteriophage of the present invention comprises ananti-CRISPR operon comprising an anti-CRISPR promoter and encoding said at least one inhibitor of a quorum sensing molecule and a sequence encoding an anti-CRISPR associated repressor.In one embodiment, the bacteriophage further encodes and expresses an anti-CRISPR (acr) protein. In one embodiment, the bacteriophage comprises thepolynucleotide sequence set forth in SEQ ID NO: 8, encoding an acr protein. In another embodiment, the bacteriophage further encodes and expresses an acr protein comprising the amino acids sequence set forth in SEQ ID NO: 9. CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading bacteriophages. Anti-CRISPR proteins interact with specific components of CRISPR-Cas systems, such as the effector nucleases, to avoid the destruction of the phage DNA. Thus, in the context of the presentinvention, the encoded anti-CRISPR (acr) protein is a defence mechanism toavoid inactivation of the bacteriophage in the host bacterium.The expression of the anti-CRISPR (acr) protein is preferably regulated by thesame promoter that direct the expression of the QS inhibitor. Thus, in one embodiment, the at least one inhibitor of a quorum sensing molecule, saidrepressor and said an anti-CRISPR (acr) protein are encoded by a poly-cistronic transcript.The anti-CRISPR operon encodes an anti-CRISPR (acr) protein and an anti-CRISPR repressor and the expression is directed by the anti-CRISPR promoter(Pacr). The sequence encoding the QS inhibitor may be inserted in the anti-CRISPR operon under control of the anti-CRISPR promoter. In oneembodiment, the nucleotide sequence encoding the at least one inhibitor of aquorum sensing molecule is inserted in the operon replacing or in partP476852PC00 17replacing the sequence encoding an endogenous anti-crispr (acr) protein. Inone embodiment, the nucleotide sequence encoding the at least one inhibitorof a quorum sensing molecule is inserted in the operon such that theexpression of the endogenous anti-crispr (acr) protein is maintained.In one embodiment, the bacteriophage comprises an endogenous anti-CRISPRoperon, wherein said operon is genetically modified to encode and expresssaid at least one inhibitor of a quorum sensing molecule. Thus, in thisembodiment, the sequence encoding the QS inhibitor is inserted an endogenous anti-CRISPR operon, where the expression of the endogenousanti-crispr (acr) protein is maintained or the sequence may be inserted toreplace (or partly replace) the sequence encoding the endogenous anti-crispr (acr) protein.The anti-CRISPR operon may also be exogenous. Thus, the exogenous operonmay be artificial, an operon from another species of bacteriophage or a modified version of the operon of the bacteriophage used for the preparation of the bacteriophage of the present invention. In one embodiment, thebacteriophage comprises an exogenous anti-CRISPR operon, wherein saidoperon is genetically modified to encode and express said at least one inhibitor of a quorum sensing molecule. In one embodiment, the bacteriophage is lytic. In another embodiment, thebacteriophage is temperate. The temperate bacteriophages is typicallyintegrated in the host genome (as a prophage) remains in a lysogenic stateuntil a lytic life cycle is induced. Thus, the temperate bacteriophage maintainsthe capacity of lysing the bacterium. The bacteriophage of the present invention is typically provided usingrecombinant techniques well known known to one skilled in the art. Thus, in apreferred embodiment, the bacteriophage is recombinant. The bacteriophage of the present invention may be based on any suitable bacteriophage forP476852PC00 18infecting a pathogenic bacterium as described herein. Thus, in oneembodiment, the bacteriophage of the present invention is recombinant.Thus, the backbone used for the preparation of the bacteriophage of thepresent invention may be a bacteriophage selected from the list consisting of Phage DMS3 (accession_no.NC_008717.1), Phage JBD44 (accession_no.NC_030929.1), and phages classified in the orders of Caudoviricetes, Kalamavirales, Mindivirales, Timlovirales, Tubulavirales, Vinavirales, and Durnavirales, and unclassified or reclassified phages. In one embodiment, the backbone used for the preparation of the bacteriophage of the present invention comprises the sequence of SEQ ID NO: 10 (Phage DMS3). In another embodiment, the backbone used for the preparation of the bacteriophage of the present invention comprises the sequence of SEQ ID NO: 11 (Phage JBD44). One further aspect of the present invention provides a method for the preparation of a bacteriophage according to the present invention, said method comprising the step of introducing at least one polynucleotidesequence encoding an inhibitor of a quorum sensing molecule in the genomeof a bacteriophage, wherein said sequence is inserted in the genome to be operably linked to a promoter, wherein said promoter is regulated by a repressor. In one embodiment, promoter is an anti-CRISPR promoter and the repressor is an anti-CRISPR associated repressor (Aca protein). In another embodiment, the bacteriophage (cloning bacteriophage) used for the preparation of the bacteriophage of the present invention comprises an anti-CRISPR operoncomprising an anti-CRISPR promoter and encoding said at least one inhibitorof a quorum sensing molecule and a sequence encoding an anti-CRISPRassociated repressor. In one embodiment, the bacteriophage (cloningbacteriophage) used for the preparation of the bacteriophage of the presentP476852PC00 19invention comprises the polynucleotide sequence of SEQ ID NO: 12 (cloningphage DMS3 (DMS3 aqs1_FSDARE)). In one embodiment, the bacteriophage of the present invention comprises the sequence set forth in SEQ ID NO: 13 (Phage_DMS3_aqs1_FSDARE_vir_qsdA). In another embodiment, the bacteriophage of the present invention comprises the sequence set forth in SEQ ID NO: 14 (Phage_DMS3_aqs1_FSDARE_vir_aqdC). In one embodiment, the bacteriophage of the present invention comprises the sequence set forth in SEQ ID NO: 15 (Phage_JBD44_Δgp48-53_gp46- 47::Pacr-qsdA-aqdC-aca-T1). In one embodiment, the bacteriophage of the present invention is a prophage. In another embodiment, the bacteriophage of the present invention comprises the sequence set forth in SEQ ID NO: 16 (Prophage_JBD44_Δgp48-53_gp46- 47::Pacr-qsdA-aqdC-T1) Encoded quorum sensing molecule inhibitor The bacteriophage of the present invention is modified to encode and expressat least one quorum sensing molecule inhibitor (also referred to as QS inhibitorherein). In one embodiment, the bacteriophage encodes and expresses one quorum sensing molecule inhibitor. In another embodiment, the bacteriophageencodes and expresses two quorum sensing molecule inhibitors. In a furtherembodiment, bacteriophage encodes and expresses a plurality of quorumsensing molecule inhibitors.In one embodiment, said at least one quorum sensing molecule inhibitor isselected from the group consisting of quorum sensing molecule inhibitordegrading enzymes, and a repressor of said quorum sensing. In embodimentsP476852PC00 20where the bacteriophage encodes more than one quorum sensing moleculeinhibitor, the quorum sensing molecule inhibitors are independently selectedfrom the group consisting of quorum sensing molecule inhibitor, degradingenzymes, and a repressor of said quorum sensing. In one embodiment, said at least one quorum sensing molecule inhibitor is a biological inhibitor that interferes directly with the quorum sensing molecule. In the context of the present invention, a biological inhibitor is a biomoleculethat can be encoded and expressed by the bacteriophage. A QS inhibitor mayalso be a biological molecule that competitively inhibit the QS signaling system.In one embodiment, the quorum sensing molecule inhibitor is a repressor ofthe quorum sensing molecule. In another embodiment, the inhibitor of thequorum sensing molecule is a synthase synthesizing a repressor of saidquorum sensing molecule, an antisense RNA or CRISPRi repressing productionof said quorum sensing molecule.In one embodiment, said at least one quorum sensing molecule inhibitor is an enzyme, such as an QS degrading enzyme. The enzyme may directly orindirectly inhibit the function of quorum sensing molecule such as by degradingor partly degrading the quorum sensing molecule. In other embodiments, the enzyme degrades or partly degrades a molecule that interact with the quorumsensing molecule and is essential for its function. In another embodiment, thequorum sensing molecule inhibitor is an enzyme that break up outermembrane vesicles that carry QS molecules (such as PQS and long chainhomoserine lactones).In one embodiment, the said at least one heterologous polypeptide is anenzyme. In one embodiment, the said enzyme is a lactonase. In oneembodiment, the said enzyme is a hydrolase. In one embodiment, the saidenzyme is a AHL acylase. In one embodiment, the enzyme is a protease.P476852PC00 21In a further embodiment, the at least one quorum sensing molecule inhibitoris an enzyme selected from the group consisting of quorum sensing molecule degrading enzymes such as alkylquinolone degradation C (AqdC), autoinducing peptide (AIP)-degrading protease, AHL-degrading enzymes such as AHL lactonases and acylases (autoinducer inactivation A (AiiA), autoinducer inactivation B (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD), autoinducer inactivation S (AiiS), attachment gene M (AttM), N-acylhomoserine lactone D (AhlD), N-acylhomoserine lactone K (AhlK), N-acylhomoserine lactone M (AhlM), N-acylhomoserine lactone S (AhlS), metallo-β-lactamase superfamily lactonase (AaL), quorum-sensingsignal degradation A (QsdA), quorum-sensing signal degradation H (QsdH),autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase(MCP), quorum-quenching lactonase from Geobacillus kaustophilus (GKL),NAHL-lactonase (QlcA), quorum signal utilization and inactivation protein(QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA),Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHLacylase B (HacB).In one embodiment, the quorum sensing molecule inhibitor is quorum-sensingsignal degradation A (QsdA) or a functional variant of QsdA. In anotherembodiment, the at least one quorum sensing molecule inhibitor comprisesthe amino acid sequence set forth in SEQ ID NO: 6 (QsdA) or a functionalvariant thereof. The variant of the QsdA is a protein that displays the sameactivity or essentially the same activity or higher activity than QsdA. In oneembodiment, the variant QsdA comprises an amino acid sequence having asequence identity of at least 80% to the sequence of SEQ ID NO: 6, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the sequence of SEQ ID NO: 6. In another embodiment, the bacteriophage comprising the polynucleotide sequence encoding said at least one quorum sensing molecule inhibitorcomprises the sequence set forth in SEQ ID NO: 4 (encoding QsdA) or aP476852PC00 22functional variant thereof. In one embodiment, the variant sequence encodingQsdA comprises a polynucleotide sequence having a sequence identity of atleast 80% to the polynucleotide sequence of SEQ ID NO: 4, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the polynucleotide sequence of SEQ ID NO: 4. In one embodiment, the quorum sensing molecule inhibitor is alkylquinolonedegradation C (AqdC) or a functional variant of AqdC. In another embodiment,the at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 7 (AqdC) a functional variant thereof. The variant of the AqdC is a protein that displays the same activity or essentially the same activity or higher activity than AqdC. In one embodiment, the variantAqdC comprises an amino acid sequence having a sequence identity of at least80% to the sequence of SEQ ID NO: 7, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the sequence of SEQ ID NO: 7. In another embodiment, the bacteriophage comprising the polynucleotide sequence encoding said at least one quorum sensing molecule inhibitorcomprises the sequence set forth in SEQ ID NO: 5 (encoding AqdC) or afunctional variant thereof. In one embodiment, the variant sequence encodingAqdC comprises a polynucleotide sequence having a sequence identity of atleast 80% to the polynucleotide sequence of SEQ ID NO: 5, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the polynucleotide sequence of SEQ ID NO: 5. In one embodiment, the anti-CRISPR repressor is encoded by a polynucleotidesequence comprising the sequence set forth in SEQ ID NO:2 or a functionalvariant of said sequence. In the context of the present invention, the variantof the repressor is a repressor that displays the same activity or essentially the same activity as said anti-CRISPR associated repressor. In oneP476852PC00 23embodiment, the variant sequence encoding the anti-CRISPR associatedrepressor comprises a sequence having a sequence identity of at least 80% tothe polynucleotide sequence of SEQ ID NO: 2, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 97%, such as at least 98%, for example 99% to the polynucleotide sequence of SEQ ID NO: 2.In another embodiment, the bacteriophage encodes and expresses twoinhibitors of quorum sensing molecule, such as two enzymes targeting at least one quorum sensing molecule. In a further embodiment, the bacteriophageencodes and expresses QsdA and AqdC, such as the N-acylhomoserinelactonase (QsdA) from Rhodococcus erythropolis and the alkyl quinolonedegradation C (AqdC) from Mycobacteroides abscessus. Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce andrelease signal molecules, quorum sensing molecule (also called autoinducers),that increase in concentration as a function of cell density. Bacteria may forexample use the quorum sensing to sense that the population density hasreached a threshold level. At this stage, the bacteria communicate through thequorum sensing molecules, which enable them to express genes for differentphenotypes, especially those responsible for their virulent behaviour. Thusupon reaching a threshold concentration, quorum sensing molecules triggercascades of signal transduction which are well described in many bacterial species and regulate processes such as biofilm formation, virulence, competence and sporulationSome bacterial species also seem to use quorum sensing to enhance eachother’s virulence, such as Burkholderia cepacia and Pseudomonas aeruginosa,which sometimes co-infect the lungs of cystic fibrosis patients. The bacteriophage of the present invention, which express at least one QS inhibitor, while maintaining the capacity of lysing the host cells is a promisingP476852PC00 24alternative to attenuating pathogenicity. While the target bacteria may escape infection by developing resistance, such phage-resistant bacteria are still targets for inhibition by the QS inhibitor, which suppress virulence. The inventors have demonstrated that the bacteriophage of the presentinvention effectively limits virulence factor production in co-cultures of phage-susceptible and phage-resistant populations (see Examples and Fig. 7).In the context of the present invention, a quorum sensing molecule is abacterially synthesized molecule that it either passively or actively releasedinto the extracellular space and involved in quorum sensing. Classes of quorumsensing molecules includes but are limited to acylated homoserine lactones, and oligopeptides. In one embodiment, the quorum sensing molecule is selected from the groupconsisting of N-acyl homoserine lactones including harveyi autoinducer 1 (HAI-1), Pseudomonas Quinolone Signal 2-heptyl-3-hydroxy-4-quinolone (PQS), 4-hydroxy-2-heptylquinoline (HHQ), 4-hydroxy-2-heptylquinoline-N-oxide(HQNO), autoinducer-2 ((3aS,6S,6aR)-2,2,6,6a-Tetrahydroxy-3a- methyltetrahydro-2H-furo[2,3-d][1,3,2]dioxaborol-2-uide), Auto inducing peptides (AIP), diffusible signal factor (DSF, cis-11-methyldodecenoicacid), methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME), cholerae autoinducer 1 (S)-3-hydroxytridecan-4-one (CAI-1), , and3,5-dimethyl-pyrazin-2-ol (DPO). In the context of the present invention, the term host cell refers to a bacterium that is susceptible to infection by the bacteriophage of the present invention. In one embodiment, the bacterium is a pathogenic bacterium. In another embodiment, the bacteriophage is capable of infecting at least one bacteriaselected from the group consisting of a Pseudomonas sp, such asPseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp.,Escherichia coli, Ralstonia solanacearum, Pseudomonas syringae,P476852PC00 25Xanthomonas campestris, Xylella fastidiosa, Dickeya dadantii, Agrobacterium tumefasciens, and Pectobacterium atrosepticum. In another embodiment, the bacterium is a probiotic bacterium. The probiotic bacterium may be used as “carrier” of the QS inhibitor. The probiotic bacterium carrying the bacteriophage of the present invention may thus be used to combat bacterial infection by sole inhibiting quorum sensing. A further aspect of the present invention provides a bacteriophage encoding and expressing at least one heterologous polypeptide, wherein the expressionof said at least one heterologous polypeptide is operably linked to an anti-CRISPR promoter, wherein said promoter is regulated by a repressor, wherein said repressor is an anti-CRISPR associated repressor. The above recombinant bacteriophage may be used to express heterologous polypeptides (such as an enzyme degrading a QS inhibitor) in a host in a conditional way without impairing the virulence of the bacteriophage as demonstrated herein.In one embodiment an anti-CRISPR promoter regulates transcription of thesequence encoding said at least one heterologous polypeptide and said anti-CRISPR associated repressor.In one embodiment, the said at least one heterologous polypeptide is anenzyme. In one embodiment, the said enzyme is a lactonase. In oneembodiment, the said enzyme is a hydrolase. In one embodiment, the saidenzyme is a AHL acylase. In one embodiment, the enzyme is a protease.In another embodiment, enzyme is selected from the group consisting ofalkylquinolone degradation C (AqdC), autoinducing peptide (AIP)-degradingproteases, AHL-degrading enzymes such as AHL lactonases and acylases (e.g. autoinducer inactivation A (AiiA), autoinducer inactivation B (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD),P476852PC00 26autoinducer inactivation S (AiiS), attachment gene M (AttM), N- acylhomoserine lactone D (AhlD), N-acylhomoserine lactone K (AhlK), N- acylhomoserine lactone M (AhlM), N-acylhomoserine lactone S (AhlS), metallo-β-lactamase superfamily lactonase (AaL), quorum-sensing signal degradation A (QsdA), quorum-sensing signal degradation H (QsdH), autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase(MCP), quorum-quenching lactonase from Geobacillus kaustophilus (GKL),NAHL-lactonase (QlcA), quorum signal utilization and inactivation protein (QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA), Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHLacylase B (HacB). In a further embodiment, said at least one heterologouspolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6(QsdA) or amino acid sequence having at least 85% identity to the amino acidsequence set forth in SEQ ID NO: 6 and / or said at least one quorum sensingmolecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 7 (AqdC) or amino acid sequence having at least 85% identity to the aminoacid sequence set forth in SEQ ID NO: 7. In yet another embodiment, thesequence encoding said at least one heterologous polypeptide comprises thesequence set forth in SEQ ID NO: 4 (encoding QsdA). In one embodiment, thesequence encoding said at least one heterologous polypeptide comprises thesequence set forth in SEQ ID NO: 5 (encoding AqdC).In one embodiment, the bacteriophage comprises an anti-CRISPR operoncomprising an anti-CRISPR promoter and encoding said at least oneheterologous polypeptide and a sequence encoding an anti-CRISPR associatedrepressor. In one embodiment, the nucleotide sequence encoding said at least oneheterologous polypeptide is inserted in the operon replacing or in partreplacing the sequence encoding an endogenous anti-crispr (acr) protein. In one embodiment, said promoter comprises the sequence set forth in SEQ ID NO: 1.P476852PC00 27In one embodiment, said anti-CRISPR repressor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO:2. In one embodiment, said anti-CRISPR repressor comprises the amino acid sequence set forth in SEQ ID NO: 3 (anti-CRISPR- associated_repressor_from_phage_DMS3) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 3.In one embodiment, the bacteriophage comprises an endogenous anti-CRISPRoperon, wherein said operon is genetically modified to encode and express said at least one heterologous polypeptide.In one embodiment, the bacteriophage comprises an exogenous anti-CRISPRoperon, wherein said operon is genetically modified to encode and express said at least one heterologous polypeptide.In one embodiment, said bacteriophage is lytic. In another embodiment, saidbacteriophage is temperate. In yet a further aspect the present invention provides a method for preparing bacteriophage of the present invention, said method comprising the steps of: (a) providing a polynucleotide encoding a bacteriophage; (b) providing polynucleotide encoding at least one inhibitor of aquorum sensing molecule and a promoter, which is regulated by a repressor;(c) inserting said polynucleotide encoding at least one inhibitor ofa quorum sensing molecule or heterologous polypeptide and said promoter inthe genome encoding said bacteriophage such that the polynucleotideencoding at least one inhibitor of a quorum sensing molecule or heterologouspolypeptide is operably linked to said promoter.In one embodiment the method further comprises inserting a polynucleotide sequence encoding said repressor such that said promoter regulatesP476852PC00 28transcription of said at least one inhibitor of a quorum sensing molecule or heterologous polypeptide and said repressor.In another embodiment, said promoter is an anti-CRISPR promoter and saidrepressor is an anti-CRISPR associated repressor.In one embodiment, said polynucleotide encoding at least one inhibitor of aquorum sensing molecule or heterologous polypeptide and a promoter, andoptionally said polynucleotide sequence encoding said repressor, is provided a single polynucleotide sequence and inserted in the polynucleotide encoding the bacteriophage. In one embodiment, said polynucleotide(s) encoding at least one inhibitor ofa quorum sensing molecule is inserted downstream of an acr promoter alreadypresent within the phage genome. Compositions comprising the bacteriophage of the present inventionIn another aspect, the present invention provides a composition comprisingthe bacteriophage of the present invention. The composition is typicallyprepared not to include ingredients that affect or significantly affect the viability of the bacteriophage. In fact, the composition may include one ormore ingredients that stabilizes the bacteriophage and increases the shelf lifeof the bacteriophage in the composition. The composition may be in dry form,semi dry or liquid form.The composition may comprise a combination of bacteriophage of the present invention. The combination of bacteriophage may be targeting the same or different bacteria. In another embodiment, combination of bacteriophage encodes different inhibitors of quorum sensing molecules. Accordingly, a composition comprising a combination of different bacteriophage may be useful for targeting a combination of pathogenic bacterial species and / orP476852PC00 29intervening with quorum sensing by inhibiting or eliminating more than one quorum sensing molecule of the system. Ingredients that stabilize the bacteriophage may be a sugar, such as a sugar selected from the group consisting of N-acetyl-D-glucosamine, 2-deoxy-D- glucose, maltose, L-rhamnose, cellobiose, and D-xylose. The titer of the bacteriophage in the composition is typically at least 105pfu / ml, such as at least 107pfu / ml, for example at least 109pfu / ml, such asin the range of 1010 - 1012 pfu / ml.In a further aspect, the composition is a pharmaceutical compositioncomprising the bacteriophage composition and at least one pharmaceuticalacceptable excipient. The pharmaceutical compositions of the presentinvention can be prepared by admixing a quantity of the bacteriophages witha pharmaceutically acceptable carrier. The pharmaceutical composition may for example be formulated for oraladministration, topical administration (such as transdermal administration oradministration to the eyes), pulmonary administration, intranasaladministration, rectal administration or intravenous administration.For example, the composition of the present invention may be foradministration in the form of an injectable composition. A typical compositionfor such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain about 10 mg of human serum albuminper millilitre of phosphate buffer containing NaCl. When the compositioncomprises a stabilizing sugar, the sugar concentration should be adapted to reach a non-toxic concentration as known to one skilled in the art.Pharmaceutically acceptable carriers / excipients include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences, 15th Ed. Easton: MackP476852PC00 30Publishing Co. pp 1405-1412 and 1461-1487 (1975) And The National Formulary XIV., 14th Ed. Washington: American Pharmaceutical Association (1975). Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers can include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobials, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the bacteriophage pharmaceutical compositions of the invention can be adjusted according to routine known in the art. Alternatively, the pharmaceutical compositions of the present invention can be in the form of liposomes, lipophilic microcapsules, dendrimers or the like for oral administration to treat systemic bacterial infections. Those skilled in the art are capable of preparing the compositions of the present invention in the form of a lipophilic microcapsule, a dendrimer or a liposome using conventional techniques known in the art. Use of anti-QS bacteriophage One aspect of the present invention provides that bacteriophage of the present invention for use as a medicament. In particular, the bacteriophage of the present invention or a composition comprising said bacteriophage is provided for use in treating bacterial-infectious diseases. Thus, in one embodiment, the bacteriophage or acomposition comprising said bacteriophage for use in the treatment of a bacterial infection. The compositions of the present invention can be used to treat mammals, suchas humans, having bacterial infections. Suitable bacteriophage-containingcompositions can be prepared that will be effective in killing, obliterating orP476852PC00 31reducing the quantity of any of the bacterial microorganisms using the guidelines presented above.The present invention further provides a method for treating bacterial infectionin a subject, comprising administering to the subject a pharmaceutical composition consisting of a therapeutically effective amount of thebacteriophage of the present or a pharmaceutically composition comprisingsaid bacteriophage and at least one pharmaceutical excipient. In the context of the present invention, the expression "treating bacterial infections," denotes either (i) killing or obliterating sufficient bacterial microorganisms to render the microorganisms ineffective in infecting the host, or (ii) reducing a sufficient quantity of bacterial microorganisms so as the render the microorganisms more susceptible to treatment using conventional antibiotics.In one embodiment, the bacteriophage is for use in the treatment of a bacterialinfection selected from the group consisting of a Pseudomonas sp infection,such as a Pseudomonas aeruginosa infection, Enterococcus faecium,Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus, Clostridium species, Bacillus cereus, Mycobacterium bovis, Brucella species, Yersinia enterocolitica, Listeria monocytogenes, Campylobacter, and Salmonella. Clostridium species, Bacillus cereus, Mycobacterium bovis, Brucella species,Yersinia enterocolitica, Listeria monocytogenes, Campylobacter, or Salmonellainfections are typically found in bovine species, porcine species and poultry. Milk and meat product obtained from the infected animals may thus be the source of infection of human beings, who have consumed or have been in contacted with infected matter. Accordingly, the bacteriophage of the present invention may be used for treating infections in the animals and / or humans infected with the pathogen.P476852PC00 32In one embodiment, the subject to be treated for the bacterial infection is a livestock, such as a cow, a sheep, a pig or a chicken. The subject may also be a pet animal, such as a dog or a cat. In another embodiment, the subject to be treat for a bacterial infection is a human subject.Bacterial infections comprising Pseudomonas sp infection, such as aPseudomonas aeruginosa infection, Enterococcus faecium, Staphylococcusaureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, orStreptococcus are typically transmitted from humans to humans either directlyor indirectly (for example through waste material comprising the pathogen). ESKAPE pathogens refers to the scientific names of six highly virulent and antibiotic resistant bacterial pathogens including: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,Pseudomonas aeruginosa, and Enterobacter spp.The bacteriophage of the present invention is a new intervention in the treatment of pathogenic bacteria that are resistant to conventional treatment with antibiotics. In one embodiment, the pathogen is a pathogenic bacterium resistant toantibiotics. In a particular embodiment, pathogen is an ESKAPE pathogensselected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonasaeruginosa and Enterobacter spp.In one embodiment, a combination of bacteriophages of the present invention is used for the treatment of for example an infection comprising more than one species of bacteria. A combination of bacteriophages may also be used toP476852PC00 33intervene with quorum sensing by inhibiting or eliminating more than onequorum sensing molecule of the system.The bacterial infection may manifest in the form a variety of bacterial infectious diseases. In one embodiment, the bacterial infection is selected from the group consisting of wounds such as burn wounds, diabetic foot ulcer, a urinary tract infection, gastrointestinal infection, infection of the eyes, pulmonary infections and blood infection. In another embodiment, the bacterial infection is selected from the groupconsisting infected medical devices / implants. Non-limiting examples ofmedical devices / implants includes pacemakers, mechanical cardiac valves,prosthesis such as hip prosthesis and knee prosthesis.The subject may be a subject that is particularly vulnerable to bacterial infection and in particular infection with an ESKAPE pathogen. Non-limiting examples of these subjects include immunocompromised subjects, preterminfants, diabetic subjects. The subject may also be suffering from anunderlying condition such as cystic fibrosis.The bacteriophage for use according to the present invention may beformulated to the particular infection to be treated. In one embodiment, the bacteriophage is formulated for oral administration, topical administration (such as administration to the eyes), pulmonary administration, rectal administration or intravenous administration.In a further aspect, the bacteriophage of the present invention is provided foruse against a plant pathogenic bacteria.P476852PC00 34The present invention further provides a method for treating bacterial infectionof a plant, comprising administering to the plant a composition consisting ofan effective amount of the bacteriophage of the present invention.In one embodiment, the plant pathogenic bacteria is selected from the groupconsisting of Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Xanthomonas axonopodis, Xanthomonas oryzae, Xylella fastidiosa, Dickeya dadantii, Dickeya solani, Agrobacterium tumefasciens, Erwinia amylovora, Pectobacterium carotovorum, and Pectobacterium atrosepticum. The bacteriophage may be formulated as a composition that is suitable for being for example dispersed over a field comprising infected crops. Forexample, a field of olive trees infected with Xylella fastidiosa.The terms “comprising”, “comprise” and “comprises” herein are intended by the inventors to be optionally substitutable with the terms “consisting of”, “consist of” and “consists of”, respectively, in every instance. The present invention is further characterized in the following non-limiting embodiments. Embodiment 1. A bacteriophage encoding and expressing at least one inhibitor of a quorum sensing molecule, wherein the expression of said at least one inhibitor of a quorum sensing molecule is operably linked to a promoter, wherein said promoter is regulated by a repressor.Embodiment 2. The bacteriophage according to embodiment 1, wherein saidpromoter regulates transcription of said at least one inhibitor of a quorum sensing molecule and said repressor.P476852PC00 35Embodiment 3. The bacteriophage according to any one of embodiments 1 or2, wherein said at least one inhibitor of a quorum sensing molecule and said repressor are encoded by a bi-cistronic or poly-cistronic transcript. Embodiment 4. The bacteriophage according to any of one the preceding embodiments, wherein said bacteriophage further encodes and expresses ananti-CRISPR (acr) protein.Embodiment 5. The bacteriophage according to embodiment 4, wherein saidat least one inhibitor of a quorum sensing molecule, said repressor and saidan anti-CRISPR (acr) protein are encoded by a poly-cistronic transcript.Embodiment 6. The bacteriophage according to any of one the preceding embodiments, wherein said promoter is an anti-CRISPR promoter and said repressor is an anti-CRISPR associated repressor. Embodiment 7. The bacteriophage according to any of one the precedingembodiments comprising an anti-CRISPR operon comprising an anti-CRISPRpromoter and encoding said at least one inhibitor of a quorum sensing molecule and a sequence encoding an anti-CRISPR associated repressor.Embodiment 8. The bacteriophage according to embodiment 7, wherein thenucleotide sequence encoding said at least one inhibitor of a quorum sensing molecule is inserted in the operon replacing or in part replacing the sequence encoding an endogenous anti-crispr (acr) protein. Embodiment 9. The bacteriophage according to any of one the preceding embodiments, wherein said promoter comprises the sequence set forth in SEQ ID NO: 1. Embodiment 10. The bacteriophage according to any of one the preceding embodiments, wherein said anti-CRISPR repressor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO:2.P476852PC00 36Embodiment 11. The bacteriophage according to any of one the preceding embodiments, wherein said anti-CRISPR repressor comprises the amino acid sequence set forth in SEQ ID NO: 3 (anti-CRISPR- associated_repressor_from_phage_DMS3) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 3. Embodiment 12. The bacteriophage according to any of one the precedingembodiments comprising an endogenous anti-CRISPR operon, wherein saidoperon is genetically modified to encode and express said at least one inhibitor of a quorum sensing molecule. Embodiment 13. The bacteriophage according to any of one the precedingembodiments comprising an exogenous anti-CRISPR operon, wherein saidoperon is genetically modified to encode and express said at least one inhibitor of a quorum sensing molecule. Embodiment 14. The bacteriophage according to any of one the preceding embodiments, wherein said bacteriophage is lytic. Embodiment 15. The bacteriophage according to any of one the preceding embodiments, wherein said bacteriophage is temperate. Embodiment 16. The bacteriophage according to any of one the preceding embodiments, wherein said at least one quorum sensing molecule inhibitor is selected from the group consisting of quorum sensing molecule inhibitor, quorum sensing molecule degrading enzymes, and a repressor of said quorum sensing molecule. Embodiment 17. The bacteriophage according to any of one the preceding embodiments, wherein said at least one quorum sensing molecule inhibitor is an enzyme selected from the group consisting of quorum sensing moleculedegrading enzymes such as alkylquinolone degradation C (AqdC),P476852PC00 37autoinducing peptide (AIP)-degrading proteases, AHL-degrading enzymes such as AHL lactonases and acylases (e.g. autoinducer inactivation A (AiiA), autoinducer inactivation B (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD), autoinducer inactivation S (AiiS), attachment gene M (AttM), N-acylhomoserine lactone D (AhlD), N- acylhomoserine lactone K (AhlK), N-acylhomoserine lactone M (AhlM), N- acylhomoserine lactone S (AhlS), metallo-β-lactamase superfamily lactonase (AaL), quorum-sensing signal degradation A (QsdA), quorum-sensing signal degradation H (QsdH), autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase (MCP), quorum-quenching lactonasefrom Geobacillus kaustophilus (GKL), NAHL-lactonase (QlcA), quorum signalutilization and inactivation protein (QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA), Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHL acylase B (HacB). Embodiment 18. The bacteriophage according to any of one the preceding embodiments, wherein said repressor of said quorum sensing molecule is selected from the group consisting of a synthase synthesizing said repressor of said quorum sensing molecule, an antisense RNA or CRISPRi repressing expression said quorum sensing molecule. Embodiment 19. The bacteriophage according to any of one the preceding embodiments, wherein said quorum sensing molecule is selected from thegroup consisting of N-acyl homoserine lactones including harveyi autoinducer1 (HAI-1), Pseudomonas Quinolone Signal 2-heptyl-3-hydroxy-4-quinolone(PQS), 4-hydroxy-2-heptylquinoline (HHQ), 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), autoinducer-2 ((3aS,6S,6aR)-2,2,6,6a-Tetrahydroxy-3a-methyltetrahydro-2H-furo[2,3-d][1,3,2]dioxaborol-2-uide), Auto inducing peptides (AIP), diffusible signal factor (DSF, cis-11-methyldodecenoicacid),methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME), cholerae autoinducer 1 (CAI-1), , and 3,5-dimethyl-pyrazin-2-ol(DPO).P476852PC00 38Embodiment 20. The bacteriophage according to any of one the preceding embodiments, wherein the sequence encoding said at least one quorum sensing molecule inhibitor comprises the sequence set forth in SEQ ID NO: 4 (encoding QsdA). Embodiment 21. The bacteriophage according to any of one the preceding embodiments, wherein the sequence encoding said at least one quorum sensing molecule inhibitor comprises the sequence set forth in SEQ ID NO: 5 (encoding AqdC). Embodiment 22. The bacteriophage according to any of one the preceding embodiments, wherein said at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 6 (QsdA) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 6. Embodiment 23. The bacteriophage according to any of one the preceding embodiments, wherein said at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 7 (AqdC) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 7. Embodiment 24. The bacteriophage according to any of one the preceding embodiments, wherein said bacteriophage is capable of infecting at least onebacteria selected from the group consisting of a Pseudomonas sp, such asPseudomonas aeruginosa, Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp.,Escherichia coli, Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Xylella fastidiosa, Dickeya dadantii, Agrobacterium tumefasciens, and Pectobacterium atrosepticum. Embodiment 25. The bacteriophage according to any of one the preceding embodiments, wherein said bacteriophage is selected from the listingP476852PC00 39consisting of Phage DMS3 (accession_no.NC_008717.1), Phage JBD44 (accession_no.NC_030929.1), and phages classified in the orders of Caudoviricetes, Kalamavirales, Mindivirales, Timlovirales, Tubulavirales, Vinavirales, and Durnavirales, and unclassified or reclassified phages. Embodiment 26. The bacteriophage according to any of one the precedingembodiments for use as a medicament.Embodiment 27. The bacteriophage according to any of one the precedingembodiments for use in the treatment of a bacterial infection.Embodiment 28. The bacteriophage according to any of one the precedingembodiments for use in the treatment of a bacterial infection selected fromthe group consisting of a Pseudomonas sp infection, such as a Pseudomonasaeruginosa infection, Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus, Clostridium species, Bacillus cereus, Mycobacterium bovis, Brucella species,Yersinia enterocolitica, Listeria monocytogenes, Campylobacter, andSalmonella Embodiment 29. The bacteriophage for use according to any one ofembodiments 27 and 28, wherein said infection is selected from the groupconsisting of infected medical devices / implants, wounds such as burn wounds, diabetic foot ulcer, a urinary tract infection, gastrointestinal infection, eye infection, pulmonary infections and blood infection. Embodiment 30. The bacteriophage for use according to any one ofembodiments 27 to 29, wherein the subject is an immunocompromisedsubject, preterm infant, diabetic subject. Embodiment 31. The bacteriophage for use according to any one ofembodiments 27 to 30, wherein the subject is a human subject.P476852PC00 40Embodiment 32. The bacteriophage for use according to any one ofembodiments 27 to 31, wherein said bacteriophage is formulated for oraladministration, topical administration (such as administration to the eyes),pulmonary administration, rectal administration, or intravenous administration. Embodiment 33. A composition comprising the bacteriophage according to anyone of embodiments 1 and 25.Embodiment 34. A pharmaceutical composition comprising the bacteriophageaccording to any one of embodiments 1 and 25 and at least onepharmaceutical acceptable excipient. Embodiment 35. Use of bacteriophage according to any of one the precedingembodiments against a plant pathogenic bacteria.Embodiment 36. The use according to embodiment 35, wherein said plantpathogenic bacteria is selected from the group consisting of Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Xanthomonas axonopodis, Xanthomonas oryzae, Xylella fastidiosa, Dickeya dadantii, Dickeya solani, Agrobacterium tumefasciens, Erwinia amylovora, Pectobacterium carotovorum, and Pectobacterium atrosepticum. Embodiment 37. A recombinant bacteriophage comprising a promoter,wherein said promoter is regulated by a repressor and a multiple cloning sitefor the insertion of a polynucleotide sequence encoding a heterologouspolypeptide, wherein the expression of said heterologous polypeptide isoperably linked to said promoter.Embodiment 38. A bacteriophage encoding and expressing at least oneheterologous polypeptide (or heterologous peptide), wherein the expressionof said at least one heterologous polypeptide is operably linked to an anti-P476852PC00 41CRISPR promoter, wherein said promoter is regulated by a repressor, wherein said repressor is an anti-CRISPR associated repressor.Embodiment 39. The bacteriophage according to embodiment 38, wherein saidanti-CRISPR promoter regulates transcription of the sequence encoding saidat least one heterologous polypeptide (or heterologous peptide) and said anti-CRISPR associated repressor. Embodiment 40. The bacteriophage according to any one of embodiments 38or 39, wherein said at least one heterologous polypeptide is an enzyme, suchas a lactonase, a hydrolase, a AHL acylase or a protease.Embodiment 41. The bacteriophage according to any one of embodiments 38 to 40, wherein said enzyme is selected from the group consisting ofalkylquinolone degradation C (AqdC), autoinducing peptide (AIP)-degradingproteases, AHL-degrading enzymes such as AHL lactonases and acylases (e.g. autoinducer inactivation A (AiiA), autoinducer inactivation B (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD), autoinducer inactivation S (AiiS), attachment gene M (AttM), N- acylhomoserine lactone D (AhlD), N-acylhomoserine lactone K (AhlK), N- acylhomoserine lactone M (AhlM), N-acylhomoserine lactone S (AhlS), metallo-β-lactamase superfamily lactonase (AaL), quorum-sensing signal degradation A (QsdA), quorum-sensing signal degradation H (QsdH), autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase(MCP), quorum-quenching lactonase from Geobacillus kaustophilus (GKL),NAHL-lactonase (QlcA), quorum signal utilization and inactivation protein (QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA), Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHL acylase B (HacB). Embodiment 42. The bacteriophage according to any one of embodiments 38to 41, wherein said at least one heterologous polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 6 (QsdA) or amino acid sequence havingP476852PC00 42at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 6 and / or said at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 7 (AqdC) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 7. Embodiment 43. The bacteriophage according to any one of embodiments 38 to 42, wherein the sequence encoding said at least one heterologouspolypeptide comprises the sequence set forth in SEQ ID NO: 4 (encodingQsdA). Embodiment 44. The bacteriophage according to any one of embodiments 38 to 43, wherein the sequence encoding said at least one heterologouspolypeptide comprises the sequence set forth in SEQ ID NO: 5 (encodingAqdC). Embodiment 45. The bacteriophage according to any one of embodiments 38 to 44, comprising an anti-CRISPR operon comprising an anti-CRISPR promoterand encoding said at least one heterologous polypeptide and a sequenceencoding an anti-CRISPR associated repressor.Embodiment 46. The bacteriophage according to embodiment 45, wherein thenucleotide sequence encoding said at least one heterologous polypeptide (orat least one heterologous peptide) is inserted in the operon replacing or in partreplacing the sequence encoding an endogenous anti-crispr (acr) protein. Embodiment 47. The bacteriophage according to any one of embodiments 38 to 46, wherein said promoter comprises the sequence set forth in SEQ ID NO: 1. Embodiment 48. The bacteriophage according to any one of embodiments 38 to 47, wherein said anti-CRISPR repressor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO:2.P476852PC00 43Embodiment 49. The bacteriophage according to any one of embodiments 38 to 48, wherein said anti-CRISPR repressor comprises the amino acid sequence set forth in SEQ ID NO: 3 (anti-CRISPR- associated_repressor_from_phage_DMS3) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 3. Embodiment 50. The bacteriophage according to any one of embodiments 38 to 49 comprising an endogenous anti-CRISPR operon, wherein said operon is genetically modified to encode and express said at least one heterologouspolypeptide (or at least one heterologous peptide).Embodiment 51. The bacteriophage according to any one of embodiments 38 to 50 comprising an exogenous anti-CRISPR operon, wherein said operon is genetically modified to encode and express said at least one heterologouspolypeptide (or at least one heterologous peptide).Embodiment 52. The bacteriophage according to any one of embodiments 38 to 50, wherein said bacteriophage is lytic. Embodiment 53. The bacteriophage according to any one of embodiments 38 to 52, wherein said bacteriophage is temperate. Embodiment 54. A method for preparing bacteriophage according to any one of embodiment 1 to 13, said method comprising the steps of: (a) providing a polynucleotide encoding a bacteriophage; (b) providing polynucleotide encoding at least one inhibitor of aquorum sensing molecule and a promoter, which is regulated by a repressor;(c) inserting said polynucleotide encoding at least one inhibitor ofa quorum sensing molecule and said promoter in the genome encoding saidbacteriophage such that the polynucleotide encoding at least one inhibitor ofa quorum sensing molecule is operably linked to said promoterP476852PC00 44Embodiment 55. The method of embodiment 54, further inserting a polynucleotide sequence encoding said repressor such that said promoter regulates transcription of said at least one inhibitor of a quorum sensing molecule and said repressor Embodiment 56. The method of embodiment 54 or 55, wherein said promoter is an anti-CRISPR promoter and said repressor is an anti-CRISPR associated repressor. Embodiment 57. The method according to any one of embodiment 54 to 56, wherein said polynucleotide encoding at least one inhibitor of a quorumsensing molecule and a promoter, and optionally said polynucleotide sequenceencoding said repressor, is provided a single polynucleotide sequence and inserted in the polynucleotide encoding the bacteriophage. Embodiment 58. The method according to any one of embodiments 54 to 57, wherein the said polynucleotide(s) encoding at least one inhibitor of a quorumsensing molecule is inserted downstream of an acr promoter already presentwithin the phage genome. Examples ResultsP. aeruginosa is often associated with hospital-acquired infections and posesan urgent global health risk. The pathogen communicates using AIs tocoordinate behavior such as biofilm formation and virulence in addition to launching anti-phage defense systems. Thus, an effective anti-bacterial strategy is to inhibit AIs, thereby disarming the pathogen without killing it. Another promising antimicrobial approach is the use of phages to infect and kill bacteria. The inventors engineered the P. aeruginosa phage JBD44 (accession no. NC_030929.1) (SEQ ID NO: 11) and phage DMS3 (accession no.P476852PC00 45NC_008717.1) (SEQ ID NO: 10) to express the N-acylhomoserine lactonaseQsdA, which cleaves AHLs (Example 1B), and the PQS dioxygenase AqdCthat cleaves PQS (Example 1D) and reduces P. aeruginosa virulence(Examples 2-3). To ensure rapid and potent qsdA and aqdC expression, theinventors cloned them under the anti-CRISPR promoter and controlled by theanti-CRISPR-associated repressor. The DMS3 phage naturally encodes the anti-QS peptide Aqs1 that binds and inhibits LasR. To exclusively assess the anti-QS effect of expressing AI-degrading enzymes from DMS3, the inventors additionally mutated the aqs1 gene from DMS3, preventing Aqs1 from binding and blocking LasR. The inventors used a Galleria mellonella, wax moth larvae burn wound modelof P. aeruginosa infection, since it closely mimics the infection dynamics ofmammalian infection models. This model offers a robust in vivo infectionsystem and it is ideal for the purpose of comparing the effectiveness of different therapies against P. aeruginosa infection.First, the inventors addressed if the anti-QS effect of the engineered phagescould rescue G. mellonella wax moth larvae from P. aeruginosa infection in alysogenic state i.e. without phage-mediated killing of P. aeruginosa(Examples 2-3). The Pacr promoter used in the engineered phages drivesearly and rapid expression upon phage infection but is then repressed and controlled by the Aca repressor. To mimic an acute phage infection expressionlevel of anti-QS genes, the inventors used the Pacr promoter but without theAca repressor to control expression in a JBD44 lysogen of P. aeruginosa UCBPP-PA14. This allows for high anti-QS gene expression in the lysogenic state of the prophage. To quantify the ability of the anti-QS constructs in theengineered phages to inhibit P. aeruginosa virulence, the inventors used arecently established G. mellonella larvae in vivo burn wound infection model.Strikingly, the QsdA and AqdC expressing anti-QS JBD44 prophage effectivelydisarmed P. aeruginosa, resulting in equal survival rates as burnwoundcontrols after 5 days (Fig. 2). In stark contrast, 90% of the larvae infectedwith P. aeruginosa carrying the wildtype (WT) JBD44 prophage died (Fig. 2).P476852PC00 46Thus, expressing AI-degrading enzymes from a prophage was sufficient to render P. aeruginosa harmless and protect G. mellonella larvae. To further investigate the ability of the anti-QS phages to rescue G. mellonellafrom P. aeruginosa infection, the inventors infected the larvae with PA14 WTculture instead of PA14 JBD44 lysogens (Examples 2-3). One hour afterinitiating PA14 infection, the inventors subjected the larvae to various DMS3phage treatments to test the effect of phage infection with the parental DMS3aqs1_FSDARE vir phage or phage infection with a 1:1 mixture of virulent DMS3phages expressing qsdA or aqdC, respectively. Similarly, to what the inventorshad observed with the anti-QS JBD44 lysogens, anti-QS phage treatment withqsdA- and aqdC-carrying DMS3 vir phages resulted in significantly highersurvival rates as compared to phage treatment with the parental DMS3 aqs1_FSDARE vir phage (Fig. 3). This further demonstrates the potential ofthe anti-QS phages to protect G. mellonella from P. aeruginosa infection.Phage-resistant mutants are naturally selected for in populations during phageexposure. Therefore, the inventors challenged the robustness of the anti-QSphages to inhibit QS activation in mixed populations of phage-susceptible and phage-resistant cells. As phage receptors perform key functions in bacteria, including virulence, the inventors created a phage-resistant mutant without deleting the phage receptor to avoid adverse effects. Instead, the inventors generated a JBD44 prophage lacking the ability to create phage particles, to provide phage-resistance. Specifically, the inventors deleted the JBD44 genesgp1-39 in a PA14 JBD44 lysogen. This strain, PA14 JBD44 Δgp1-39, wasresistant to JBD44 phage infection, unlike the WT PA14 strain (Fig. 4). Likewise, the inventors created a PA14 DMS3 aqs1_FSDARE Δgp26-48 lysogen, resistant to DMS3 vir infection. To confirm that it was resistant, DMS3vir was applied in a line on a vertical line on a plate and PA14 WT and the PA14DMS3 aqs1_FSDARE Δgp26-48 lysogen were streaked across the line of phage, resulting in killing of PA14 WT only (Fig. 5). The inventors then testedthe ability of the DMS3 anti-QS phage to degrade QS molecules in a mixedpopulation of phage-susceptible and phage-resistant P. aeruginosa cells.P476852PC00 47Specifically, the inventors infected co-cultures of phage-susceptible and phage-resistant cells at a 9-to-1 ratio with a multiplicity of infection (MOI) of10 and quantified the relative QS molecule levels. 3O-C12-HSL and PQS levelswere significantly reduced in response to anti-QS phage infection (Example6). The inventors additionally tested the ability of the JBD44 anti-QS phage toeffectively silence and / or kill mixed populations of phage-susceptible andphage-resistant P. aeruginosa cells. Specifically, the inventors infected co-cultures of phage-susceptible and phage-resistant cells at a 9-to-1 ratio withan MOI of 5 and incubated for 5.5 h to achieve phage infection of susceptiblecells, while allowing phage-resistant cells to grow, reach high cell densities to activate QS, and produce the QS-activated virulence factor pyocyanin (Example 7). Remarkably, the anti-QS phage significantly inhibited pyocyanin accumulation by 7.8-fold and reduced the cell density of the mixed populations by 2.5-fold compared to the WT phage (Fig. 7), implying that the anti-QS phage enabled sufficient QsdA and AqdC enzyme production to degrade 3OC12-HSL and PQS and thereby ultimately limiting pyocyanin accumulation. Thus, the anti-QS phage effectively inhibited the growth of mixed populations of phage-susceptible and phage-resistant cells in addition to disarming the emerging phage-resistant population, underlining that phages encoding quorum sensing molecule degrading enzymes may be highly potent for phage therapy.The inventors tested whether the ability of the anti-QS phages to reduceaccumulation of 3O-C12-HSL and PQS in phage-susceptible and phage-resistant P. aeruginosa co-cultures is reflected in reduced virulence. Theinventors infected G. mellonella with a mixture of phage-susceptible andphage-resistant P. aeruginosa cells (Example 8). One hour after initiatinginfection, the inventors subjected the larvae to phage treatments with theparental DMS3 aqs1_FSDARE vir phage or the 1:1 mixture of virulent DMS3aqs1_FSDARE vir phages expressing qsdA or aqdC, respectively. The anti-QSphage treatment with qsdA- and aqdC-expressing DMS3 aqs1_FSDARE virphages resulted in significantly higher survival rates as compared to phage treatment with the parental DMS3 aqs1_FSDARE vir phage (Fig. 8). ThisP476852PC00 48demonstrates that the anti-QS phages remain effective, even againstpopulations with 10% phage-resistant P. aeruginosa cells. Although phage-resistant cells survive phage infection, anti-QS phages manage to disarm themby releasing the anti-QS enzymes QsdA and AqdC from infected phage- susceptible cells. Phage infection of P. aeruginosa can trigger a specific phage defense mediated by a stress response in which the infected cells overproduce PQS. Healthy nearby P. aeruginosa cells which are motile through a process known asswarming sense the PQS signal released by the infected cells, and these motileswarm cells redirect to avoid colliding with the infected cells. The inventors tested the effect of the anti-QS phages to inhibit PQS production during phage- infection of bacterial colonies and thereby avoid repulsion of uninfected cellsin a swarming assay. Whereas infection by the phage that does not inhibit thePQS-caused repulsion of the uninfected swarm cells 4 out of 4 times, the anti-QS phage-infected colonies were unable to repulse healthy swarm cells, which collided with the uninfected swarms in every scenario (Example 8). Thus, the anti-QS phages effectively inhibit a physical phage defense mechanism, known as the collective stress response, by limiting PQS accumulation. Discussion The inventors present engineered anti-QS phages as precision antimicrobials that combine the unconventional treatment options of quorum quenching andphage therapy to treat P. aeruginosa infections in G. mellonella wax mothlarvae (Fig. 2-3 and 8). This synergistic combination allows specific pathogenkilling alongside production and release of the anti-QS enzymes QsdA and AqdC at the site of infection. This approach may enhance the antimicrobial effect of phages by silencing the bacteria evolving immunity to the phages, asAI degrading enzymes released from infected phage-sensitive bacteriadegrade the extracellular AIs emitted by any phage-immune bacteria (Fig. 6),hindering activation of virulence of the remaining cells (Fig. 7-8). Additionally,the anti-QS phages inhibit phage defenses, allowing for more efficient phagespread and infection (Table 2). In a patient setting, this may pave the wayP476852PC00 49for natural clearance of residual phage-resistant mutants by the human immune system and / or healthy microbiota.The inventors discovered that the anti-QS enzymes QsdA and AqdC expressedfrom a prophage as well as from virulent phages disarm P. aeruginosa andimprove survival of P. aeruginosa-infected G. mellonella larvae (Fig. 2-3),even in the presence of phage-resistant P. aeruginosa cells (Fig. 8), and thatthe anti-QS phages effectively limit virulence factor production in co-cultures of phage-susceptible and phage-resistant populations (Fig. 7). While previous studies have shown that expressing quorum quenching enzymes fromplasmids limits P. aeruginosa virulence effectively, applying purified quorumquenching enzymes to treat P. aeruginosa. This limited success could additionally be due to the anti-QS enzymes being degraded or extensivelydiluted in the animal model before reaching the site of infection. The inventors’approach circumvents these limitations by enabling in situ amplification of the “drug” at the site of infection, using a phage specific for the pathogen, in addition to providing phage-mediated clearance of the targeted pathogen. As QS controls virulence properties in a broad range of pathogens infecting humans, animals, and plants, the concept of combining quorum-modulating proteins and phage therapy may be generalized to target other bacterial pathogens beyond the human pathogen P. aeruginosa described here. The inventors showed that QsdA can degrade a broad range of N-acylhomoserine lactones (Fig.9), therefore this enzyme can reduce quorum sensing in a broad range of Gram-negative species. The principle of arming phages with the ability to manipulate their host bacteria for reduced bacterial virulence and enhanced bacterial killing may pave the way for implementing effective phage therapy to mitigate the potential global disaster of a post antibiotic area. Methods Example 1P476852PC00 50Bacterial growth conditions. P. aeruginosa and E. coli strains were grownat 37 °C with 300 rpm shaking in Luria-Bertani (LB) broth or on LB agar plates containing 1.5% agar (wt / vol). When appropriate, overnight cultures were diluted in fresh LB medium to a lower OD600. Bacterial strains, plasmids, andphages used in this study are listed in Table 1.DNA manipulations. JBD44 and DMS3 phages were armed with genes encoding the N-acylhomoserine lactonase QsdA from Rhodococcuserythropolis (accession no. AP008957.1) and the PQS dioxygenase AqdC fromMycobacteroides abscessus (accession no. CP050978.1) using homologousrecombination. For phages encoding both enzymes, qsdA and aqdC were placed in tandem and separated by 3 bp (GTT). For the DMS3 phage, the constructs were inserted downstream of the anti-CRISPR promoter, while deleting the type I-E anti-CRISPR gene (DMS3-30) but leaving the anti- CRISPR-associated repressor gene (DMS3-31) intact in lysogens carrying DMS3 prophages. For the JBD44 phage, the anti-CRISPR expression system was used to control transcription of the anti-QS constructs. These wereinserted between JBD44 gp46 and gp47 and the Lambda T1 transcriptionalterminator was included immediately downstream of the inserts. Details are outlined below. Plasmids were extracted using NucleoSpin® Plasmid EasyPure kit (Machery- Nagel, Ref.740727.250). Plasmids for cloning were digested with FastDigest™restriction enzymes (Thermo Scientific™) and dephosphorylated using FastAPThermosensitive Alkaline Phosphatase (Thermo Scientific™, Ref. EF0654)according to manufacturer’s instructions. Linearized plasmids were gel- purified using NucleoSpin® Gel and PCR Clean-up kit (Machery-Nagel, Ref. 740609.250). For cloning purposes, PCRs were carried out using the Q5® HighFidelity DNA Polymerase (New England BioLabs, Ref. M0491L). For otherpurposes, PCRs were performed using the DreamTaq DNA Polymerase (Thermo Scientific™, Ref. EP0702). PCR products were purified using NucleoSpin® Gel and PCR Clean-up kit (Machery-Nagel, Ref. 740609.250). Desired inserts were assembled with digested plasmids using T4 DNA LigaseP476852PC00 51(Thermo Scientific™, Ref. EL0016) or Gibson assembly using NEBuilder® HiFi DNA Assembly (New England BioLabs, Ref. M5520AA) according tomanufacturer’s instructions. Plasmids were recovered in One Shot™ TOP10Chemically Competent Cells (Invitrogen, Ref. C404003) according tomanufacturer’s instructions or in E. coli SM10 λpir using TSS chemicalcompetence. Plasmids were confirmed by PCR and sequencing (Eurofinsand / or Plasmidsaurus). Sanger sequencing of pEXG2-based plasmids was done using primers NMHK250+251. Sanger sequencing of crRNAs and repairtemplate in pAB01 was done using primers NHMK197+198 and 225+226,respectively.The pEXG2-based plasmids were conjugated into P. aeruginosa strains bymating. Exconjugants were selected on LB containing 30 µg ml-1gentamicin and 100 µg ml-1irgasan and cultured at 37 °C overnight. Colonies were grown in LB at 37 °C for 2 h, plated on LB containing 15% sucrose and no NaCl andcultured at 37 °C overnight. Mutants were confirmed by PCR and sequencing(Eurofins). The pAB01-based plasmids were electroporated into P. aeruginosa PA14 pre-washed in 300 mM sucrose, and recovered in LB at 37 °C for 1 h and thenplated on LB containing 50 µg ml-1gentamicin.Plasmid pEXG2-DMS3-aqs1_FSDARE was constructed by PCR amplifyingchosen regions from DMS3 phage using primer pairs 119+120 and 121+122 designed with 50 bp overlaps. The fragments were fused using PCR andinserted into BamHI- and HindIII-digested and dephosphorylated pEXG2 usingGibson assembly. The resulting mutations cause a change in Aqs1 amino acid sequence position 39-44 from YRDALD to FSDARE in phage DMS3. The DMS3 prophages were confirmed by PCR and sequencing using primers 19+20 (Eurofins).Plasmid pEXG2-DMS3-∆acrI-E::qsdA-aqdC was constructed by PCR amplifyingchosen regions from DMS3 phage using primer pairs 21+83 and 171+26. TheP476852PC00 52synthesized genes qsdA (TAG Copenhagen) and aqdC (Eurofins) were PCR amplified using primer pairs 85+82 and 81+172, respectively. The fragments were designed with 50 bp overlaps, fused using PCR and finally inserted intoBamHI- and HindIII-digested and dephosphorylated pEXG2 using Gibsonassembly. DMS3 prophages were confirmed by PCR and sequencing using primers 27+28+129+172 (Eurofins).Plasmid was constructed by PCR amplifyingchosen regions from pEXG2-DMS3-∆acrI-E::qsdA-aqdC using primer pairs21+22 and 23+26 designed with 50 bp overlap. The fragments were fusedusing PCR and inserted into BamHI- and HindIII-digested anddephosphorylated pEXG2 using Gibson assembly. DMS3 prophages wereconfirmed by PCR and sequencing using primers 27+28 (Eurofins). was constructed by PCR amplifyingchosen regions from pEXG2-DMS3-∆acrI-E::qsdA-aqdC using primer pairs21+183 and 184+26 designed with 50 bp overlap. The fragments were fusedusing PCR and inserted into BamHI- and XhoI-digested and dephosphorylatedpEXG2 using Gibson assembly. DMS3 prophages were confirmed by PCR andsequencing using primers 27+28 (Eurofins).Plasmid 48 was constructed by PCR amplifying chosenregions from DMS3 phage using primer pairs 150+151 and 152+153 designed with 50 bp overlaps. The fragments were fused using PCR and inserted intoBamHI- and HindIII-digested and dephosphorylated pEXG2 using Gibsonassembly. DMS3 prophages were confirmed by PCR and sequencing using primers 154+155 (Eurofins).Plasmid was constructed by using primers 215+216 and plasmidpHERD30T as template in site-directed mutagenesis PCR. Briefly, primers with overlapping regions containing the crRNA insert were used to generate a synthetic plasmid from the template pHERD30T plasmid. The Dam-methylated template plasmid was selectively degraded using DpnI digestion leaving theP476852PC00 53synthetic plasmid intact. The protocol is based on the paper by Liu and Naismith. The Q5 PCR reactions containing 5% DMSO, the GC enhancer and primers at 0.4 µM each were run on a gradient with annealing temperatures of 55-70 °C and 6 min extension time. Subsequently, the reactions were pooled and DpnI-digested at 37 °C for 4 h. Then, the synthetic plasmid wastransformed into E. coli as described earlier.Plasmid crRNA-DMS3-cI was constructed by phosphorylating primers217+218 using T4 Polynucleotide Kinase (Thermo Scientific™, Ref. EK0031) according to manufacturer’s instructions. Then, the primers were brought to 50 mM NaCl final concentration using a 4 M NaCl stock and annealed at 98 C for 5 min, then gradually cooling down 2 degrees at a time with 30 sec at each temperature. Then, 30 nM of annealed primers were inserted into 40 ng BbsI- digested and dephosphorylated pAB01 using T4 ligase at room temperature overnight.Plasmid DMS3-∆cI was constructed by PCR amplifying chosen regionsfrom phage DMS3 using primer pairs 223+227 and 228+229 designed with 50 bp overlap. The fragments were fused using PCR and inserted into NheI-digested and dephosphorylated pAB01-crRNA-DMS3-cI using Gibsonassembly. This template can delete bp position 154-380 of DMS3 cI repressor (DMS3-1).Virulent DMS3 were constructed by infecting 1:1,000 back-dilutedovernight cultures of P. aeruginosa PA14 pAB01-DMS3-∆cI with 200 µl ofsterile-filtered supernatants from overnight cultures of various DMS3 lysogens and cultured in 2 ml LB containing 50 µg ml-1gentamicin and 0.1% arabinose at 37 °C for 5 h. To isolate single plaques, plate lysates were created using500 µl supernatant and 200 µl uninfected P. aeruginosa PA14 pAB01-DMS3-∆cI overnight culture in soft agar containing 50 µg ml-1gentamicin and 0.1% arabinose and incubated at 37 °C overnight. Virulent DMS3 phages were confirmed by sequencing with primers #6+35 and re-streaked on lawns ofPA14 pAB01-DMS3-∆cI.P476852PC00 54Cloning of JBD44 prophages and phages was carried out following similarprinciples and protocols as described above for DMS3 phages. Phage infection assaysP. aeruginosa PA14 cultures were challenged with DMS3 aqs1_FSDARE vir oranti-QS variants. The phages were mixed in a 1:1 ratio for the dual phage treatment. At OD600~0.14, phage-susceptible PA14 was infected with a 10- fold excess of phages (multiplicity of infection = 10). (A) OD600 was measuredto monitor bacterial growth post phage infection and supernatants weresampled and 0.2-µm sterile-filtered for QS molecule measurements.Luminescence reporter assays – QS molecule measurementsTo evaluate the relative abundances of the QS molecules 3O-C12-HSL, C4- HSL, and PQS in sterile-filtered culture supernatants, E. coli strains carryingan arabinose inducible pBAD expression vector and a pCS26 lux reporter wereused. In these strains, arabinose induces production of either the 3O-C12-HSL-responsive LasR, C4-HSL-responsive RhlR, or PQS-responsive PqsR QSreceptors driving the expression from the lasB, rhlA, or the pqsA promoters,respectively, upstream of the luxCDABE operon in pCS26. To evaluate therelative abundances of the QS molecules C4-HSL, C6-HSL, 3O-C6-HSL, C8- HSL, 3O-C8-HSL, C10-HSL, and 3O-C12-HSL, an E. coli reporter with thegadW promoter upstream of the luxCDABE operon in pCS26 was used, takingadvantage of the native E. coli HSL-responsive SdiA QS receptor. Thus, these strains sense the QS molecules and produce luminescence as a result. Briefly, overnight cultures of the E. coli strains carrying the respective plasmids were 100-fold diluted in LB containing 100 µg ml-1kanamycin, and for the E. colistrains also carrying pBAD 100 µg ml-1 ampicillin and 0.1% arabinose wasadded. 10 µl P. aeruginosa sterile-filtered culture supernatants was added in a total volume of 200 µl per well. For PQS measurements, the P. aeruginosa supernatants were 100-fold diluted, whereas for measurements of C4-HSL, C6-HSL, 3O-C6-HSL, C8-HSL, 3O-C8-HSL, C10-HSL, and 3O-C12-HSL, the supernatants were undiluted. The measurements were carried out in black-P476852PC00 55well, clear-bottom 96-well plates with intermittent shaking at 30 °C and OD600and luminescence was measured using a Synergy H1 plate reader (BioTekInstruments Inc.) with software Gen5 v.3.05.11. The values are reported asRLU (lum / OD600) (relative light units). Phage proliferation Briefly, the engineered phages were purified by re-streaking single plaques on PA14 lawns three times, resuspended in SM buffer, and proliferated using plate lysates with PA14 lawns. In more detail, the single plaques were resuspended in 500 µl SM buffer and after three rounds of re-streaking, 300 µl of the resuspended plaque solution was mixed with 50 µl PA14 overnight culture and 5 ml molten soft agar, subsequently poured onto LB agar plates and incubated at 37 °C overnight. Next, 5 mL SM buffer was added to the infected lawns and incubated for 3-5 h at room temperature. The phages were then collected, 0.2-µm sterile-filtered and enumerated using standard plaque assay on lawnsof PA14 or PA14 pAB01-DMS3-∆cI containing 50 and 200 ul overnight culture,respectively. For lawns of PA14 pAB01-DMS3-∆cI, 50 µg ml-1gentamicin and 0.1% arabinose were embedded in the soft agar.Examples 2-3 - Larvae burn wound infection assayGalleria mellonella wax moth larvae (raised antibiotics free) were ordered froma local pet shop. Once delivered, larvae were immediately stored at 12 °C for no longer than 10 days. Before running an experiment, the larvae were sortedaccording to size and placed in petri dishes with 2 larvae per dish. The larvalbodies were sterilized using 70% ethanol and placed back at 12 °C briefly until further use. A burn wound was created using the head of a stainless-steel nailwith a diameter of 2 mm. The nail was heated until red using a Bunsen burnerand let to cool down for 4 sec before gently applying the nail head to the back of the larvae for a total of 4 sec. To infect the fresh wounds, 10 µl of OD = 1standardized inoculum of bacterial overnight culture was slowly pipetted ontothe wound. In addition to untreated larvae, larvae with an uninfected woundwere included as a control. As indicated, burn wounds were treated with a totalP476852PC00 56of 109 PFU (plaque forming units) of phage one hour after bacterial infection.The larvae were placed in the 37 °C incubator for up to 5 days and monitored for survival at regular intervals.Example 4 - Phage susceptibility test in a liquid infection assayP. aeruginosa PA14 and PA14 lysogen JBD44 Δgp1-39 were treated with eitherSM buffer solvent control or with a MOI 10 of WT JBD44 phage. OD600 was measured at 37 °C in a plate reader over a 24 h period.Example 5 - Cross streak assay phage susceptibility testA phage lysate of DMS3 vir was applied in a vertical line across the surface of an agar plate. One dry, a single colony of PA14 and the PA14 DMS3aqs1_FSDARE Δgp26-48 lysogen were streaked perpendicularly across the lineof phage lysate, exposing these cells to the phage. The plate was incubated overnight at 37 °C.Example 6 – Phage infection of phage-susceptible and phage-resistantco-cultures and QS molecule measurementPhage-susceptible (PA14 WT) and phage-resistant cells (PA14 DMS3aqs1_FSDARE Δgp26-48 lysogen) were co-cultured at a ratio of 90:10 andtreated with DMS3 aqs1_FSDARE vir phage, as well as phages expressingeither of the anti-QS enzymes, or a 1:1 mixture of aqdC- and qsdA-expressingphages. At OD600~0.14 PA14 co-cultures were infected with a 10-fold excess of phages (multiplicity of infection = 10). (A) OD600 was measured to monitor bacterial growth post phage infection. The relative abundance of 3O-C12-HSL,C4-HSL, and PQS was quantified by E. coli bioreporters, as described inExample 1, producing relative light units as a response to the QS molecules.The values are reported as RLU (lum / OD600) (relative light units).P476852PC00 57Example 7 - Phage infection of phage-susceptible and phage-resistantco-cultures and virulence factor measurementPA14 and PA14 JBD44 Δgp1-39 overnight cultures were mixed in a 90:10 ratiobased on OD600, back-diluted to OD600=0.01 in LB and grown at 37 °C shaking with 300 rpm. At OD600=0.15, the mixed co-cultures were infected with eitherJBD44 WT phage or phage JBD44 ∆gp48-53 gp46-47::Pacr-qsdA-aqdC-aca ata MOI of 5. After 5.5 h post infection, pyocyanin levels in supernatants as wellas cell densities were measured at OD695 and OD600, respectively, using a Synergy H1 plate reader (BioTek Instruments Inc.) with software Gen5 v.3.05.11.Example 8 – Swarm assaySwarming petri dishes were prepared with 21 ml of M9 minimum medium supplemented with 1 mM MgSO4, 0.2% glucose, 0.5% Casamino Acids, and 0.5% agar. Petri dishes were dried for 1 h on the bench and for an additional 30 min in a laminar flow hood with the lids off. 5 µl of overnight culture was spotted in the center of the dish, or mixed with 1 µl phage lysate and spottedat satellite positions 2.8 cm from the center spot. Plates were incubatedovernight at 37°C in a box containing 10 mL MQ water to maintain humidity (see data in Table 2).Example 9 - Larvae burn wound infection assayGalleria mellonella wax moth larvae (raised antibiotics free) were ordered froma local pet shop. Once delivered, larvae were immediately stored at 12 °C for no longer than 10 days. Before running an experiment, the larvae were sortedaccording to size and placed in petri dishes with 2 larvae per dish. The larvalbodies were sterilized using 70% ethanol and placed back at 12 °C briefly until further use. A burn wound was created using the head of a stainless-steel nail with a diameter of 2 mm. The nail was heated until red using a Bunsen burner and let to cool down for 4 sec before gently applying the nail head to the back of the larvae for a total of 4 sec. PA14 and phage resistant PA14 JBD44 Δgp1-P476852PC00 5839 overnight cultures were mixed in a 90:10% ratio based on OD600. Freshwounds were treated with DMS3 aqs1_FSDARE vir phage alone or infectedwith bacteria and treated with phages as follows: To infect the fresh wounds, 10 µl of OD = 1 standardized inoculum of the mixed bacterial overnight cultures were slowly pipetted onto the wound. Next the infected larvae were treated withDMS3 aqs1_FSDARE vir or with both DMS3 aqs1_FSDARE qsdA vir and DMS3aqs1_FSDARE aqdC vir. The larvae were placed in the 37 °C incubator for 5days and monitored for survival at regular intervals. See data in Figure 8.Example 10 – QsdA activity against acylhomoserine lactonesThe homoserine lactone-deficient strain PA14 ∆lasI ∆rhlI carrying eitherpHERD30T empty vector control or pHERD30T-qsdA expressing qsdA under an arabinose inducible promoter, were cultured at 37 °C for 4 h in LB containing gentamicin (50 µg mL-1), 0.1% arabinose, MOPS buffer (50 mM, pH=7),ZnSO4·7H2O (10 µM), 1 µM N-acylhomoserine lactones: C4-HSL, C6-HSL, 3O-C6-HSL, C8-HSL, 3O-C8-HSL, C10-HSL, and 3O-C12-HSL. The relative abundance of C4-HSL, C6-HSL, 3O-C6-HSL, C8-HSL, 3O-C8-HSL, C10-HSL,and 3O-C12-HSL was quantified by E. coli bioreporters, as described inExample 1, producing relative light units as a response to the QS molecules. The values are reported as RLU (lum / OD600) (relative light units). Table 1 Strains, phages, and plasmids used in this study Strain,Description Sourcephage, or plasmid UCBPP-PA14 Wildtype P. aeruginosa George O’Toole,Geisel School of Medicine at Dartmouth University, Hanover, NH NMHK342 PA14 JBD44 lysogen This studyP476852PC00 59JJM101 PA14 JBD44 Δgp48-53 gp46-This study 47::Pacr-qsdA-aqdC-T1JJM178 / 242 PA14 JBD44 Δgp48-53 gp46-This study 47::Pacr-qsdA-aqdC-aca-T1JJM133 PA14 JBD44 Δgp1-39 This studySMC3884 PA14 DMS3 lysogen Budzik JM, et al.2004. J Bacteriol 186:3270-3.JJM113 PA14 DMS3 aqs1_FSDARE This studyJJM227 PA14 DMS3 aqs1_FSDAREThis study ∆acrIE::qsdAJJM228 PA14 DMS3 aqs1_FSDAREThis study ∆acrIE::aqdCJJM146 PA14 DMS3 ∆gp26-48 This studyJJM148 PA14 DMS3 aqs1_FSDARE ∆gp26-48 This studyJJM313 PA14 ∆lasI ∆rhlI pHERD30T This studyJJM314 PA14 ∆lasI ∆rhlI pHERD30T qsdA This studyE. coli SM10thi thr leu tonA lacY supE recA::RP4- Laboratory stock λpir 2-Tc::Mu Plasmid Allelic exchange vector with pBR pEXG2 origin, gentamicin resistance, sacBpJJM109 Plasmid pEXG2-JBD44-∆gp48-53 This studypJJM93 Plasmid pEXG2-JBD44-gp46-This study 47::Pacr-qsdA-aqdC-T1pJJM174 Plasmid pEXG2-JBD44-gp46-This study 47::Pacr-qsdA-aqdC-aca-T1pJJM132 Plasmid pEXG2-JBD44 ∆gp1-39 This studypJJM112 Plasmid pEXG2-DMS3-aqs1_FSDARE This studypJJM239 Plasmid pEXG2-DMS3-∆acrIE::qsdA This studypJJM185 Plasmid pEXG2-DMS3-∆acrIE::aqdC This studypJJM142 Plasmid pEXG2-DMS3-∆gp26-48 This studypJJM245 Plasmid pAB01. pHERD30T-basedarabinose-inducible expressionP476852PC00 60vector containing a type I-F crRNA insertion site instead of multiple cloning site. Gentamicin resistance.pJJM247 Plasmid pAB01-crRNA-DMS3-cI.This study pAB01 containing crRNA targeting cI repressor in DMS3 phage.pJJM262 Plasmid pAB01-DMS3-∆cI This studyE. coli E. coli pCS26 lasB::lux pBAD lasR, 3O-C12-Paczkowski et al., HSL reporter Journal of Biological Chemistry, 2017. 292(10): p. 4064- 4076.E. coli E. coli pCS26 rhlA::lux pBAD rhlR, C4-HSL Paczkowski et alreporter Journal of Biological Chemistry, 2017. 292(10): p. 4064- 4076.E. coli E. coli pCS26 pqsA::lux pBAD pqsR, PQSEichler AH et al. reporter bioRxiv 2025.04.11.648453E. coli pCS26 pgadW::lux, broad HSL reporter This workPhage JBD44 Temperate phage Phee A, et al.,Efficacy of bacteriophage treatment on Pseudomonas aeruginosa biofilms. J Endod 39:364-9. 2013.Phage JJM178 Phage JBD44 Δgp48-53 gp46-This study 47::Pacr-qsdA-aqdC-aca-T1P476852PC00 61Phage DMS3 Mu-like temperate phage Budzik JM, et al. JBacteriol 186:3270-3. 2004. Phage JJM113Phage DMS3 aqs1_FSDARE vir This studyvir Phage JJM227 Phage DMS3 aqs1_FSDARE This study vir∆acrIE::qsdA virPhage JJM228 Phage DMS3 aqs1_FSDARE This study vir∆acrIE::aqdC virTable 2 Anti-QS phages block phage-infection mediated stressresponse Satellite colony # Swarm collisions with phage-infected colony DMS3 aqs1_FSDARE vir 0 / 4DMS3 aqs1_FSDARE vir qsdA 3 / 3DMS3 aqs1_FSDARE vir aqdC 4 / 4DMS3 aqs1_FSDARE vir qsdA + 4 / 4 DMS3 aqs1_FSDARE vir aqdC in a 1:1 ratio

Claims

1. P476852PC00 62Claims Claim 1. A bacteriophage encoding and expressing at least one inhibitor of a quorum sensing molecule, wherein the expression of said at least one inhibitor of a quorum sensing molecule is operably linked to a promoter, wherein said promoter is regulated by a repressor. Claim 2. The bacteriophage according to claim 1, wherein said promoter regulates transcription of said at least one inhibitor of a quorum sensing molecule and said repressor. Claim 3. The bacteriophage according to any of one the preceding claims,wherein said promoter is an anti-CRISPR promoter and said repressor is ananti-CRISPR associated repressor. Claim 4. The bacteriophage according to any of one the preceding claims comprising an anti-CRISPR operon comprising an anti-CRISPR promoter and encoding said at least one inhibitor of a quorum sensing molecule and a sequence encoding an anti-CRISPR associated repressor, optionally whereinsaid operon further encodes an anti-CRISPR (acr) protein.Claim 5. The bacteriophage according to claim 4, wherein the nucleotide sequence encoding said at least one inhibitor of a quorum sensing molecule is inserted in the operon replacing or in part replacing the sequence encoding an endogenous anti-crispr (acr) protein. Claim 6. The bacteriophage according to any of one the preceding claims, wherein said promoter comprises the sequence set forth in SEQ ID NO:

1. Claim 7. The bacteriophage according to any of one the preceding claims, wherein said anti-CRISPR repressor comprises the amino acid sequence set forth in SEQ ID NO: 3 or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 3.P476852PC00 63Claim 8. The bacteriophage according to any of one the preceding claims, wherein said at least one quorum sensing molecule inhibitor is selected from the group consisting of quorum sensing molecule inhibitor, quorum sensing molecule degrading enzymes, and a repressor of said quorum sensing molecule. Claim 9. The bacteriophage according to any of one the preceding claims, wherein said at least one quorum sensing molecule inhibitor is an enzyme selected from the group consisting of quorum sensing molecule degradingenzymes such as alkylquinolone degradation C (AqdC), autoinducing peptide(AIP)-degrading proteases, AHL-degrading enzymes such as AHL lactonasesand acylases (e.g. autoinducer inactivation A (AiiA), autoinducer inactivationB (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD),autoinducer inactivation S (AiiS), attachment gene M (AttM), N-acylhomoserine lactone D (AhlD), N-acylhomoserine lactone K (AhlK), N-acylhomoserine lactone M (AhlM), N-acylhomoserine lactone S (AhlS),metallo-β-lactamase superfamily lactonase (AaL), quorum-sensing signal degradation A (QsdA), quorum-sensing signal degradation H (QsdH), autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase(MCP), quorum-quenching lactonase from Geobacillus kaustophilus (GKL),NAHL-lactonase (QlcA), quorum signal utilization and inactivation protein (QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA), Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHL acylase B (HacB). Claim 10. The bacteriophage according to any of one the preceding claims, wherein said quorum sensing molecule is selected from the group consistingof N-acyl homoserine lactones including harveyi autoinducer 1 (HAI-1),Pseudomonas Quinolone Signal 2-heptyl-3-hydroxy-4-quinolone (PQS), 4- hydroxy-2-heptylquinoline (HHQ), 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), autoinducer-2 ((3aS,6S,6aR)-2,2,6,6a-Tetrahydroxy-3a- methyltetrahydro-2H-furo[2,3-d][1,3,2]dioxaborol-2-uide), Auto inducingP476852PC00 64peptides (AIP), diffusible signal factor (DSF, cis-11-methyldodecenoicacid), methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3- OH PAME), cholerae autoinducer 1 (CAI-1), and 3,5-dimethyl-pyrazin-2-ol (DPO). Claim 11. The bacteriophage according to any of one the preceding claims, wherein said at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 6 (QsdA) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ IDNO: 6 and / or said at least one quorum sensing molecule inhibitor comprisesthe amino acid sequence set forth in SEQ ID NO: 7 (AqdC) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

7. Claim 12. The bacteriophage according to any of one the preceding claims for use as a medicament. Claim 13. The bacteriophage according to any of one the preceding claims for use in the treatment of a bacterial infection, such as an infection is selected from the group consisting of infected medical devices / implants, wounds such as burn wounds, diabetic foot ulcer, a urinary tract infection, gastrointestinal infection, eye infection, pulmonary infections and blood infection, optionally wherein the bacterial infection is selected from the group consisting of aPseudomonas sp infection, such as a Pseudomonas aeruginosa infection,Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Streptococcus, Clostridium species, Bacillus cereus, Mycobacterium bovis, Brucella species, Yersinia enterocolitica, Listeria monocytogenes, Campylobacter, and Salmonella. Claim 14. A composition comprising the bacteriophage according to any one of claims 1 and 11, such as a pharmaceutical composition comprising theP476852PC00 65bacteriophage according to any one of claims 1 and 11 and at least onepharmaceutical acceptable excipient. Claim 15. Use of bacteriophage according to any of one the preceding claims against a plant pathogenic bacteria, such as a plant pathogenic bacteria selected from the group consisting of Ralstonia solanacearum, Pseudomonas syringae, Xanthomonas campestris, Xanthomonas axonopodis, Xanthomonas oryzae, Xylella fastidiosa, Dickeya dadantii, Dickeya solani, Agrobacterium tumefasciens, Erwinia amylovora, Pectobacterium carotovorum, and Pectobacterium atrosepticum. Claim 16. A method for preparing bacteriophage according to any one of claims 1 to 13, said method comprising the steps of: (a) providing a polynucleotide encoding a bacteriophage; (b) providing polynucleotide encoding at least one inhibitor of aquorum sensing molecule and a promoter, which is regulated by a repressor;(c) inserting said polynucleotide encoding at least one inhibitor ofa quorum sensing molecule and said promoter in the genome encoding saidbacteriophage such that the polynucleotide encoding at least one inhibitor ofa quorum sensing molecule is operably linked to said promoterClaim 17. The method of claim 16, further inserting a polynucleotide sequence encoding said repressor such that said promoter regulates transcription of said at least one inhibitor of a quorum sensing molecule and said repressor Claim 18. The method of claim 16 or 17, wherein said promoter is an anti- CRISPR promoter and said repressor is an anti-CRISPR associated repressor. Claim 19. The method according to any one of claims 16 to 18, wherein said polynucleotide encoding at least one inhibitor of a quorum sensing molecule and a promoter, and optionally said polynucleotide sequence encoding said repressor, is provided a single polynucleotide sequence and inserted in the polynucleotide encoding the bacteriophage.P476852PC00 66Claim 20. The method according to any one of claims 16 to 18, wherein thesaid polynucleotide(s) encoding at least one inhibitor of a quorum sensingmolecule is inserted downstream of an acr promoter already present withinthe phage genome. Claim 21. A bacteriophage encoding and expressing at least one heterologouspolypeptide (or at least one heterologous peptide), wherein the expression ofsaid at least one heterologous polypeptide is operably linked to an anti-CRISPRpromoter, wherein said promoter is regulated by a repressor, wherein said repressor is an anti-CRISPR associated repressor.Claim 22. The bacteriophage according to claim 21, wherein said an anti-CRISPR promoter regulates transcription of the sequence encoding said atleast one heterologous polypeptide (or at least one heterologous peptide) andsaid anti-CRISPR associated repressor. Claim 23. The bacteriophage according to any one of claims 21 or 22, whereinsaid at least one heterologous polypeptide is an enzyme.Claim 24. The bacteriophage according to any of one the preceding claims, wherein said enzyme is selected from the group consisting of alkylquinolonedegradation C (AqdC), autoinducing peptide (AIP)-degrading proteases, AHL-degrading enzymes such as AHL lactonases and acylases (e.g. autoinducer inactivation A (AiiA), autoinducer inactivation B (AiiB), autoinducer inactivation C (AiiC), autoinducer inactivation D (AiiD), autoinducer inactivation S (AiiS), attachment gene M (AttM), N-acylhomoserine lactone D (AhlD), N- acylhomoserine lactone K (AhlK), N-acylhomoserine lactone M (AhlM), N- acylhomoserine lactone S (AhlS), metallo-β-lactamase superfamily lactonase (AaL), quorum-sensing signal degradation A (QsdA), quorum-sensing signal degradation H (QsdH), autoinducers degrading hydrolase (AidH), phosphotriesterase-like lactonase (MCP), quorum-quenching lactonasefrom Geobacillus kaustophilus (GKL), NAHL-lactonase (QlcA), quorum signalP476852PC00 67utilization and inactivation protein (QuiP), pyoverdine synthesis Q (PvdQ), AHL acylase A (HacA), Phosphotriesterase-Like Lactonase SacPox, lactonase SsoPox, and AHL acylase B (HacB). Claim 25. The bacteriophage according to any of one the preceding claims,wherein said at least one heterologous polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 6 (QsdA) or amino acid sequence having atleast 85% identity to the amino acid sequence set forth in SEQ ID NO: 6 and / orsaid at least one quorum sensing molecule inhibitor comprises the amino acid sequence set forth in SEQ ID NO: 7 (AqdC) or amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 7.

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