WO2019162302A1

WO2019162302A1 - TRUNCATED PLANTARICIN NC8β FOR USE IN THE TREATMENT OR PROPHYLAXIS OF A BACTERIAL INFECTION

Info

Publication number: WO2019162302A1
Application number: PCT/EP2019/054166
Authority: WO
Inventors: Torbjörn BENGTSSON; Hazem KHALAF
Original assignee: Bengtsson Torbjoern; Khalaf Hazem
Priority date: 2018-02-20
Filing date: 2019-02-20
Publication date: 2019-08-29

Abstract

In the invention, a polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID NO 3 is provided. Also provided is a pharmaceutical composition a polypeptide X. The pharmaceutical composition may further comprise a polypeptide Y having 15 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with SEQ ID NO 4. The pharmaceutical composition may be used in the treatment or prophylaxis of a bacterial infection.

Description

TRUNCATED PLANTARICIN \C8|5 FOR USE IN THE TREATMENT OR PROPHYEAXIS OF A BACTERIAE INFECTION

Field of the Invention

This invention pertains in general to the field of treatment of infections. More particularly the invention relates to a use of a truncated b-chain of the bacteriocin PLNC8 αβ for the prevention and/or treatment of infections.

Background of the Invention

Hospital-acquired infection (HAI), also known as a nosocomial infection, is an infection that is acquired in a hospital or other health care facility. Such an infection can be acquired in hospital, nursing home, rehabilitation facility, outpatient clinic, or other clinical settings. Infection is spread to the susceptible patient in the clinical setting by various means. Health care staff can spread infection, in addition to contaminated equipment, bed linens, or air droplets. It is estimated that 6 million patients in the EU and USA contract a HAI per year, resulting in up to 150 000 deaths annually.

Prevention of HAI often includes hospital sanitation protocols regarding uniforms, equipment sterilization, washing, and other preventive measures. Thorough hand washing and/or use of alcohol rubs by all medical personnel before and after each patient contact is one of the most effective ways to combat nosocomial infections. More careful use of antimicrobial agents, such as antibiotics, is also considered vital.

Among the categories of bacteria most known to infect patients are the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter), including MRS A (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin- resistant Enterococcus), Streptococcus spp and Escherichia coll. Development of new effective antimicrobial strategies in the treatment of infections caused by antibiotic- resistant bacteria presents one of the major challenges in medicine today. Since most infections are caused by pathogens that live protected in complex biofilms, antibacterial substances need a good ability to penetrate or dissolve biofilm. Such ability is usually limited/lacking in traditional antibiotics, which must therefore be compensated with very high concentrations, often 100-1000 times higher than the doses required for bactericidal effects on planktonic bacteria. This overdose contributes to accelerated development of antibiotic resistance and severe cytotoxic effects. Furthermore, infections are often associated with high proteolytic activity caused by both bacteria and the body's immune system, which means that antimicrobial agents may quickly become inactivated.

It is known that bacteriocins constitute a promising potential alternative or complement to traditional antibiotics and have several advantages such as low risk of resistance development, limited effects on normal flora and beneficial effects on human tissue. Bacteriocins are a group of bacterially produced peptides used to fight other bacteria. Bacteriocins may have a net positive charge and express amphipathic structures that interact with negatively charged microbial membranes and kill microbes usually through pore-forming mechanisms. These mechanisms are more difficult to evade by developing resistance, compared to metabolic enzymes, which usually are targets for conventional antibiotics.

Thus, there is a need for alternative method of antibiotic therapy in the prevention or treatment of bacteria and bacterial infections, especially spread of antibiotic resistance in health care (e.g. methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin- resitant Enterococcus (VRE)).

Summary of the Invention

Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above mentioned problems.

According to a first aspect of the invention, a polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with YTLGIKIL W S AYKH (SEQ ID NO 3) is thus provided. The polypeptide X is a truncated form of the b-chain of the bacteriocin PLNC8 αβ .

Such a polypeptide has been shown to have antimicrobial activity. The antimicrobial activity is i.a. mediated by lysis and fragmentation of the cell wall of the bacteria.

According to one embodiment, the polypeptide X comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising

SVPTSVYTLGIKILWSAYKH SEQ ID NO 5

VPTSVYTLGIKILWSAYKH SEQ ID NO 6 PTSVYTLGIKILWSAYKH SEQ ID NO 7 TSVYTLGIKILWSAYKH SEQ ID NO 8 SVYTLGIKILWSAYKH SEQ ID NO 9 V YTLGIKILW S AYKH SEQ ID NO 10 YTLGIKIL W S AYKH SEQ ID NO 3 YTLGIKILW S AYKHR SEQ ID NO 11 YTLGIKILW S AYKHRK SEQ ID NO 12 YTLGIKILW S AYKHRKT SEQ ID NO 13 YTLGIKILW S AYKHRKTI SEQ ID NO 14 YTLGIKILW S AYKHRKTIE SEQ ID NO 15 YTLGIKILW S AYKHRKTIEK SEQ ID NO 16 YTLGIKILW S AYKHRKTIEKS SEQ ID NO 17 YTLGIKILW S AYKHRKTIEKSF SEQ ID NO 18 YTLGIKILW S AYKHRKTIEKSFN SEQ ID NO 19 YTLGIKILWSAYKHRKTIEKSFNK SEQ ID NO 20

YTLGIKILW S AYKHRKTIEKSFNKG SEQ ID NO 21 YTLGIKILWSAYKHRKTIEKSFNKGF SEQ ID NO 22 YTLGIKILWSAYKHRKTIEKSFNKGFY SEQ ID NO 23 YTLGIKILW S AYKHRKTIEKSFNKGF YH SEQ ID NO 24

According to another embodiment, at least 90% of the amino acids in said polypeptide X are D-amino acid residues.

Such peptides are more stable and less sensitive to proteolytic cleavage compared to their corresponding L-variants.

According to a second aspect of the invention, a pharmaceutical composition comprising a polypeptide X according to the invention is also provided.

The pharmaceutical composition may be formulated for administration as a single dose or as multiple doses, such as two, three, four, five or even more doses.

According to one embodiment, the pharmaceutical composition may further comprise a polypeptide Y having 15 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with DLTTKLWSSWGYYLG (SEQ ID NO 4). The polypeptide Y is a truncated form of the a-chain of the bacteriocin PLNC8 αβ .

The addition of a truncated form of the a-chain of the bacteriocin PLNC8 αβ has the effect that the antibacterial effect is enhanced. This may be seen as lysis of bacterial cells. According to one embodiment, the polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:

DLTTKLWS S WG Y YLGKKARWNLKHP Y VQ SEQ ID NO 25 DLTTKLWS SWGY YLGKKARWNLKHP YV SEQ ID NO 26 DLTTKLWS SWGY YLGKKARWNLKHP Y SEQ ID NO 27 DLTTKLWS SWGY YLGKKARWNLKHP SEQ ID NO 28 DLTTKLWS S WG Y YLGKKARWNLKH SEQ ID NO 29 DLTTKLW S S W GYYLGKKARWNLK SEQ ID NO 30 DLTTKLW S S WGYYLGKKARWNL SEQ ID NO 31. DLTTKLW S S WGYYLGKKARWN SEQ ID NO 32 DLTTKLW S S W GYYLGKKARW SEQ ID NO 33 DLTTKLW S S W GYYLGKKAR SEQ ID NO 34 DLTTKLWSSWGYYLGKKA SEQ ID NO 35 DLTTKLWSSWGYYLGKK SEQ ID NO 36 DLTTKLWSSWGYYLGK SEQ ID NO 37 DLTTKLWSSWGYYLG SEQ ID NO 4

According to another embodiment, at least 90% of the amino acids in peptide X and/or peptide Y are D-amino acid residues.

According to yet another embodiment, the pharmaceutical composition further comprises at least one antibiotic.

The peptide(s) and the antibiotic act synergistically and enhance the effect of each other.

According to a further embodiment, the antibiotic is selected from the group consisting of antibiotics that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.

According to one embodiment, peptide X and peptide Y are present in a in a molar ratio of from between 5:1 to 1 :20, preferably 1 :1 to 1 :7, most preferably 1 :1.

According to another embodiment, the pharmaceutical composition comprises between 0.1 to 50 mM of peptide X and/or peptide Y.

According to yet another embodiment, the pharmaceutical composition comprises the antibiotic in an amount of between 0.0019 and 50 pg/ml. According to one embodiment, the pharmaceutical composition is formulated as a solution, a cream, a gel, or an ointment or formulated in immobilized form as a coating on a device.

Furthermore, the pharmaceutical composition is formulated as a composition, which when applied to a surface forms a coating substantially coating the part of the device on which the pharmaceutical composition is applied. The coating may be resistant or substantially resistant to water and certain solvents. The coating may also be resistant or substantially resistant to abrasion and/or wear. Thus, when applied to e.g. a handle, the coating will not need to be applied after each use of the handle, but will resist abrasion and/or wearing for a longer time period, such as several hours up to several days or weeks, depending on the frequency of interaction with other objects.

According to yet another embodiment, the pharmaceutical composition is formulated as a gel, wherein the gel further comprises gelatine and glycerol. The gel may comprise hyaluronic acid.

According to another embodiment, the pharmaceutical composition is formulated in immobilized form as a coating on a device, wherein the device is chosen from the group consisting of a wound dressing, an orthopedic implant, a dental implant, a urinary catheter and an urinary stent.

In this way, the device is provided with an antibacterial coating, which exerts its effect on the surroundings during a longer period of time, such as hours, days or even weeks. This may be advantageous, since the antibiotic effect is only exerted locally and not systemically. Furthermore, infections that are commonly associated with implants, catheters and stents may be counteracted locally, without the need of administrating antibiotics systemically.

According to a third aspect of the invention, a pharmaceutical composition for use in the treatment or prophylaxis of a bacterial infection, wherein the pharmaceutical composition is a pharmaceutical composition according to the invention is also provided.

The bacterial infection may be caused by gram-negative bacteria.

The bacterial infection may be caused by gram-positive bacteria.

According to one embodiment,, the bacterial infection is caused by

Staphylococcus spp (including MRSA, MRSE), Streptococcus spp (e.g. S. mutans, S. constellatus, S. anginosus), Enterococcus faecium (including VRE), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or Escherichia coli. According to one embodiment, the bacterial infection is caused by

Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus. According to one embodiment, the bacterial infection is caused by Staphylococcus spp and/or Streptococcus spp.

The bacterial infection may be caused by Escherichia coli.

The bacterial infection may be caused by Enterococcus ssp.

The bacterial infection may be caused by Pseudomonas aeruginosa.

The bacterial infection may be caused by Porphyromonas gingivalis.

Such bacteria are a common cause of hospital-acquired infection (HAI).

Among the categories of bacteria most known to infect patients are the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter ), including MRS A (Methicillin-resistant Staphylococcus aureus) and VRE (Vancomycin-resistant Enterococcus), Streptococcus spp and Escherichia coli. .Thus, one advantage of the present invention is that infections caused by bacteria which are resistant to

conventional antibiotics may be treated.

According to another embodiment, the composition is administered locally on the site of infection, such as topically.

According to a fourth aspect of the invention, a use of a pharmaceutical composition according to the invention in coating at least part of a device to limit colonization of bacteria on the surface of the device is also provided.

According to one embodiment, the device is a medical device, such as a prosthesis or a wound dressing.

According to another embodiment, the bacteria are Staphylococcus spp

(including MRSA, MRSE), Streptococcus spp (e.g. S. mutans, S. constellatus, S.

anginosus), Enterococcus faecium (including VRE), Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or

Escherichia coli.

According to another embodiment, the bacterial infection is caused by

Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.

According to another embodiment, the bacteria are Staphylococcus spp and/or Streptococcus spp.

According to a fifth aspect of the invention, a polypeptide for use in the treatment or prophylaxis of a bacterial infection, wherein the polypeptide is a polypeptide according to the invention, is also provided. Brief Description of the Drawings

These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which

Figure 1 shows PLNC8 αβ markedly inhibits the growth and survival of different strains of S. aureus and S. epidermidis. Different Staphylococcus species were cultured for 20 h in the presence of increasing concentrations of PLNC8 αβ (1 : 1).

S. epidermidis was generally more susceptible to PLNC8 αβ than S. aureus. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for Staphylococcus species in response to PLNC8 αβ.

Figure 2. The molar ratio of PLNC8 a and PLNC8 b is critical for optimal antimicrobial activity. S. epidermidis ATCC 12228 was exposed to different molar ratios of PLNC8 a and b for 20 h. A molar ratio of 1 : 1 between PLNC8 a and PLNC8 b is most efficient at inhibiting and killing S. epidermidis. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for different molar ratios of PLNC8 a and b. *Thc highest total concentration of the peptides was kept constant at 50 mM, while the concentrations of PLNC8 a and b individually were altered to obtain different molar ratios.

Figure 3. PLNC8 αβ is effective at disrupting S. epidermidis biofilms. The biofilm positive strain S. epidermidis RP62A was allowed to form biofilms followed by removal of suspended bacteria and then incubation with PLNC8 αβ, PLNC8 a or PLNC8 b for 1 h. A- Absorbance measurement of detached biofilms. B- Crystal violet staining of the remaining attached biofilms. PLNC8 αβ is most efficient and rapid at disrupting biofilms of S. epidermidis.

Figure 4. Membrane disrupting and antimicrobial activity of PLNC8 a and b with L- or D -amino acids.

A- CF release was recorded after exposure of liposomes with increasing concentrations of L- or D-variants of PLNC8 a, b or αβ (1 :1). B- S. epidermidis ATCC 12228 was incubated with increasing concentrations of PLNC8 a and b, alone or in combination (1 : 1), for 20 h. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for PLNC8 a, b and αβ are indicated.

Figure 5. The L- and D-variant of PLNC8 αβ rapidly permeabilize the plasma membrane of S. epidermidis. The uptake of Sytox Green by S. epidermidis ATCC 12228 after treatment with 5mM of L-PLNC8 αβ, D-PLNC8 αβ or scrambled-PLNC8 αβ for 2 min, compared to untreated bacteria (C).

Figure 6. The D-form of PLNC8 αβ is more stable and less sensitive to proteolytic cleavage. The peptides (100mM) a) L-PLNC8 α b) D-PLNC8 α c) E-REM38β d) ϋ-REM38b were treated with Trypsin (5mM) in Ammonium Bicarbonate buffer (50mM) for 16 h at 37°C before being acified (2.5% TFA), dried, suspended in H20 +0.1% TFA, desalted (ZipTip) and analyzed by MALDI-ToF MS. Number above the peaks indicate molecular weights (Da) and number in brackets sequences of amino acids. Full-length a- and b-peptide, 1-29 and 1-34, respectively.

Figure 7. (A) Both the L- and D-form of PLNC8 αβ display a low hemolytic activity. Human erythrocytes were incubated with different concentrations (0.5-50 mM) of L- or D-variant of PLNC8 α, b or αβ (1 :1) for 1 h. In (B) this is shown for and truncated forms α1-15, α1-22, b7-20, b1-20, b7-34. Hemolytic activity was determined after incubation of human erythrocytes with the peptides for 1 h.

Figure 8. Amino acid sequences of truncated peptides of PLNC8 a and PLNC8 b.

Figure 9. Antimicrobial activities of truncated forms of PLNC8 b. In A.

disruption of the membrane and release of (6)-carboxyfluorescein (CF) from liposomes was obtained with the b-peptides 1-34 (full-length), 7-34, 1-20 and 7-20. In B, when combined with a full length PLNC8 a peptide, effects were also obtained with the other truncated peptides, although at higher concentrations. In C. amino acid sequences of truncated peptides of L-PLNC8 b. In D. quantification of 50% CF release with truncated L-PLNC8 b, with and without L-PLNC8 a In E. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of truncated PLNC8 b, in the absence or presence of full-length α-peptide, against S. epidermidis ATCC 12228. Growth of S. epidermidis was inhibited by the full-length b1-34, b7-34, b1-20 and b7- 20.

Figure 10. Antimicrobial activities of truncated forms of PLNC8 a. (A) Release of (6)-carboxyfluorescein (CF) from liposomes was obtained with α1-22 and full-length α1-29 (B). When combined with a full length PLNC8 b peptide, effects were also obtained with the other truncated peptides, although at higher concentrations. (C)

Amino acid sequences of truncated peptides of L-PLNC8 a. (D) Quantification of 50% CF release by truncated L-PLNC8 a peptides, with and without L-PLNC8 b. (E) Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of truncated PLNC8 a against S. epidermidis ATCC 12228. Growth and survival of S. epidermidis was inhibited by a 1-29 and a 1-22 in combination with the b- peptide.

Figure 11. Antimicrobial activity of a combination of truncated PLNC8 a and PLNC8 b. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of a combination of truncated PLNC8 a and PLNC8 b against S. epidermidis ATCC 12228. The inhibition of growth and survival of S. epidermidis by b1-20 and b7-20, respectively, was not further enhanced by a co-incubation with α1-22 or α1-l5.

Figure 12. Morphological effects of PLNC8 αβ on S. epidermidis using TEM and SEM. PLNC8 a caused massive bleb formation and PLNC8 b induced bacterial lysis shown by an extracellular release of intracellular content. PLNC8 αβ was most efficient causing complete bacterial lysis. The truncated forms of PLNC8 b, bΐ -20 and b7-20, induced fragmentation of the bacterial cell wall and in combination with PLNC8 α S. epidermidis went through lysis.

Figure 13. PLNC8 αβ in a formula is effective against S. epidermidis and retains its activity after long-term storage. Bacterial lysis was visualized by studying the uptake of Sytox Green by S. epidermidis ATCC 12228 exposed to a gel containing different concentrations (5-100 mM) of PLNC8 αβ. The activity of the formula with 100 mM PLNC8 αβ was also tested on blood-agar plates with S. epidermidis, at time zero and after long-term storage at 4°C. A gradient of PLNC8 αβ was created by spreading the gel over the agar surface with a plastic loop. Inhibition of bacterial growth is demonstrated by the translucent areas.

Figure 14. PLNC8 αβ is effective against heterogeneous strains of S.

epidermidis. S. epidermidis isolated from prosthetic joint infections, including heterogeneous glycopeptide intermediate S. epidermidis (hGISE), was exposed to L- PLNC8 αβ for 20 h and MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were determined.

Figure 15. PFNC8 αβ acts synergistically with antibiotics. Synergistic antimicrobial effects between antibiotics and F- or D-PFNC8 αβ. S. epidermidis (strain 154) was exposed to F-PFNC8 αβ or D-PFNC8 αβ (3.1 mM), a serial dilution of teicoplanin, vancomycin, rifampicin and gentamicin, alone or in their combination with 3.1 mM F-PFNC8 αβ or D-PFNC8 αβ.

Figure 16. PFNC8 b and truncated PFNC8 a markedly amplify the inhibitory effects of teicoplanin against S. epidermidis. Synergistic antimicrobial effects between teicoplanin and PFNC8 αβ. S. epidermidis (strain 154) was exposed to a serial dilution of teicoplanin or full-length/truncated PLNC8 αβ alone or in their combination (serial dilution of teicoplanin and 6.25 mM full-length/truncated PLNC8 αβ), and

Figure 17. PLNC8 αβ markedly permeabilizes and kills different species of Streptococcus. Streptococcus spp (S. mutans (Sm), S. constellatus (Sc) and S. anginosus (Sa)) were treated with 5mM PLNC8 αβ for 2 min, followed by analysis of uptake of Sytox Green. S. constellatus and S. anginosus were more susceptible to PLNC8 αβ than S. mutans.

Figure 18. PLNC8 αβ causes rapid membrane permeabilization on liposomes. PLNC8 b and PLNC8 αβ (1 : 1), but not PLNC8 a, of both the (A) L- form and (B) in form, caused complete lysis of liposomes after 2 min, and

In figure 19, CD-spectroseopy of PLNC8 αβ. CD measurements of (A) L- PLNC8 αβ and (B) D-PLNC8 αβ (100mM each) without (dashed) and with (solid) liposomes (0.5mg/ml, ~660mM) in PBS. Three repeats with PBS as background.

Liposome containing samples were incubated for at least 30 min prior to measurements.

Description of embodiments

The following description focuses on an embodiment of the present invention applicable to combating infection, and especially Hospital-acquired infection (HAI) (but also other types of infections) using peptides derived from a L. plantarum NC8 bacteriocin optionally used together with antibiotics. However, it will be appreciated that the invention is not limited to this application but these peptides may be applied to many other uses, including for example disinfection and coating of surfaces.

There are several problems associated with combating infections, such as Hospital-acquired infection (HAI). These include: Inadequate treatment strategies for many severe and serious bacterial infections; Development and spread of antibiotic resistance in health care (e.g. methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE)); Large costs for society for prevention and treatment of infectious diseases (e.g. annual hospital costs of treating healthcare- associated infections (HAIs) in US is estimated to 40 billion dollar and in Sweden to 6.5 billion SEK): Human suffering from infectious diseases (annually approx. 6 million patients with HAIs in US and EU and 150 000 die).

These problem are not trivial to approach, since they include aspects such as intractable infections in the form of bio films, high proteolytic activity in infections antagonizing the action of antibacterial agents, limited stability and activity of antibacterial agents, chronic infection and inflammation, and slow and complicated wound healing.

It was envisaged by the present inventors that specific Lactobacillus species indeed may be able to contribute to solving these problems, from its ability to suppress pathogens primarily through expression and secretion of certain bacteriocins.

Lactobacillus plantarum is a highly versatile lactic acid bacterium found in saliva and gastrointestinal tract as well as fermented vegetables, meat and dairy products. L. plantarum NC8 has been used as a model strain in many laboratories worldwide, and is a naturally plasmid-free L. plantarum strain. L. plantarum NC8 has previously been shown to produce a two-peptide bacteriocin, PLNC8 αβ, classified as a class lib bacteriocin. The inventors have previously shown that PLNC8 αβ is efficient against the periodontal pathogen Porphyromonas gingivalis and stimulates cell proliferation (1,2).

The idea of the invention is to exploit the antibacterial effects of bacteriocin PFNC8c 3 for the prevention and treatment of acute and chronic infections, such as periodontitis, wound infections, implant-associated infections and urinary tract infections. Products based on bacteriocins can be of enormous importance in health care, with improved public health and a positive impact on the social economy.

Since development and spread of antibiotic resistance in health care primarily concerns methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), the effect of PLNC8αβ on different strains of S. aureus and S. epidermidis were studied. As can be seen in Fig. 1 and Table 1, PLNC8αβ markedly inhibited the growth and the survival of all bacterial strains (Fig. 1).

A bio film is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharides, proteins and extracellular DNA.

Gradients of nutrients and oxygen exist from the top to the bottom of bio films and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Thus, it is of importance that the antimicrobial agent can penetrate the biofilm to expose the biofilm bacteria to the antimicrobial agent and there exert antibacterial effect.

Indications are that e.g. Staphylococcus biofilms are not totally impervious to antibiotics, and certain fluorescently tagged antimicrobials (such as daptomycin) have been shown to penetrate the biofilms of S. aureus and S. epidermidis by diffusion.

In the invention, it is shown that PLNC8 αβ is effective at disrupting S.

epidermidis bio films (figure 3). Further, it was envisioned that the bio film penetration of PLNC8 αβ could be optimized through modifying the bacteriocins. There are several different options for peptide modification. It was hypothesized that if key structural motifs in the PLNC8 αβ could be maintained, PLNC8 αβ bateriocin key functionality could be maintained, while the resulting shorter peptide would benefit from better diffusion properties due to its restricted size. Thus, truncated forms of PLNC8 αβ were developed to investigate whether these truncated forms were able to retain antibacterial activities similar to the native bacteriocin.

Truncation of a polypeptide will result in structural and chemical changes of the molecule, why truncated peptides of PLNC8 a and PLNC8 b, respectively, were constructed in sequences of 6-7 amino acids, to correspond to the number of amino acids required for formation of an alpha helix (shown in Fig. 8). The effects of truncated PLNC8 a and PLNC8 b were evaluated in both a liposome system (resembling bacteria) and on S. epidermidis. Truncated peptides of PLNC8 b were shown to have antimicrobial activity. The antimicrobial activity is i.a. mediated by lysis and fragmentation of the cell wall of the bacteria. As shown in figure 9, such effects could be seen, revealed by liposome release of (6)-carboxyfluorescein (CF), for truncated b-peptides with as little as 14 amino acids (amino acids 7-20). Truncated PLNC8 b peptides of the invention are referred to as polypeptide X.

According to a first aspect of the invention, a polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with YTLGIKIL WSAYKH (SEQ ID NO 3) is provided. The polypeptide X is a truncated form of the b-chain of the bacteriocin PLNC8 αβ .

It was found that fragments with potent antimicrobial activity were PLNC8 b fragments comprising amino acids 1-34 (full-length), 7-34, 1-20 and 7-20. It seems that peptide b-sequences b7-13 and b14-20, or parts thereof, are crucial for the antibacterial effects of PLNC8 b. Furthermore, fragments retaining the b7-13 and b14-20 sequences were even more efficient when combined with b1-6. Thus, the peptide b1-20 is the most effective in inhibiting S. epidermidis. It was surprisingly found that growth of S.

epidermidis was most efficiently inhibited by either sequence b1-20 (SEQ ID NO 5) or b7-20 (SEQ ID NO 3), respectively, and these truncated peptides were as effective, or even more effective, than the full-length native PLNC8 b (1-34) (Fig. 9).

S VPT S V YTLGIKIL W S AYKH SEQ ID NO 5 VPTSVYTLGIKILWSAYKH SEQ ID NO 6 PTSVYTLGIKILWSAYKH SEQ ID NO 7 TSVYTLGIKILWSAYKH SEQ ID NO 8 SVYTLGIKILWSAYKH SEQ ID NO 9 V YTLGIKILW S AYKH SEQ ID NO 10 YTLGIKIL W S AYKH SEQ ID NO 3 YTLGIKILW S AYKHR SEQ ID NO 11 YTLGIKILW S AYKHRK SEQ ID NO 12 YTLGIKILW S AYKHRKT SEQ ID NO 13, YTLGIKILW S AYKHRKTI SEQ ID NO 14, YTLGIKILW S AYKHRKTIE SEQ ID NO 15 YTLGIKILW S AYKHRKTIEK SEQ ID NO 16 YTLGIKILW S AYKHRKTIEKS SEQ ID NO 17 YTLGIKILW S AYKHRKTIEKSF SEQ ID NO 18 YTLGIKILW S AYKHRKTIEKSFN SEQ ID NO 19 YTLGIKILWSAYKHRKTIEKSFNK SEQ ID NO 20 YTLGIKILW S AYKHRKTIEKSFNKG SEQ ID NO 21 YTLGIKILWSAYKHRKTIEKSFNKGF SEQ ID NO 22 YTLGIKILWSAYKHRKTIEKSFNKGFY SEQ ID NO 23 YTLGIKILW S AYKHRKTIEKSFNKGF YH SEQ ID NO 24

These shortened forms of the bacteriocins diffuse more rapidly into the biofilm due to their limited size.

Similarly, truncated forms of PLNC8 a were further probed, as shown in Fig. 10. Truncated PLNC8 a peptides of the invention are referred to as polypeptide Y.

When combining the truncated PLNC8 b fragments with a PLNC8 a peptide, it was revealed that a combination of truncated b7-34, b7-20, bΐ -20, or b1-34 showed effect on MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) against S. epidermidis, as shown in figure 9. As can be seen, truncated b7-20, bΐ -20 were especially effective, both in themselves and in combination a PLNC8 a peptide.

When combining the truncated PLNC8 a fragments with a PLNC8 b peptide, it was revealed that a combination of truncated α1-29, α1-22, α9-l5, α9-22 or α9-29 showed effect on MIC and MBC against S. epidermidis, as shown in figure 10. Of the truncated fragments, α1-22 was especially effective. It was also noted that a fragment as short as α9-15 show effect together with the PLNC8 b peptide.

Furthermore, it is shown in figure 11, that α1-l5 with b 1 -20 or b7-20 and α1- 22 with b1-20 or b7-20 showed effect on MIC and MBC against S. epidermidis.

Especially effective was the combination of PLNC8 a peptide α1-22 in combination with PLNC8 b peptide b1-20.

According to one embodiment, the polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:

DLTTKLWSSWGYYLGKKARWNLKHPYVQ SEQ ID NO 25 DLTTKLWS S WG Y YLGKKARWNLKHP YV SEQ ID NO 26 DLTTKLWS SWGY YLGKKARWNLKHP Y SEQ ID NO 27 DLTTKLWS SWGY YLGKKARWNLKHP SEQ ID NO 28 DLTTKLWS S WG Y YLGKKARWNLKH SEQ ID NO 29 DLTTKLW S S W GYYLGKKARWNLK SEQ ID NO 30 DLTTKLW S S WGYYLGKKARWNL SEQ ID NO 31. DLTTKLW S S WGYYLGKKARWN SEQ ID NO 32 DLTTKLW S S W GYYLGKKARW SEQ ID NO 33 DLTTKLW S S W GYYLGKKAR SEQ ID NO 34 DLTTKLWSSWGYYLGKKA SEQ ID NO 35 DLTTKLWSSWGYYLGKK SEQ ID NO 36 DLTTKLWSSWGYYLGK SEQ ID NO 37 DLTTKLWSSWGYYLG SEQ ID NO 4

DLTTKLWSSWGYYLGKKARWNLKHPYVQ SEQ ID NO 25 DLTTKLWS SWGY YLGKKARWNLKHP YV SEQ ID NO 26

DLTTKLWS SWGY YLGKKARWNLKHP Y SEQ ID NO 27 DLTTKLWS SWGY YLGKKARWNLKHP SEQ ID NO 28 DLTTKLWS SWGY YLGKKARWNLKH SEQ ID NO 29 DLTTKLW S S W GYYLGKKARWNLK SEQ ID NO 30 DLTTKLW S S WGYYLGKKARWNL SEQ ID NO 31.

As such, the innovation pertains to truncated peptides from plantaracin NC8 αβ (PLNC8 αβ) a and b peptides (e.g. comprising the functional motifs in a1-15, α1-22, bΐ -20 and b7-20). It was found that antibacterial properties are retained, or even enhanced, while higher diffusion rates in bacterial films are obtained when peptide size decreases. This since the diffusion coefficients increase strongly as the system size increases, enabling smaller peptides to penetrate further into a biofilm. In a gel, such as a bacterial biofilm, diffusion is even more affected by particle size, since larger particles will also have a higher risk of becoming entrapped in pores of the gel.

In table 2 are summarized the truncated sequences containing the motifs of the optimized fragments. Sequence identity (%SI) as described herein may be assessed by any convenient method. Programs that compare and align pairs of sequences, like ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), or BLASTP (Devereux et al., Nucleic Acids Res., 12:387, 1984) can be used for this purpose. If no such resources are at hand, according to one embodiment, sequence identity (%SI) can be calculated as (%SI) = 100% * (Nr of identical residues in pairwise alignment) / (Length of the shortest sequence).

Table 2: SEQUENCE LIST

Bacterial biofilms may also seek to combat antibiotics by a reaction with the antimicrobial agent. Similarly, infections are often associated with high proteolytic activity caused by both bacteria and the body's immune system, which means that antimicrobial peptides or proteins may be inactivated.

In the invention, it was hypothesized that the inactivation through proteolytic activity often targets specific sequence motifs. Hypothetically bacteriocin modifications altering the susceptibility to these target attacks could increase the lifetime, and thereby the effect, of the bacteriocin. However, such modifications risk altering the molecular structure of the peptide, which may affect the peptide function.

Under the hypothesis that a structurally stable structure might be provided if the whole peptide was modified, all L-amino acids of the peptides were replaced by D- amino acids (all alpha amino acids but glycine can exist in either of two enantiomers). It was also hypothesized that this could affect proteolytic cleavage of the peptides and thus increase efficacy. The effects of the L- and D-variants of PLNC8 αβ were tested on both a liposome system (resembling bacteria) and on S. epidermidis. The D-variant of PLNC8 αβ was almost as effective in destroying liposomes and inhibiting/killing S. epidermidis as the L-variant (Fig. 4). The perturbation of the plasma membrane of S. epidermidis was equally rapid (2 min) for the L- and D-variant, respectively, of PLNC8 αβ (Fig. 5).

To analyze whether PLNC8 αβ with D-amino acids is more stable and less sensitive to proteolytic cleavage compared to the L-variant of PLNC8αβ; D- PLNC8α , D-PLNC8b, L-PLNC8α and T-PLNC8b were exposed to trypsin and the presence of proteolytic fragments was analyzed with MALDI-TOF mass spectrometry (Fig. 6). While trypsin generated several fragments of both the a- and b-peptide of L-PLNC8, no obvious fragmentation was observed of the a- and b-peptide of D-PLNC8. Thus, the D- variants are more resistant to trypsin-mediated degradation than the L-variants.

Furthermore, to clarify whether PLNC8 αβ (the L- and D-variant) exerts cytotoxic effects, lysis of erythrocytes isolated from human whole blood was investigated. However, no hemolytic activity was observed (Fig. 7). As such, there is no toxic effect on human cells.

Thus, according to another embodiment, at least 90% of the amino acids in said polypeptide X are D-amino acid residues.

The advantages of the D-variants are increased stability and less sensitivity to proteolytic cleavage compared to their corresponding L-variants. This results in a longer lifetime of the D-variant peptides and thus prolonged antibacterial effect.

Non-natural or modified amino acids may also be introduced that enable convenient coupling chemistries, including click-chemistry approaches. The

bacteriocins can also be modified with either N- or C-terminal azide groups to enable copper-free click reaction with e.g. cyclooctyne conjugated polymers. Using

biodegradable polymers such as hyaluronic acid (HA), the release rate will be dependent on the hydrolysis rate of the biopolymer backbone and can be tuned to a certain extent by using different polymers. Interestingly, hyaluronidase is expressed by S. aureus, as a virulence factor, degrading polysaccharides between cells and thereby enabling spreading of the infection. Thus, if the biodegradeable polymer is HA, the release rate of the peptides will increase in the presence of S. aureus.

PLNC8o$ is a two peptide bacteriocin, so in order to investigate the role of the PLNC8 a chain and PLNC8 b chain, respectively, in the inhibitory and bactericidal action of the bacteriocin, the effects of different molar ratios between the peptides on S. epidermidis were studied. It was found that a molar ratio of 1 : 1 is most efficient at inhibiting and killing S. epidermidis (Fig. 2). However, a ratio of between PLNC8 a chain and PLNC8 b chain of 1 : 1 to 1 :7 also showed a good effect.

Thus, in one embodiment, the first and second peptides are present in a in a molar ratio of from between 5 : 1 to 1 :20, preferably 1 :1 to 1 :7, most preferably 1 :1.

Further, it was probed if the antibmicrobial effect could be enhanced using combination therapy. In combination therapy, combinations of antimicrobial agents are utilized for the prevention of the development of resistance and to shorten the length of treatment time. It was investigated whether combinations of PLNC8 αβ together with different traditional antibiotics would be effective in the treatment of S. epidermidis. In Fig. 15, results are summarized for PLNC8 αβ together with Rifampicin and

teicoplanin. Here it was surprisingly found that PLNC8 αβ decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (Fig. 15). A combination of PLNC8 αβ and rifampicin was found to be even more effective. MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 αβ or D-PLNC8 αβ. Furthermore, L-PLNC8 αβ decreased MIC and MBC of gentamicin 15-30 fold against S. epidermidis. L-PLNC8 αβ or D-PLNC8 αβ lowered MIC and MBC of vancomycin 2-fold. (Fig. 15 and Table 3).

Table 3 : Antimicrobial effect is enhanced using PLNC8 αβ combination therapy

These results demonstrate a surprisingly strong synergistic effect, with up to hundred- fold decrease of MIC and MBC of the antibiotic against specific bacteria. Without being bound to theory, this may be due to the membrane permeabilizing effect of PLNC8 αβ, which may damage bacterial membranes and thus facilitate passage for the antibiotics, which thus more easily reach their intracellular targets (e.g., ribosomes, RNA polymerase). The consequence is that the concentration of antibiotics can be significantly lowered with reduced problems of both antibiotic resistance and cytotoxic side effects.

The synergistic antimicrobial effect between PLNC8 αβ and traditional antibiotics against resistant strains of Staphylococcus is shown in table 4 below. Here the effects of vancomycin or teicoplanin combined with L-PLNC8 αβ against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant

Staphylococcus epidermidis (MRSE) are shown.

Table 4. Synergistic antimicrobial effect between PLNC8 αβ and traditional antibiotics against resistant strains of Staphylococcus.

This shows that combination therapy with PLNC8 αβ and antibiotics is an efficient treatment strategy. This was further shown during trials using ESKAPE pathogens and Escherichia coli, one of the leading causes of nosocomial infections throughout the world. The acronym ESKAPE includes six pathogenic bacterialspecies (Enterococcusfaecium, Staphylococcusaureus, Klebsiellapneumoniae,

Acinetobacterbaumannii, Pseudomonasaeruginosa and Enterobacter). These bacteria have become resistant to multiple antibiotics and are associated with higher rates of morbidity and mortality, indicating the need for new strategies to prevent and treat these types of infections. ESKAPE pathogens are prioritized by WHO to promote research and development of new antimicrobials, since multidrugresistance is a serious threat to global public health. Infections caused by these pathogens are often hospital-acquired, and pose a particular threat to patients requiring medical devices, such as catheters, ventilators and implants.

As can be seen in table 5, PLNC8αβ alone does not affect the growth of E.coli, however a sub-MIC concentration of the peptides significantly enhanced the effects of different antibiotics.

Similarly, PLNC8αβ alone is both inhibitory and bactericidal against

Enterococcus faecium, and addition of sub-MIC concentrations significantly enhanced the effects of different antibiotics. This is shown in table 6 below.

Also, in table table 7, it is shown that although PLNC8αβ alone does not affect the growth of Pseudomonas aeruginosa, addition of sub-MIC concentration of the peptides enhanced the effects of different antibiotics.

Table 5. PLNC8αβ markedly enhances the inhibitory and bactericidal effects of antibiotics against Escherichia coli

*Peptide concentration in combination with antibiotics is 10 mM Table 6. PLNC8αβ markedly enhances the inhibitory and bactericidal effects of antibiotics against Enterococcus faecium.

*Peptide concentration in combination with antibiotics is 1.5 mM Table 7. PLNC8αβ markedly enhances the inhibitory and bactericidal effects of antibiotics against Pseudomonas aeruginosa.

*Peptide concentration in combination with antibiotics is 15 mM

Many hospital-acquired bacterial infections are found in superficial infections and severe infections associated with chronic wounds and insertion of medical devices, including catheters and prosthetic joint implants. This may subsequently increase the risk for development of life-threatening conditions, such as sepsis.

The antimicrobial activities of truncated forms of PLNC8 a were further probed, as shown in Fig. 16. PLNC8 b together with truncated PLNC8 a, PLNC8 a together with truncated PLNC8 b and truncated PLNC8 a together with truncated PLNC8 b markedly amplify the inhibitory effects of teicoplanin against S. epidermidis.

As demonstrated above, the peptide(s) and the antibiotic act synergistically and enhance the effect of each other.

To verify the synergy effect of peptides of the bacteriocin NC8 αβ together with antibiotics, antibiotics from the three largest groups of antibiotics were selected and tested; antibiotics that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis. The combination of antibiotics and PLNC8 αβ provides a powerful synergistic effect, and reduces (up to 100 times) MIC and MBC for antibiotics from the three classes. As can be seen in Fig. 15 teicoplanin, vancomycin, rifampicin, and gentamicin were evaluated. Of these, synergistic effects were (from high to low) rifampicin [100 fold], gentamicin [15-30 fold] teicoplanin [10 fold] and vancomycin [2 fold]. The highest effect was thus shown for a combination of PLNC8 αβ and rifampicin.

According to a further embodiment, the antibiotic is selected from the group consisting of antibioticts that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.

In one further embodiment, the antibiotic is selected from a group consisting of rifampicin, gentamicin, teicoplanin and vancomycin.

In one further embodiment, the antibiotic is selected from a group consisting of rifampicin, gentamicin, and teicoplanin.

The composition may comprise between 10 nM to 50 mM of the first peptide and/or of the second peptide. As shown in Fig. 15, concentrations within the micromolar range effectively reduce S. epidermidis in the presence of an antibiotic.

As can be seen in Fig. 15, MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 αβ or D-PLNC8 αβ, resulting in an effective amount already at 0.0019 pg/ml.

Thus, in one embodiment, the pharmaceutical composition comprises the antibiotic in an amount of at between 0.002 pg/ml to 50 pg/ml, such as at least 0.01 pg/ml to 5 pg/ml, such as at least 0.1 pg/ml to 1 pg/ml, such as at least 0.8 pg/ml. Thus, in one embodiment, the pharmaceutical composition comprises the antibiotic in an amount of at least 0.78 pg/ml, such as at least 0.097 pg/ml such as at least 0.0019 pg/ml, such as at least 0.0097 pg/ml.

Thus, in one embodiment, the antibiotic is vancomycin in an amount of at least 0.78 pg/ml.

In one embodiment, the antibiotic is teicoplanin in an amount of at least 0.097 pg/ml.

In one embodiment, the antibiotic is rifampicin in an amount of at least 0.0019 pg/ml.

In one embodiment, the antibiotic is gentamicin in an amount of at least 0.0097 pg/ml.

In one embodiment, the antibiotic is a combination of at least two of vancomycin, teicoplanin, rifampicin and gentamicin.

Traditionally, one may think of antibiotics treatment as administered orally. Such treatment may lead to unwanted side effects, such as affecting or even destroying the protective flora or stimulating the development of antibiotics resistance. Such treatment may also lead to changes in the intestinal bacterial composition, which may result in superinfection by fungi and other infective organisms.

Peptides X and Y (truncated NC8 αβ bacteriocins) may beneficially be administered locally, in the form of a solution, a cream, a gel or in immobilized form (as described further under coating below).

The formulations may further include a solvent and/or a variety of excipients, for instance to stabilize the peptides and suppress aggregation, such as solubilizers, surfactants, bulking agents (such as carbohydrates), thickeners (such as polymers) to increase solution viscosity, preservatives, vehicles, salts or sugars to stabilize proteins and to obtain physiological tonicity and osmolality and/or buffering agents to control pH.

Thus, in one embodiment, the composition is formulated as a solution, a cream, a gel, or an ointment or formulated in immobilized form as a coating on a device.

In another embodiment, the pharmaceutical composition is for use in the treatment or prophylaxis of a bacterial infection.

In one embodiment, the composition is administered locally on the site of infection, such as topically.

To be able to treat local infections, e.g. chronic wounds, peptides from PLNC8 αβ may be linked or associated with a supporting material. To test this, PLNC8 αβ was loaded in a formula (gel) consisting of gelatin and glycerol. PLNC8 αβ in the gel rapidly lysed S. epidermidis and the PLNC8 αβ-containing gel totally inhibited the growth of the bacteria on agar plates (Fig. 13). The activity of PLNC8 αβ in the gel was stable after long-term storage at 4°C for at least 180 days.

Thus, in one embodiment, the composition is formulated as a gel, wherein the gel further comprises gelatine and glycerol.

The effect of formulating the composition as a gel is to provide a localized, long-term antibacterial effect.

In one embodiment, the bacterial infection is caused by Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.

In on further embodiment, the bacterial infection is caused by Staphylococcus spp and/or Streptococcus spp.

Bacterial infection and inflammation is sometimes linked to implants, caused by the bacterial adherence and colonization in the implant area. Treatment may include removing dead tissue, antibiotics, and improved hygiene. Preventive measures include polishing the implant surface, to minimize bacterial adherence, which is a time consuming and costly procedure. Implant coating or treatment with antibacterial material would minimize these incidences and avoid the high-cost of producing a highly polished surface on implant.

Thus, a coating comprising the X and Y peptide of the invention (i.e. truncated bacteriocin NC8 αβ), possibly together with an antibiotic, may be used to impart bacterial resistance to a coating for an implant.

Similarly, such a coating may be used for any medical device, or part of a medical device, where bacterial colonization on the surface should be prevented.

The medical device may also be a band-aid comprising the first and second peptide (i.e. pepides X and Y) and/or antibiotic of the invention. This would help facilitate local administration on a wound or infection site. The bacteriocin and antibiotic may either be tethered to a polymeric scaffold via a flexible linker or physically entrapped in a biopolymeric matrix, its bactericidal property will be retained, or even improved because of its high local concentration

In one embodiment, the composition is formulated in immobilized form as a coating on a device, wherein the device is chosen from the group consisting of a wound dressing, an orthopedic implant, a dental implant, a urinary catheter and an urinary stent. In one embodiment, a pharmaceutical composition is used in coating at least part of a device to limit colonization of bacteria on the surface of the device.

In one further embodiment, the device is a medical device, such as a prosthesis or a wound dressing.

According to one embodiment,, the bacterial infection is caused by

Staphylococcus spp (including MRSA, MRSE), Streptococcus spp (e.g. S. mutans, S. constellatus, S. anginosus), Enterococcus faecium (including VRE), Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or Escherichia coli.

In one embodiment, the bacteria are Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus. In one further embodiment, the bacteria are Staphylococcus spp and/or Streptococcus spp.

Conclusions

Thus, it has been shown that truncated b in combination with full length or truncated a, truncated a and in combination with truncated or full length b, have a rapid and direct effect on different pathogens without expressing any toxic effects on surrounding human cells. In addition, it was found that the combination enhances, 2-130 fold, the effect of and sensitivity to antibiotics. Substitution of L-amino acids of PLNC8α/β by D-amino acids does not change the anti-bacterial effects of the bacteriocin. However, the D-form of PLNC8α/β is much more stable against proteolytic cleavage and is thus adapted for a therapeutical use in vivo.

The data indicates that the use of truncated b in combination with full length or truncated a, truncated a in combination with truncated or full length b, optionally in D/L form, optionally together with antibiotics, is very well suited for the prevention or treatment of infections. This combination can be administered locally in soluble form in gels (ointments, creams) and in immobilized form, e.g. on wound dressings, orthopedic implants, dental implants, urinary catheters and stents, and act antibacterially with no cytotoxic side effects.

Such a combination provides the following advantages: It acts very fast (seconds to minutes); is effective and very potent (nano-micromolar doses); has a wide anti-bacterial spectrum - both against gram-negative and gram-positive bacteria;

facilitates and/or enhances the absorption, activity and efficacy of different conventional antibiotics; enables the use of lower doses of antibiotics, which reduces resistance development; enables treatment of complex infections caused by multiple pathogens including multiresistant bacteria, such as MRSA, in suspension or biofilm; low or no effects on normal flora; In addition, such a combination has low or no cytotoxic effects; is simple and stable; offers cheap production.

Today there is no method of counteracting and treating chronic infections, for example caused by bacterial bio films. Treatment of bio films with antibiotics is very ineffective and costly, and there is also a risk that the protective normal flora is affected and that antibiotic resistance develops. Here it has been shown that truncated peptides of PLNC8αβ can effectively attack different pathogens, in suspension or bio film. The truncation also results in higher diffusion rates into bio films. It has also been shown that the peptides act synergistically with antibiotics. Thus, treatment according to the invention constitutes a more specific, potent and direct anti-bacterial treatment of troublesome infections and associated diseases, and thus lead to less human suffering and greater health-economic effects compared to current forms of treatment.

The invention can be implemented in any suitable form or any combination of forms. Although the present invention has been described above with reference to (a) specific embodiment(s), it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims, e.g. different than those described above.

In the claims, the term "comprises/comprising" does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements may be implemented. Additionally, although individual features may be included in different claims, these may advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an",“first”,“second” etc do not preclude a plurality. Experimental Section

Bacterial culture conditions

Staphylococcus aureus CCUG 35601 (MRSA, Culture Collection, University of Gothenburg) and Staphylococcus aureus ATCC 29213 (MSSA, ATCC, Manassas, VA). Staphylococcus epidermidis ATCC 12228 (ATCC, Manassas, VA), RP62A, N15 and 10 clinical isolates of Staphylococcus epidermidis that have previously been characterized. Isolated Escherichia coli, Enterococcus faecium (including VRE), Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter spp and Acinetobacter baumannii were obtained from Orebro University hospital. Isolated Streptococcus mutans, Streptococcus constellatus and Streptococcus anginosus were obtained from Malmo University. The bacteria were grown on Luria-Bertani (LB) agar plates, supplemented with 5% defibrinated horse blood, and incubated at 37°C overnight. Single colonies were inoculated into 5 ml of LB broth and incubated on a shaker (300 rpm) at 37°C overnight. The bacterial concentration was determined by viable count and adjusted to correlate with approximately 10⁹ CLU/ml.

Peptide synthesis

All chemicals were bought from Sigma Aldrich unless otherwise noted and used without further purification. PLNC8α (H2N-

DLTTKLW S S WGYYLGKKARWNLKHP YV QL -COOH), PLNC8β (H2N- SVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH-COOH), scrambled-PLNC8α (H2N-TWLKY GHGDAKLWSWSKPLNLTFRY QYVK-COOH), scramblcd-PLNC8β (H2N-LKLWNTYGTFSRFYTSKSEVKIAHGIKSIHVPYK-COOH), and truncated forms of PLNC8α and PLNC8β were synthesized using conventional Fmoc chemistry on a Quartet automated peptide synthesizer (Protein Technologies, Inc) in a 100 pmol scale. Peptide elongation was performed using a four-fold excesses of amino acid (Iris biotech gmbh) and activator (TBTU, Iris biothech gmbh) and using an eight- fold excesses of base (DIPEA). Fmoc removal was accomplished by treatment with

Piperidine (20% in DMF, v/v). All peptides were cleaved from their solid support using a mixture of TFA, triisoproylsilane and water (95:2.5:2.5, v/v/v) for 2 h before being, filtered, concentrated and precipitated twice in cold diethylether. Crude peptides were purified on a C- 18 reversed phase column (Kromatek HiQ-Sil C18HS) attached to a semi preparative HPLC system (Dionex) using an aqueous gradient of acetonitrile (10- 46%) containing 0.1% TFA. Mass identity of all peptides was confirmed by MALDI- ToF MS (Applied biosystems) using a-cyano-4-hydroxycinnamic acid as matrix. To study the effects and stability of D-forms of PLNC8α and PLNC8β, the I form of amino acids was substituted with the D-form of amino acids during peptide synthesis. The sensitivity to proteolytic cleavage of D-PLNC8α , D-PLNC8β, L- PLNC8α and L-PLNC8β was analyzed by exposing the peptides to Trypsin for l6h, whereafter the presence of proteolytic fragments was determined with MALDI-TOF mass spectrometry.

Liposome preparation

Liposomes were prepared by dry film formation, hydration and finally extrusion through a polycarbonate membrane to form monodisperse large unilamellar vesicles. The lipids l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1- palmitoyl-2-oleoyl-sn-glycero-3 -phosphatidylcholine (POPC) (Avanti Polar Lipids, Alabaster, USA) were mixed at molar ratios 1 :99, 5:95 and 10:90 while dissolved in chloroform. A dry lipid film was formed by evaporation of the chloroform by nitrogen flow and overnight lyophilization. The film was hydrated with either 10 mM phosphate buffer (PB) pH 7 or 10 mM phosphate buffer saline (PBS) pH 7, and the solution was vortexed for 1 min and put on a shaker for 1 h before extruded 21 times through a 100 nm pore-sized polycarbonate membrane. For fluorescence leakage assay the lipid film was hydrated with buffer (PBS) containing self-quenching concentration (50 mM) of 5(6)-carboxyfluorescein (CF) (Sigma Aldrich) and liposomes were prepared as described above. Removal of unencapsulated CF was done by gel filtration using a PD- 25 column (GE Healthcare, Uppsala, Sweden) and liposomes with encapsulated CF were eluted with PBS.

Fluorescence leakage assay

Leakage of the liposome encapsulated fluorophore CF due to additions of the bacteriocins was recorded using a fluorescence plate reader (Infinite 200, Tecan, Austria) where λ_ex = 485 nm and

= 520 nm. CF was encapsulated at self-quenching concentration, and CF release results in an increased fluorescence signal. Liposomes were diluted to 25 mM (total lipid concentration) in PBS, followed by additions of 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 mM of the L-form or D-form of PLNC8 a and b, separately and combined, and truncated forms of PLNC8 a, separately and combined with PLNC8 b, and truncated forms of PLNC8 b, separately and combined with PLNC8 a. In order to estimate the maximum release from each sample a final addition of 0.5 % Triton X-100 was made at the end of all measurements and the total amount of CF (100% release) was estimated after 15 min incubation. The CF release is presented as percentage release for each time interval (measurements taken every minute). The percentage CF release is calculated as 100 x (F - F₀)/(F_{T -} F₀) where Fo is the initial fluorescence intensity of CF before peptide addition, F is the fluorescence intensity of CF at time point t and F_T is the maximum fluorescence after the addition of Triton X-100. Results are shown in Fig. 4.

Antimicrobial activity of PLNC8 ab

The broth microdilution method was used to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Two-fold serial dilutions of the peptides were used and the final concentrations ranged from 0.097-50 mM. The final concentrations of the antibiotics vancomycin and teicoplanin ranged from 0.097-50 Lig/ml, while rifampicin ranged from 0.0019-1 μg/ml and gentamicin 0.0097-5 Lig/m 1. The effect of bacteriocin-antibiotic combinations was accomplished by using the same concentration series of antibiotics with a constant concentration of bacteriocins (3.1 mM) in all the wells. The MIC was determined visually and spectroscopically (620 nm) as the first concentration that completely inhibited bacterial growth. All concentrations that resulted in complete inhibition of bacterial growth were cultured (10 mΐ) on blood- agar plates, and the lowest concentration where no growth was observed on agar represented the MBC. All experiments were repeated at least three times.

Microscopy

The fluorescent dye Sytox® Green, which can only cross damaged membranes and fluoresce upon binding to nucleic acids, was used to study the antimicrobial activity of PLNC8 αβ on S. epidermidis, S. aureus (MSSA, MRSA) and Streptococcus spp. . The bacteria were washed and resuspended in Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KC1, 1.2 mM MgS0₄, 1.7 mM KH₂P0₄, 8.3 mM Na₂HP0₄, and 10 mM glucose, pH 7.3) and incubated in the presence or absence of different combinations of PLNC8 αβ in 96-well microtiter plates for 2 min. Images were captured with Olympus BX41 at 40x magnification.

Electron microscopy was used to visualize the damage of bacteria caused by PLNC8 αβ. Briefly, bacteria were pelleted and washed with Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KC1, 1.2 mM MgS0₄, 1.7 mM KH₂P0₄, 8.3 mM Na₂HP0₄, and 10 mM glucose, pH 7.3). The bacteria were then treated with different concentrations of PLNC8 αβ in a ratio of 1 : 1 for 5 min, followed by fixation in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.3. Critical point drying was applied for specimens for SEM coated with Gold using a Sputter coater. Specimens for TEM were washed in 0.1M phosphate buffer, postfixed in 2% osmium tetroxide in 0.1M phosphate buffer for 2 hours and embedded into LX- 112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Munster, Germany). Representative images of three independent experiments can be seen in Fig. 12.

Circular Dichroism (CD) spectroscopy

Bacteriocins are often unstructured in solution but typically adopt a more ordered secondary structure when bound to the bacterial cell membrane as a result of membrane partitioning. Circular dichroism spectroscopy measurements were performed on a Chirascan (Applied Photophysics, United Kingdom) using a 1 mm cuvette at room temperature. A wavelength scan of 195-280 nm was recorded 3 times for each sample, averaged and baseline corrected using PB buffer (pH 7.4, 10 mM). In all samples, the concentration of each peptide was 30 mM, prepared in PB buffer. In experiments with liposomes the final lipid concentration was 660 mM (0.5 mg/ml). To compensate for the different total peptide concentrations used, the averaged data were converted to mean residue ellipticity (MRE).

Proteolytic degradation

Full length PLNC8 αβ (100 mM) in both L-and D-form was subjected to Trypsin (0.125 mg/ml, ~5 mM) in ammonium bicarbonate buffer (50 mM, pH 8.5) for 16 hours at 37 °C. Sample solutions were acidified by adding 2.5 % TFA and dried in an exicator at room temperature. Samples were resuspended in MQ-water containing 0.1% TFA, desalted using ZipTip-Cl8 columns (Millipore) and analyzed using MALDI-ToF MS (UltraflexXtreme, Bruker Daltonics) with α-cyano-4-hydroxycinnamic acid as matrix.

Hemolysis

The hemolytic activity of the peptides was investigated by collecting blood from healthy volunteers in heparinized vacutainers. The blood was centrifuged at 600 xg for 5 min and the erythrocyte pellet was washed three times in PBS. The cells were then suspended in PBS and added to 96-well plates (15% erythrocyte suspension/well), containing the peptides with two-fold serial dilution. The plates were incubated for 1 h at 37°C followed by centrifugation for 5 min at 900 xg and measurement of the supernatants at 540 nm. Haemolytic activity (%) was calculated by subtracting the negative control from all values and normalization against the positive control (0.5% Triton X-100), that was set to 100%. All experiments, each in duplicate, were repeated three times.

Bacterial biofilms

S. epidermidis RP62A was inoculated into 5 ml of LB broth and incubated on a shaker at 37°C overnight. The bacterial culture was diluted 1 :100 into fresh media and 100 mΐ of bacterial suspension per well was added in a 96-well microtiter plate and incubated statically at 37°C for 20 h. The wells were washed three times by submerging the plate into a container with distilled water to remove unattached cells. Fresh LB media was added to each well (100 mΐ) followed by addition of the peptides in different concentrations. The plate was incubated statically for 1 h. Detached material in the wells were transferred to a new microtiter plate for absorbance measurements at 620 nm. The remaining attached bio films were stained with 0.1% crystal violet for 15 min before the plate was washed four times in distilled water as mentioned above and allowed to dry at room temperature for 2 h. The crystal violet was solubilized in 30% acetic acid for 15 min and the absorbance quantified at 550 nm. Each experiment, with three replicates, was repeated three times.

Statistical analysis

All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA with Bonferroni's post hoc test was used for the comparisons between the different treatments. P-values are referred to as *p<0.05;

**p<0.0l; ***p<0.00l .

Ethics statement

This work deals with clinical bacterial isolates from human infections. No tissue material or other biological material was stored from the patients, only

subcultured bacterial isolates. Swedish law does not require ethical approval for work with bacterial isolates from humans. All information regarding these isolates was anonymized. Results

The effects of PLNC8αβ on different strains of S. aureus and S. epidermidis were studied (Table 1). PLNC8αβ markedly inhibited the growth and the survival of all bacterial strains (Fig. 1).

In addition, PLNC8 αβ permeabilized and killed Streptococcus spp (Fig. 17),

Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecium (Table 5-7).

In order to investigate the role of PLNC8 a and PLNC8 b, respectively, in the inhibitory and bactericidal action of the bacteriocin, the effects of different molar ratios between the peptides on S. epidermidis were studied. It was found that a PLNC8 a to PLNC8 b molar ratio of 1 : 1 is most efficient at inhibiting and killing S. epidermidis (Fig. 2). However, ratios between 1 :1 and 1 :7 were also found to be effective.

Since most bacteria grow and are a part of complex biofilms, where they often are more resistant against antibiotic treatment compared to when they exist in a planktonic state, the effects of PLNC8 αβ on biofilms consisting of S. epidermidis were tested. It was found that PLNC8 αβ efficiently disrupted the biofilms and killed the bacteria (Fig. 3). Also the a and b peptide of PLNC8 exerted by themselves, although at higher concentrations, disruptive effects on the biofilms.

The antibacterial activity of PLNC8 αβ may in vivo be restricted by proteolytic activity exerted by proteases from both bacteria and human cells. In order to circumvent the problem with a proteolytic cleavage of PLNC8 αβ, the L-form of amino acids, that normally occurs in peptides such as PLNC8 αβ, was substituted with the D-form of amino acids. The effects of the L- and D-variants of PLNC8 αβ were tested on both a liposome system (resembling bacteria) and on S. epidermidis. It was found that the D- variant of PLNC8 αβ was almost as effective in destroying liposomes and inhibiting and/or killing S. epidermidis as the L-variant (Fig. 4). Furthermore, the perturbation of the plasma membrane of S. epidermidis was equally rapid (2 min) for the L- and D- variant, respectively, of PLNC8 αβ (Fig. 5).

To analyze whether PLNC8 αβ with D-amino acids is more stable and less sensitive to proteolytic cleavage compared to the L-variant of PLNC8c 3; D-PLNC8α , D-PLNC8β, L-PLNC8α and L-PLNC8β were exposed to trypsin and the presence of proteolytic fragments was analyzed with MALDI-TOF mass spectrometry (Fig. 6). While trypsin generated several fragments of both the a- and b-peptide of L-PLNC8, no obvious fragmentation was observed of the a- and b-peptide of D-PLNC8. The truncated forms of PLNC8β, β1 -20 and b7-20, most efficiently inhibited the growth of S. epidermidis (more effective than the full-length native PLNC8 b (1-34) (Fig. 9)) and, as shown in figure 6, were also restistant to further proteolytic cleavage.

To clarify whether PLNC8 αβ (the L- and D-variant) exerts cytotoxic effects, lysis of erythrocytes isolated from human whole blood was investigated. However, no hemolytic activity was observed (figure 7).

Truncated forms of PLNC8 αβ express antibacterial activities similar to the native bacteriocin or are even more effective. Truncated peptides of PLNC8 a and PLNC8 b, respectively, were constructed in sequences of 6-7 amino acids corresponding to the number of amino acids needed for formation of an alpha helix (figure 8). The effects of truncated PLNC8 a and PLNC8 b were tested on both a liposome system (resembling bacteria) and on S. epidermidis. Disruption of the liposome membranes, revealed by release of (6)-carboxyfluorescein (CF), was obtained with the b-peptides 1- 34 (full-length), 7-34, 1-20 and 7-20 (figure 9). When combined with PLNC8 a, effects were also obtained with the other truncated peptides, although at higher concentrations.

Interestingly, growth of S. epidermidis was most efficiently inhibited by either sequence bΐ -20 or b7-20, and these truncated peptides were more effective than the full- length native PLNC8 b (1-34) (Fig. 9).

The peptide b-sequences b7-13 and b14-20 are crucial for the effects of PLNC8 b and are more efficient when combined with b1-6. Thus, the peptide b1-20 is most effective in inhibiting S. epidermidis.

The truncated form 1-22 of the α-peptide and the full-length α-peptide (1-29) disrupted the membrane of the liposomes, revealed by a release of carboxyfluorescein (figure 10). However, the different truncated forms of the α-peptide had no significant effects on S. epidermidis. In combination with the b-peptide, α1-22 exerted inhibitory and bactericidal effects (Fig. 10).

To be able to treat local infections, e.g. chronic wounds, PLNC8 αβ was used with a supporting material. PLNC8 αβ was loaded in a formula (gel) consisting of gelatin and glycerol. PLNC8 αβ in the gel rapidly lysed S. epidermidis and the PLNC8 αβ-containing gel totally inhibited the growth of the bacteria on agar plates (Fig. 13). The activity of PLNC8 αβ in the gel was stable after long-term storage at 4°C for at least 180 days.

Heterogeneous glycopeptide intermediate S. epidermidis (hGISE) is common in prosthetic joint infections (PJIs). Glycopeptide treatment, such as treatment with vancomycin and teicoplanin, is not sufficient in many cases of PJIs. We found that PLNC8 αβ effectively inhibits different strains of S. epidermidis isolated from PJIs, including S. epidermidis (hGISE) (figure 14). The D-form of PLNC8 αβ is almost as effective as the L-form in inhibiting strain S. epidermidis 154 (Fig. 15).

Combination therapy is utilized both to prevent the development of antibiotic resistance and to shorten the length of treatment. The effect in the treatment of S.

epidermidis of the combination of L-PLNC8 αβ or D-PLNC8 αβ with different antibiotics belonging to different classes was also shown: the cell wall synthesis inhibitors vancomycin and teicoplanin, the nuclic acid synthesis inhibitor rifampicin and the protein synthesis inhibitor gentamicin.

Both L-PLNC8 αβ and D-PLNC8 αβ decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (Fig. 15). A combination of PLNC8 αβ and rifampicin was even more effective. MIC and MBC of rifampicin was lowered more than lOO-fold when treating S. epidermidis in the presence of L-PLNC8 αβ or D-PLNC8 αβ (Fig. 15). Furthermore, L-PLNC8 αβ decreased MIC and MBC of gentamicin 15 -30 fold against S. epidermidis. However, L-PLNC8 αβ or D-PLNC8 αβ lowered MIC and MBC of vancomycin 2-fold.

A combination of the truncated α-peptide 1-22 with full-length b-peptide decreased MIC and MBC of teicoplanin more than lO-fold against S. epidermidis (figure 15), i.e. the same effects as with PLNC8 αβ (figure 14). α1-22 and b1-20 lowered MIC and MBC of teicoplanin approximately 4-fold, however, full-length α- peptide and b1-20 had no effects (figure 16). As can be seen in table 8 below, the full- length and truncated PLNC8 b and PLNC8 a markedly amplify the inhibitory and bactericidal effects of teicoplanin and rifampicin against S. epidermidis.

A combination of the truncated α-peptide 1-22 with full-length b-peptide decreased MIC of rifampicin approximately 4-fold against S. epidermidis. α1-22 and b1-20, respectively full-length α-peptide and b1-20, have two-fold effect (Fig. 16).

In figure 17. PLNC8 αβ markedly permeabilizes and kills different species of Streptococcus. Streptococcus spp (S. mutans (Sm), S. constellatus (Sc) and S. anginosus (Sa)) were treated with 5mM PLNC8 αβ for 2 min, followed by analysis of uptake of Sytox Green. S. constellatus and S. anginosus were more susceptible to PLNC8 αβ than S. mutans. References

1) Khalaf, H., Nakka S., Sanden, C., Svard, A., Scherbak, N., Hultenby, K., Aili, D., Bengtsson, T. (2016) Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis, BMC Microbiology, 18:88.

2) Bengtsson, T., Zhang, B., Selegard, R., Wiman, E., Aili, D., Khalaf, H.

(2017). Dual action bacteriocin PLNC8 αβ through inhibition of Porphyomonas gingivalis infection and promotion of cell proliferation. Pathogens and Disease, 2017 Jun 12. Doi: l0.l093/femspd/ftx064

Claims

1. A polypeptide X having 14 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with

YTLGIKILW S AYKH SEQ ID NO 3.

2. The polypeptide X according to claim 1 , wherein the polypeptide X

comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:

SVPTSVYTLGIKILWSAYKH SEQ ID NO 5 VPTSVYTLGIKILWSAYKH SEQ ID NO 6 PTSVYTLGIKILWSAYKH SEQ ID NO 7 TSVYTLGIKILWSAYKH SEQ ID NO 8 SVYTLGIKILWSAYKH SEQ ID NO 9 V YTLGIKILW S AYKH SEQ ID NO 10 YTLGIKIL W S AYKH SEQ ID NO 3 YTLGIKILW S AYKHR SEQ ID NO 11 YTLGIKILW S AYKHRK SEQ ID NO 12 YTLGIKILW S AYKHRKT SEQ ID NO 13 YTLGIKILW S AYKHRKTI SEQ ID NO 14 YTLGIKILW S AYKHRKTIE SEQ ID NO 15 YTLGIKILW S AYKHRKTIEK SEQ ID NO 16 YTLGIKILW S AYKHRKTIEKS SEQ ID NO 17 YTLGIKILW S AYKHRKTIEKSF SEQ ID NO 18 YTLGIKILW S AYKHRKTIEKSFN SEQ ID NO 19 YTLGIKILWSAYKHRKTIEKSFNK SEQ ID NO 20 YTLGIKILW S AYKHRKTIEKSFNKG SEQ ID NO 21 YTLGIKILWSAYKHRKTIEKSFNKGF SEQ ID NO 22 YTLGIKILWSAYKHRKTIEKSFNKGFY SEQ ID NO 23 YTLGIKILW S AYKHRKTIEKSFNKGF YH SEQ ID NO 24

3. The polypeptide X according to claim lor 2, wherein at least 90% of the amino acids in said polypeptide X are D-amino acid residues.

4. A pharmaceutical composition comprising a polypeptide X according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, further comprising a polypeptide Y having 15 to 28 amino acids and comprising an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with

DLTTKLWSSWGYYLG SEQ ID NO 4.

6. The pharmaceutical composition according to claim 5, wherein the

polypeptide Y comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity (%SI) with an amino acid sequence selected from the group comprising:

7. The pharmaceutical composition according to any one of claims 4 to 6,

wherein at least 90% of the amino acids in peptide X and/or peptide Y are D- amino acid residues.

8. The pharmaceutical composition according to any one of claims 4 to 7, further comprising at least one antibiotic.

9. The pharmaceutical composition according to any one of claims 4 to 8,

wherein the antibiotic is selected from the group consisting of antibioticts that inhibit bacterial cell wall synthesis, antibiotics that inhibit nucleic acid synthesis and antibiotics that inhibit protein synthesis.

10. The pharmaceutical composition according to any one of claims 5 to 9,

wherein peptide X and peptide Y are present in a molar ratio of from between 5: 1 to 1 :20, preferably 1 : 1 to 1 :7, most preferably 1 : 1.

11. The pharmaceutical composition according to any one of claims 4 to 10, wherein the pharmaceutical composition comprises between 100 nM to 50 mM of peptide X and/or peptide Y.

12. The pharmaceutical composition according to any one of claims 8 to 11 , wherein the pharmaceutical composition comprises the antibiotic in an amount of between 0.002 pg/ml to 50 pg/ml, such as at least 0.01 pg/ml to 5 pg/ml, such as at least 0.1 pg/ml to 1 pg/ml, such as at least 0.8 pg/ml.

13. The pharmaceutical composition according to any one of claims 4 to 12, wherein the pharmaceutical composition is formulated as a solution, a cream, a gel, or an ointment or formulated in immobilized form as a coating on a device.

14. The pharmaceutical composition according to claim 13, wherein the

composition is formulated as a gel, wherein the gel further comprises gelatine and glycerol.

15. The pharmaceutical composition according to claim 13, wherein the

composition is formulated in immobilized form as a coating on a device, wherein the device is chosen from the group consisting of a wound dressing, an orthopedic implant, a dental implant, a urinary catheter and an urinary stent.

16. A pharmaceutical composition for use in the treatment or prophylaxis of a bacterial infection, wherein the pharmaceutical composition is a

pharmaceutical composition according to anyone of claims 4 to 15.

17. The pharmaceutical composition for use according to claim 16, wherein the bacterial infection is caused by Staphylococcus spp (including MRSA, MRSE), Streptococcus spp (e.g. S. mutans, S. constellatus, S. anginosus ), Enterococcus faecium (including VRE), Klebsiella pneumoniae,

Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and/or Escherichia coli.

18. The pharmaceutical composition for use according to claim 16, wherein the bacterial infection is caused by Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.

19. The pharmaceutical composition for use according to any one of claims 16 to 18, wherein the composition is administered locally on the site of infection, such as topically.

20. Use of a pharmaceutical composition according to any one of claims 4 to 15 in coating at least part of a device to limit colonization of bacteria on the surface of the device.

21. Use of a pharmaceutical composition according to claim 20, wherein the device is a medical device, such as a prosthesis or a wound dressing.

22. Use of a pharmaceutical composition according to claim 20 or 21 , wherein the bacteria are Staphylococcus spp (including MRSA, MRSE),

Streptococcus spp (e.g. S. mutans, S. constellatus, S. anginosus ),

Enterococcus faecium (including VRE), Klebsiella pneumoniae,

23. Use of a pharmaceutical composition according to claim 20 or 21 , wherein the bacteria are Staphylococcus spp, Streptococcus spp, such as S. mutans, S. constellatus, S. anginosus.

24. A polypeptide for use in the treatment or prophylaxis of a bacterial infection, wherein the polypeptide is a polypeptide according to anyone of claims 1 to 3.