FIELD OF THE INVENTION:

The present invention relates to methods for inhibiting or reducing Pseudomonas biofilm formation, in particular Pseudomonas aeruginosa biofilm. More specifically, the present invention relates to cationic polymers and their use in treating Pseudomonas aeruginosa bio films present in endotracheal tube (ETT) of mechanically ventilated patients. BACKGROUND OF THE INVENTION:

Many bacteria switch between a single-cell motile lifestyle and multi-cellular, sessile adhesive states forming biofilm, resulting in a protected growth mode which allows cells to survive and thrive in hostile environments (Hall-Stoodley, Let ah, Nat Rev Microbiol, 2004: 2, 95-108). In most environments, bacteria are thought to reside predominantly in biofilms (Costertonei al., 1995), in contrast to planktonic or free-swimming cells typically studied in the laboratory. Pseudomonas aeruginosa has been shown to form biofilms on a number of surfaces, including the tissues of the cystic fibrosis lung (Govanand Deretic, 1996) and on abiotic surfaces such as contact lenses and catheter lines (Nickel et al., 1985; 1989; Millerand Ahearn, 1987; Fletcher et al., 1993). This ubiquitous organism is also the cause of nosocomial infections in immunocompromised patients and individuals with severe burns (Bodeyei al., 1983).

Ventilator-associated pneumonia (VAP) is the most frequent hospital acquired infection in patients requiring mechanical ventilation and its estimated incidence is 10-20%. In addition to being an independent factor for mortality; VAP is associated with longer intensive care unit and hospital stays, prolonged mechanical ventilation, and higher costs (1, 2). Pseudomonas aeruginosa is the main pathogen of nosocomial pneumonia (3).

Ventilator-associated tracheobronchitis (VAT) is also common in critically ill patients. This infection represents an intermediate process between colonization of lower respiratory tract and VAP. VAT is characterized by increased purulent sputum production and lower respiratory tract inflammation resulting in difficult weaning and prolonged duration of mechanical ventilation (4).

Bacterial biofilm is universally present within the endotracheal tube (ETT) of mechanically ventilated patients, representing a potential source of infection (5). Biofilm on the ETT is a complex structure made of pathogens enclosed within a self-produced polymeric matrix, and respiratory secretions. The accumulation of biofilm and secretions within the ETT progressively obstructs its lumen, particularly in patients on long-term mechanical ventilation.

In the majority of patients who developed VAP, a strict association between pathogens cultured from the ETT biofilm and the lower respiratory tract has been demonstrated (6). A laboratory animal study demonstrates that following intubation with an ETT previously colonized by mature P. aeruginosa biofilm, there is a consistent translocation of pathogens into the airways (7). Indeed, the inspiratory flow interacts with the biofilm surface, which become unstable and eventually particles may be disseminated into the airways (8).

Biofilm plays a role in the development of VAP or in the relapse of VAP after treatment. Indeed, biofilm is an adaptive survival advantage for bacteria as it increases bacterial resistance to antimicrobials (9). Of note is that the effects of systemic antibiotic treatment on ETT biofilm treatment or prevention are very limited. Antibiotics do not eradicate ETT biofilm (10, 11), and only dedicated prototypes have shown removal of biofilm and secretions from within the ETT (12). However, those prototypes required disconnection of the ventilator for a period of time longer than standard suctioning.

Overall, after treatment of patients who developed VAP or VAT with systemic antibiotic, one can assume that the whole lung is initially sterilized from the responsive pathogen; however, systemic antibiotics are inefficient in eradicating an established biofilm on ETT. Thus, VAP or VAT may relapse from bacterial disseminated from the ETT biofilm towards the lower respiratory tract, giving rise to infection.

Preventing or treating the formation of microbial biofilm on the ETT seems mandatory to reduce the incidence of VAP. Previous attempts to treat microbial biofilm on the ETT are not satisfactory. Nebulized antibiotic has been proposed but there is an increasing trend to minimize patient exposure to antibiotics. The overuse of antibiotics in ICUs is well documented and the efficacy of antibiotic prophylaxis for VAP remains controversial, particularly in the actual context of increasing resistance of bacteria to antibiotics. Dedicated devices have been investigated to treat microbial biofilm on the ETT, but are time-consuming and complicate options that increase the work burden of nurse-care.

Accordingly, there is a need to develop new compounds that will be suitable for inhibiting Pseudomonas adhesion and aggregation and bio films formation, and new drugs that will be suitable for preventing or treating infection from a biofilm. In this way, it has been suggested that characterization of new anti-bacterial compounds for inhibiting Pseudomonas adhesion and aggregation and biofilms formation and for treatment or prevention of infection from a biofilm may be highly desirable. It was known in the Art that cationic polymers and in particular poly-L-lysine have the capacity to form electrostatic complexes with electronegativity charged compounds. a-poly-L- lysine and positively charged amino acids have also been described in the art for their antimicrobial properties (Dubois et ah, 2013).

SUMMARY OF THE INVENTION:

Therefore, the present invention relates to a method of inhibiting or reducing Pseudomonas biofilm formation on a surface comprising the step of applying to the surface an amount of a cationic polymer.

In a particular embodiment cationic polymers that can be used in the present invention have the following formula (I):

wherein · R is NH2 or NH linked to a histidine residue or other molecules including charged amino acids, a gluconoyl residue, a glycosyl residue or a PEG moiety, • i is the degree of polymerization comprised between 10 and 75, preferably between 10 and 50, and more preferably between 20 and 50, for example between 20 and 40 or between 30 and 50.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have now found that cationic polymers such as poly-aL-lysine have the following properties: i/ they "condensate" the biofilm present in a medical device as endotracheal tube of mechanically ventilated patients, ii/ they act as aeruginosa biofilm agents. The inventors demonstrated that cationic polymers efficiently and rapidly eliminate bacteria from biofilms.

Altogether these results provide new insights for treating biofilm on surface such as endotracheal tube, using cationic polymers of the invention as main active principle ingredient for inhibiting Pseudomonas aeruginosa biofilm.

Methods and uses of the invention

A first aspect of the invention relates to a method of inhibiting or reducing Pseudomonas biofilm formation on a surface comprising the step of applying to the surface an amount of a cationic polymer according to the invention.

In a particular embodiment, the cationic polymers that can be used in the present invention have the following formula (I):

wherein

• R is NH2 or NH linked to a histidine residue or other molecules including charged amino acids, a gluconoyl residue, a glycosyl residue or a PEG moiety,

• i is the degree of polymerization comprised between 10 and 75, preferably between 10 and 50, and more preferably between 20 and 50, for example between 20 and 40 or between 30 and 50.

In one specific embodiment, said cationic polymers for use according to the invention comprise a sufficient amount of lysine residues (whether histidinylated or not) condensate the Pseudomonas aeruginosa biofilm present in a surface and/or to act as anti-Pseudomonas aeruginosa biofilm agents. By "amount" or "sufficient amount" "or dosage level" is intended to be an amount of cationic polymers of the invention, that, when applied brings about a positive response with respect to condensate the Pseudomonas aeruginosa biofilm present in a surface and/or to act as anti-Pseudomonas aeruginosa biofilm agents. Actual dosage levels of the cationic polymer of the present invention may be varied so as to obtain an amount of the cationic polymer which is effective to achieve the desired anti-biofilm response for a particular surface, mode of application (solution or by aerosolization). Typically for ETT surface, the dosage when cationic polymers of the invitation are applied in solution is between 1 to 10 mL, preferably between 3 to 7mL more preferably 5mL. Typically for ETT surface, the dosage when cationic polymers of the invitation are applied by aerosolization is between 10 to 1 ΟΟΟμί, preferably between 100 to 300μί more preferably 200μΕ.

The effect of antibiofilm activity can be measured by monitoring condensation and reduction of the bacterial biofilm present in a surface prior to and after application with the compositions according to the invention, using in vitro assays (Susceptibility assay) adapted from different assays (George A. 0'Toole,Microtiter Dish Biofilm Formation Assay, J Vis Exp. 2011; (47): 2437).

In one more embodiment, the cationic polymers of formula (I) have a ratio of grafting of at least 10%, 20%, 30%, 40%, 50%, or 60%, preferably comprised between 10% and 60%, or between 10%> and 55%, or between 10%> and 40%>, more preferably between 10%> and 35%. For example, the cationic polymers of the present invention have a ratio of grafting of between at least 10% and 60%, and a degree of polymerization i between 20 and 50 or between 20 and 40.

More specifically, the cationic polymers of the present invention may have a ratio of grafting of between at least 10%> and 60%>, or between 10%> and 55%, or between 10% and 40%, and a degree of polymerization i between 30 and 40.

For ease of reading, specific cationic polymers of formula (I) according to the invention will be named hereafter as pLK(XX)His(YY), wherein XX refers to the average degree of polymerisation of the cationic polymers and YY refers to the average number of histidine grafted to lysine residues. In specific embodiments, the cationic polymers of the present invention are pLK36-

Hisl9, pLK36-His8, pLK72-His31, pLK72-Hisl7, pLK30-His4.5 and pLK30-His8. Lysyl (non-histidinylated) residues of the cationic polymers of the present invention may optionally be further at least partially replaced by other known positively charged amino acids, including without limitation histidine, arginine or ornithine. As used herein, the term "positively charged" refers to the side chain of the amino acids which has a net positive charge at a pH of 7.0.

A cationic polymer according to the invention typically may include at least 50% (per monomeric unit), 60% or at least 70%> of positively charged amino acid residues, preferably at least 50%>, 60%>, 70%> (per monomeric unit) of lysine residues, for example between 50%> and 90%) (per monomeric unit) of lysine residues.

Parameters such as the number of monomers (e.g. number of amino acids), the type of monomeric units (e.g. type of amino acids) and the percentage of positively charged monomeric units (e.g percentage of positively charged amino acids in a polyaminoacid) may be optimized by measuring the efficacy of the final structure in an in vitro assay for assessing condensatethe bacterial biofilm present in a surface and/or to act as anti-Pseudomonas aeruginosa biofilm agents, as disclosed in the Examples below (Susceptibility assay of Pseudomonas aeruginosa biofilm).

In particular cationic polymers for use according to the present invention include poly- L-lysine, without derivatized or substituted with histidine or neutral residue.

For example, derivatization of lysyl residues of poly-L-lysine with histidine is described in Midoux, P. and Monsigny, M. (Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjugate Chem. 1999, 10, 406-411). Methods for synthesizing the cationic polymers of the present invention are also described in WO2015078995.

Equivalent amino acids may also be used in the cationic polymers of the invention, including amino acids having side chain modifications or substitutions, the final polymer retaining its advantageous property of acting as anti-Pseudomonas aeruginosa biofilm agents.

In particular, (D) or (L) amino acids may be used, or chemically modified amino acids, including amino acid analogs such as penicillamine (3-mercapto-D-valine), naturally occurring non-proteogenic amino acids and chemically synthesized compounds that have properties known in the art to be characteristic of an amino acid.

Cationic polymers useful for this invention can be produced using technique well known in the Art, including either chemical synthesis or recombinant DNA techniques. Cationic polypeptides can be synthesized using Solid Phase Peptide Synthesis techniques with tBoc or Fmoc protected alpha-amino acids (Scholz, C.et ah, J Control Release, 2011). Alternatively, polycationic polypeptides can be produced using recombinant DNA techniques (See Coliganei al, Current Protocols in Immunology, Wiley Intersciences, 1991, Unit 9; US Pat. No. 5,593,866). In specific embodiments, the cationic polymers may be PEGylated. PEGylation is the process of covalent attachment of polyethylene glycol polymer chains to another molecule. Polyethylene glycol (PEG) molecules may be added onto cationic polymers in order to limit DNA complexes aggregation, adsorption of proteins and to lower aggregate as well as polymer cytotoxicity (Ogris, M.et al, P. Gene Ther, 1999; Toncheva, V. et al, Biochim Biophys Acta, 1998 ; Choi, Y. et al., 1999). The covalent attachment of PEG to cationic polymers may facilitate and does not compromise administration of said cationic polymers into the airways in the form of an aerosol (Dailey, L. A., et al, J Control Release, 2004).

The covalent attachment of PEG moiety onto cationic polymer can be performed by two ways leading either to a PEG-grafted-polymer or a block copolymer.

For example, PEG-grafted polylysine (PEG-g-pLK) is prepared by reaction of the N- hydroxysuccinimide derivative of the methoxypolyethylene glycol (mPEG)propionic acid (for instance of 5000 Da)with the ε-amino group of the lysyl residues of pLK (Mockey, M., et al, Cancer Gene Ther,2007).

For example, PEG-pLK(XX)His(YY) block copolymer can be prepared either by :

-(i) reaction between equal molar ratios of pLK(XX)His(YY) containing a cysteinyl residue at its C-terminal end with methoxy-PEG-maleimide as described in Ziady, A.G.ei al.

(Mol Ther., 2003);

- (ii) ring opening polymerization of N ^£ -trifluoroacetyl-L-lysine N-carboxyanhydride with the co-NFh terminal group of a-methoxy-ro-amino PEG as described in Itaka, K., et al. (J Control Release, 2010).

In other specific embodiments, the cationic polymers in accordance with the present invention are glycosylated. For example, derivatization of lysyl residues of poly-L-lysine with mannose, galactose or lactose is described in (Erbacher, P. et al (Bioconjug Chem,1995); Erbacher, P., et al. (Hum Gene Ther, 1996).Mannosyl-PEG, galactosyl-PEG or lactosyl-PEG may be grafted on poly-L-lysine as described in (Sagara, K. et al. (J Control Release,2002).

In other specific embodiments, the cationic polymers in accordance with the present invention are gluconoylated in order to decrease the number of positive charges and the cytotoxicity. For example, derivatization of lysyl residues of poly-L-lysine with β- gluconolactone is described in (Erbacher, P. et al(Biochim Biophys Acta, 1997).

The term "bacterial biofilm" has its general meaning in the art and refers to structured communities or aggregates of bacterial cells in which cells adhere to each other and/or to a living or inert (non-living) surface. These adherent cells are frequently embedded within a self- produced matrix of extracellular polymeric substance. Biofilms represent a prevalent mode of microbial life in natural, industrial and hospital settings. Biofilms can contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae. According the method of the invention, the biofilm is produced by Pseudomonas bacteria. The term "Pseudomonas bacteria" has its general meaning in the art and refers to bacteria that occur normally or pathogenically in lung of humans and other animals. The term "Pseudomonas bacteria" refers to but it is not limited to gram-negative bacteria Pseudomonas, e.g; a bacterium of the Pseudomonas aeruginosa group such as P. aeruginosa group P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borbori, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, .

In particular, the Pseudomonas biofilm according to the invention is Pseudomonas aeruginosa biofilm.

Pseudomonas aeruginosa is a common Gram-negative bacteria that can cause disease in animals, including humans. It is citrate, catalase, and oxidase positive. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, its versatility enables the organism to infect damaged tissues or those with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and kidneys, the results can be fatal (Balcht, et al (Informa Health Care, 1994). Because it thrives on moist surfaces, this bacterium is also found on and in medical equipment, including catheters, causing cross- infections in hospitals and clinics.

As used herein the term "surface" refers to any surface where bacteria (e.g. Pseudomonas bacteria) are liable to grow on.

In some embodiments, the surface is an artificial surface or is a biological surface. Typically, artificial surfaces include but are not limited to surfaces that can be used for medical, sanitary, veterinary, food preparation (e.g. food industry), agribusiness or agronomic purposes. Typically, the material is made of plastic, metal, glass or polymers. In some embodiments, the surface is any surface that constitutes an environment wherein development of enteric bacteria is not desirable (e.g. hospitals, intensive care units, dental offices...). For example, the surface is a surface of hospital furniture, non implantable and implantable devices or medical tools that are liable to be in contact with patients.

In some embodiments, the cationic polymer of the present invention is applied to a surface of a material.

As used herein, the term "material" denotes any material for any purposes, including but not limiting to, research purposes, diagnostic purposes, and therapeutic purposes. Typically the material is a natural material or is an artificial material (i.e. a man-made material). The material can be less or more solid, less or more flexible, can have less or ability to swell... In some embodiments, the material is an artificial material. Typically the material is selected form the group consisting of membranes, scaffold materials, films, sheets, tapes, patches, meshes or medical devices. In some embodiments, the material is biocompatible material. As used herein, the term

"biocompatible" generally refers having the property or characteristic of not generating injury, toxicity or immunological reaction to living tissues. Accordingly, the material does not substantively provoke injury, toxicity or an immunological reaction, such as a foreign body reaction or inflammatory response (in particular excessive inflammatory response), upon for example implantation of the material in a subject.

In some embodiments, the material is biodegradable. The term "biodegradable" as used herein is defined to include both bioabsorbable and bioresorbable materials. In particular, by "biodegradable", it is meant that the materials decompose, or lose structural integrity under body conditions (e.g., enzymatic degradation or hydrolysis) or are broken down (physically or chemically) under physiologic conditions in the body such that the degradation products are excretable or absorbable by the body. Typically the material may be made from any biocompatible polymer. The biocompatible polymer may be synthetic or natural. The biocompatible polymer may be biodegradable, non-biodegradable or a combination of biodegradable and non-biodegradable. Representative natural biodegradable polymers which may be used include but are not limited to polysaccharides, such as alginate, dextran, chitin, hyaluronic acid, cellulose, collagen, gelatin, fucans, glycosamino-glycans, and chemical derivatives thereof (substitutions and/or additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art); and proteins, such as albumin, casein, zein, silk, and copolymers and blends thereof, alone or in combination with synthetic polymers.

Synthetically modified natural polymers which may be used include but are not limited to cellulose derivatives, such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, and chitosan. Examples of suitable cellulose derivatives include methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, and cellulose sulfate sodium salt. These are collectively referred to herein as "celluloses."

Representative synthetic degradable polymers suitable for use include but are not limited to polyhydroxy acids prepared from lactone monomers, such as glycolide, lactide, caprolactone, ε- caprolactone, valerolactone, and δ-valerolactone, as well as pluronics, carbonates (e.g., trimethylene carbonate, tetramethylene carbonate, and the like); dioxanones (e.g., 1,4- dioxanone and p-dioxanone), 1 ,dioxepanones (e.g., l,4-dioxepan-2-one and 1,5- dioxepan-2- one), and combinations thereof. Polymers formed therefrom include: polylactides; poly(lactic acid); polyglycolides; poly(glycolic acid); poly(trimethylene carbonate); poly(dioxanone); poly(hydroxybutyric acid); poly(hydroxyvaleric acid); poly(lactide-co-(s- caprolactone-)); poly(glycolide-co-(8-caprolactone)); polycarbonates; poly(pseudo amino acids); poly(amino acids); poly(hydroxyalkanoate)s; polyalkylene oxalates; polyoxaesters; polyanhydrides; polyortho esters; and copolymers, block copolymers, homopolymers, blends, and combinations thereof. Some non-limiting examples of suitable non-bioabsorbable materials include but are not limited to polyolefms, such as polyethylene and polypropylene including atactic, isotactic, syndiotactic, and blends thereof; polyethylene glycols; polyethylene oxides; ultra high molecular weight polyethylene; copolymers of polyethylene and polypropylene; polyisobutylene and ethylene-alpha olefin copolymers; fluorinated polyolefms, such as fluoroethylenes, fluoropropylenes, fluoroPEGSs, and polytetrafluoroethylene; polyamides, such as nylon and polycaprolactam; polyamines; polyimines; polyesters, such as polyethylene terephthalate and polybutylene terephthalate; aliphatic polyesters; polyethers; polyether-esters, such as polybutester; polytetramethylene ether glycol; 1 ,4-butanediol; polyurethanes; acrylic polymers and copolymers; modacrylics; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl alcohols; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvmylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyaryletherketones; polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as etheylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; alkyd resins; polycarbonates; polyoxymethylenes; polyphosphazine; polyimides; epoxy resins; aramids, rayon; rayon- triacetate; spandex; silicones; and combinations thereof. In some embodiment, the material is a mesh, in particular a surgical mesh. As used herein, the term "mesh" is intended to include any element having an openwork fabric or structure, and may include but is not limited to, an interconnected network of wire- like segments, a sheet of material having numerous apertures and/or portions of material removed, or the like. As used herein the term "surgical mesh" is used to a mesh suitable for use in surgical procedures, such as, for example, meshes that do not require suturing to the abdominal wall. Surgical meshes, which are used to reinforce weakened areas of abdominal, pelvic, or thoracic tissues, or to replace a portion of internal structural soft tissue that has neither been damaged nor removed surgically, can also be made to have anti-adhesion properties. Surgical mesh drug eluting delivery devices can include one or more therapeutic agents provided with a drug eluting mesh wrap implant placed adjacent to medical devices and internal tissue as described therein. The meshes are available in various single layer, multi-layer, and 3 -dimensional configurations made without bioabsorbable adhesion coatings and films. The meshes are most often constructed of synthetic non-absorbable polymer materials, such as polyethylene, polytetrafluoroethylene, and polypropylene, and can include a carrier having a therapeutic agent attached thereto, incorporated within, or coated thereon. Typically four different material groups have become available for hernia repair and abdominal wall reconstruction: PP, PTFE, ePTFE and Polyester (POL) (Yilmaz Bilsel, IlkerAbci The search for ideal hernia repair; mesh materials and types International Journal of Surgery 10 (2012) 317e321). PP is a hydrophobic polymer of carbon atoms with alternating methyl moieties. This material is flexible, strong, easily cut, readily integrated by surrounding tissues and resists infection. The monofilament nature provides large pores facilitating fibrovascular ingrowth, infection resistance and improved compliance. PP remains the most popular material in mesh hernia repair. PTFE is a chemically inert synthetic fluoropolymer which has a high negative charge, therefore water and oils do not adhere to it. This material does not incorporate into human tissue and becomes encapsulated. Poor tissue incorporation increases hernia recurrence and an infected PTFE mesh must be explanted. PTFE is micro porous, which allows bacteria passage but prevents macrophage passage; therefore the body cannot clear the infection.8 and 9 PTFE was expanded to be improved, and it became a uniform, fibrous and micro porous structure with improved strength called ePTFE. Although it is not incorporated into tissue and has a high incidence of seroma formation, ePTFE remains inert and produces little inflammatory effects, which allows it to be placed directly on viscera. POL is a carbon polymer of terepthalic acid and can be fashioned into strong fibers suitable to be woven into a prosthetic mesh. It is a hydrophilic material and is degraded by hydrolysis. The mesh structure for this surgical application serves as a drug eluting delivery apparatus for local therapeutic delivery within the body. Affixing the carrier and or coating directly onto the surgical mesh makes it easier to handle the device without the drawbacks of film, namely tearing, folding, and rapid dissolving when contacting body fluids, and the lack of fixation or anchoring means. Non-absorbable mesh structures generally provide more handling strength and directional placement control during installation than bio-absorbable or bio-dissolvable polymer films.

In some embodiments, the material is an implant. Regular improvements have been made to facilitate the use of implants. These include: preformed or precut implants adapted to different techniques (4D Dome®; Ultrapro Plug®, Perfix plug®) for the plug techniques; different pre-cut prostheses to allow the passage of the spermatic cord (Lichtenstein technique); meshes that assume the anatomical contours of the inguinal region for the pre -peritoneal technique (ex. Swing Mesh 4A®, 3D Max®). In particular, the implant is designed to facilitate its implantation. Implants furnished with either an auto-adhesive cover (example: Swing Contact®, Adhesix®, Progrip®) or with thermo-inducted staples (example: Endorollfix®); Three-dimensional implants theoretically limiting the possibility of migration (example: UHS®, Ultrapro®, 3D patch®, PHS®); Implants adapted to laparoscopic maneuvering, for example, pre-rolled to facilitate the passage in the trocar (example: Endoroll®), or with pre- inserted cardinal point sutures (example: Parietex®) may be suitable.

In some embodiments, the material is a bioprosthesis. The bioprostheses used in abdominal wall surgery derive from animal (xenogenic prostheses from porcine (dermis or intestinal mucosa) or bovine (pericardium) origin, reticulated or not) or human (allogenic) tissues. They are constituted by type I, III or IV collagen matrixes as well as sterile acellular elastin produced by decellularization, sterilization and viral inactivation, in order to enhance integration and cellular colonization of the prosthesis by the host tissues. Comercial examples include but are not limited to Tutopatch®, SIS®, Tissue Science® process, Surgiguard®, Strattice®, CollaMend®, Permacol® , Surgisis®, XenMatrix®, Veritas® (non-reticulated bovine pericardial bioprosthesis), Protexa (porcine dermis), Alloderm®, Flex HD® Acellular Hydrated Dermis and AlloMaxTM (formerly NeoformTM) (acellular collagen matrix derived from human dermis.

In some embodiments, the material is an orthopaedic implant. Typically, orthopaedic implant include but are not limited to prosthetic knees, hips, shoulders, fingers, elbows, wrists, ankles, fingers and spinal elements.

In some embodiments, the material is a medical device. The medical device can be implanted at a variety of locations in the body including many different subcutaneous and sub- muscular locations.

In some embodiments, the medical devices include those used to sense and/or affect bodily function upon implantation and/or for carrying out various other functions in the body. These can be but are not limited to pacing devices, defibrillators, implantable access systems, monitors, stimulators including neurostimulators, ventricular assist devices, pain pumps, infusion pumps and other implantable objects or systems or components thereof, for example, those used to deliver energy and/or substances to the body and/or to help monitor bodily function. Representative examples include cardiovascular devices (e.g., implantable venous catheters, venous ports, tunneled venous catheters, chronic infusion lines or ports, including hepatic artery infusion catheters, pacemakers and pacemaker leads); neurologic/neurosurgical devices (e.g., ventricular peritoneal shunts, ventricular atrial shunts, nerve stimulator devices, dural patches and implants to prevent epidural fibrosis post-laminectomy, devices for continuous subarachnoid infusions); gastrointestinal devices (e.g., chronic indwelling catheters, feeding tubes, portosystemic shunts, shunts for ascites, peritoneal implants for drug delivery, peritoneal dialysis catheters, and suspensions or solid implants to prevent surgical adhesion); genitourinary devices (e.g., uterine implants, including intrauterine devices (IUDs) and devices to prevent endometrial hyperplasia, fallopian tubal implants, including reversible sterilization devices, fallopian tubal stents, artificial sphincters and periurethral implants for incontinence, ureteric stents, chronic indwelling catheters, bladder augmentations, or wraps or splints for vasovasostomy, central venous catheters; prosthetic heart valves, ophthalmologic implants (e.g., multino implants and other implants for neovascular glaucoma, drug eluting contact lenses for pterygiums, splints for failed dacrocystalrhmostomy, drug eluting contact lenses for corneal neovascularity, implants for diabetic retinopathy, drug eluting contact lenses for high risk corneal transplants); cochlear implants; otolaryngology devices (e.g., ossicular implants, Eustachian tube splints or stents for glue ear or chronic otitis as an alternative to transtempanic drains); dental implants, plastic surgery implants (e.g., breast implants or chin implants), catheter cuffs and orthopedic implants (e.g., cemented orthopedic prostheses) and tracheal tube. Implantable sensors for monitoring conditions such as blood pH, ion concentration, metabolite levels, clinical chemistry analyses, oxygen concentration, carbon dioxide concentration, pressure, and glucose levels are also included. Blood glucose levels, for example, may be monitored using optical sensors and electrochemical sensors.

In a particular embodiment, the medical device is a tracheal tube, more particularly endotracheal tube.

A tracheal tube is a catheter (or a probe) that is inserted into the trachea for the primary purpose of establishing and maintaining a patent airway. Indeed, the two main objectives of the tracheal tube insertion are: (i) to ensure the adequate exchange of oxygen and carbon dioxide, (ii) to protect the airways from inhalation of the oropharynx or gastric contents.

Many different types of tracheal tubes are available, suited for different specific applications:

An endotracheal tube is a specific type of tracheal tube that is nearly always inserted through the mouth (orotracheal) or nose (nasotracheal).

A tracheostomy tube is another type of tracheal tube that is inserted into a tracheostomy stoma (following a tracheotomy) to maintain a patent lumen. A tracheal button is a rigid plastic cannula that can be placed into the tracheostomy after removal of a tracheostomy tube to maintain patency of the lumen.

Endotracheal and tracheostomy tubes have a wide range of internal and external diameters and lengths according to the clinical context: premature baby, newborn, infant, adult. Endotracheal and tracheostomy tubes may have (or not): a cuff at the distal extremity, a subglottic suction line, a preformed shape, a spiral wire embedded in the wall of the tube (to reinforced the tube). Tracheostomy tubes may have extra fenestrations to improve weaning and phonation. Typically, biological surfaces include but are not limited to plant or animal surface. In some embodiments, the surface is a tissue surface. In some embodiments, the cationic polymer of the present invention is applied to at least one tissue surface selected from the group consisting of skin tissue, hair tissue, nail tissue, corneal tissue, tongue tissue, oral cavity tissue, esophageal tissue, anal tissue, urethral tissue, vaginal tissue, urinary epithelial tissue, salivary gland tissue, mammary gland tissue, lacrimal gland tissue, sweat gland tissue, prostate gland tissue, bulbourethral gland tissue, Bartholin's gland tissue, uterine tissue, respiratory and gastrointestinal tract goblet cell tissue, gastric mucosal tissue, gastric gland tissue, pancreatic tissue, spleen tissue, pulmonary tissue, pituitary gland tissue, thyroid gland tissue, parathyroid gland tissue, testicular tissue, ovarian tissue, respiratory gland tissue, gastrointestinal gland tissue, adrenal gland tissue, renal tissue, liver tissue, adipose tissue, duct cell tissue, gall bladder tissue, epidydimal tissue, vas deferens tissue, blood vessel tissue, lymph gland tissue, lymphatic duct tissue, synovial tissue, serosal tissue, squamous tissue, cochlear tissue, choroid plexus tissue, ependymal tissue, dural tissue, pia-arachnoid tissue, sclera tissue, retinal tissue, iris tissue, ciliary tissue, dental tissue, otic tissue, ligament tissue, tendon tissue, elastic cartilage tissue, fibrocartilage tissue, hyaline cartilage tissue, bone marrow tissue, intervertebral disc tissue, compact bone tissue, cancellous bone tissue, skeletal muscle tissue, cardiac muscle tissue, smooth muscle tissue, cardiac valve tissue, pericardial tissue, pleural tissue, peritoneal tissue, blood cell tissue, neuronal tissue, glial tissue, sensory transducer cell tissue, pain sensitive tissue, autonomic neuron tissue, peripheral nervous system tissue, cranial nerve tissue, ocular lens tissue, germ cell tissue, thymus tissue, placental tissue, fetal membrane tissue, umbilical tissue, stem cell tissue, mesodermal tissue, ectodermal tissue, endodermal tissue, autologous tissue, allograft tissue or a combination thereof. In a particular embodiment, the present invention relates to a method of inhibiting or reducing Pseudomonas aeruginosa biofilm formation on a surface comprising the step of applying to the surface ancationic polymer with the following formula 1

wherein

R is NH2

• i is the degree of polymerization comprised between 10 and 75, preferably between 10 and 50, and more preferably between 20 and 50, for example between 20 and 40, or between 30 and 40.

In some embodiments, the method of the present invention further comprises the step of applying at least one antimicrobial agent.

The term "antimicrobial agent" has its general meaning in the art and refers to antibacterial agent, antiprotozoal agent or antifungal agent such as described in US2013/0029981. The antimicrobial agent may be a biocide, an antibiotic agent or another specific therapeutic entity. Suitable antibiotic agents include, without limitation, penicillin, quinoline, vancomycin, sulfonamides, ampicillin, ciprofloxacin, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, and sulfisoxazole. Examples of antimicrobial agents include but are not limited to antibacterial agent, antiprotozoal agent or antifungal agent, a biocide, an antibiotic agent or another specific therapeutic agent. Suitable antibiotic agents include, without limitation, penicillin, quinoline, vancomycin, sulfonamides, ampicillin, ciprofloxacin, teicoplanin, telavancin, bleomycin, ramoplanin, decaplanin, and sulfisoxazole.

Typically, the cationic polymer of the invention is applied to the surface using conventional techniques. Coating, dipping, spraying, spreading and solvent casting are possible approaches. More particularly, said applying is manual applying, applicator applying, instrument applying, manual spray applying, aerosol spray applying, syringe applying, airless tip applying, gas-assist tip applying, percutaneous applying, surface applying, topical applying, internal applying, enteral applying, parenteral applying, protective applying, catheter applying, endoscopic applying, arthroscopic applying, encapsulation scaffold applying, stent applying, wound dressing applying, vascular patch applying, vascular graft applying, image-guided applying, radiologic applying, brush applying, wrap applying, or drip applying.

In some embodiments, the cationic polymer of the present invention is applied in solution to a surface.

In some embodiments, the cationic polymer of the present invention is applied to a surface using aerosol spray applying (or aerosolization).

As used herein, the term "aerosolization" is the process or act of converting some physical substance into the form of particles small and light enough to be carried on the air i.e. into an aerosol.

In some embodiments, the method of the invention is particular suitable for preventing the development of Pseudomonas bacteria growth on the surface and thus for preventing any contamination or infection that can be driven by said bacteria.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Anti-bio film activity of pLK against PAK-LUX strain of P. aeruginosa. For each experiment, PAK-Lux biofilm was treated with PBS, pLK 10 μΜ or pLK 100 μΜ. (A) Visualization of P. aeruginosa (PAK-Lux) bio films formed in vitro in 96-wells plate (by bioluminescence measurement) after treatment. (B) Visualization of PAK-Lux biofilms formed in vitro in sterile EET (by bioluminescence measurement) after treatment.

Figure 2: Action of pLK against PAK-LUX biofilm. Scanning electron micrographs of PAK-LUX biofilms formed in vitro in sterile EET after treatment with either PBS, pLK 10 μΜ or pLK 100 μΜ (Magnification x 5,000 and x 20,000).

Figure 3: Action of pLK against patient EET biofilm. Scanning electron micrographs of patient EET biofilms (collected during hospitalization) after treatment with either PBS, pLK 10 μΜ or pLK 100 μΜ (Magnification x 5,000 and x 20,000). Figure 4: Anti-biofilm activity of pLK against patient EET biofilms. Patient EET bio films (n = 7) were treated with either PBS (white), pLK 10 μΜ (grey) or pLK 100 μΜ (black) during 2 minutes. Percentage of CFU/100 was determined after treatment. A first count corresponding to live bacteria in the biofilm outer layer was determined after vortex/sonication /vortex (step 1) (A) and a second count corresponding to live bacteria in the biofilm inner layer was evaluated on the remaining ETT biofilm (step 2) (B).

EXAMPLE:

Material & Methods

Chemical agent

Poly-L-Lysine (pLK) was purchased from Sigma (St. Quentin Fallavier, France) unless otherwise stated. pLK was diluted in IX phosphate buffered saline (PBS, pH 7.4) (Gibco, Invitrogen, Life Technologies, Saint-Aubin, France) and a fresh stock was made for each experiment. pLK was used at 10 and 100 μΜ.

Endotracheal tube ( ETT) collection

ETT were purchased from Covidien (Mallinckrodt™ TaperGuard Tracheal Tube, Mansfield, USA). We collected ETTs from mechanically-ventilated patients with current or former P. aeruginosa respiratory infection, and extubated due to clinical improvement, change in the ETT for technical reasons, or patient death. This study was approved by the French bioethics authorities (L'Espace de Reflexion Ethique Region Centre) and was conducted in accordance with the ethical standards of the Helsinki Declaration. All patients (or their relatives) included in this study were personally informed by a written document about the collection of used-ETT, as well as their right to object to the study and obtain access to the data, according to articles L.l 121-1 and Rl 121-2 of the French Public Health Code.

Strains of P. aeruginosa

Two reference strains of Pseudomonas aeruginosa were used to establish proof of concept: PAK and PAK-Lux, a luminescent strain, kindly supplied by Reuben Ramphal (USA). Clinical strains of P. aeruginosa were collected from ETT and isolated on Cetrimide Agar plate (Biomerieux, Marcy l'Etoile, France). Prior to use, all bacteria were stored in Luria-Bertani Broth medium (LB) (Amresco, Solon, USA) with 10% glycerol and frozen at -80°C.

Culture of bacteria

For all experiments, strains of P. aeruginosa were grown to exponential phase in LB medium with aeration, at 37°C.

Formation of biofilm in 96-well microplate P. aeruginosa (PAK-Lux) was used to form biofilm in 96-well microplate. Hundred microliters of a 1/100 dilution of an exponential phase culture in LB medium (0.01 optical density) were inoculated into the wells of a 96-well plate. Microplate was incubated for 48 h at 37°C. After incubation, each well was washed with PBS and visualized regarding their luminescence with a Tecan Infinite M200 plate reader (Tecan, Lyon, France). The data presented were derived from a single experiment which was performed in triplicate.

96-well microplate biofilm susceptibility assay

Biofilms were allowed to form as described above with PAK-Lux. After incubation, the 96-well microplates were rinsed with sterile PBS (pH 7.2) and placed in contact with various concentrations of pLK diluted in LB medium (0, 10 and 100 μΜ), during 24 h, at 37°C. Then, each well was washed with sterile PBS and luminescence was measured. The data presented were derived from a single experiment which was performed in triplicate.

Susceptibility assay of P. aeruginosa biofilm in ETT

An original protocol was established to evaluate pLK anti-biofilm activity, with the idea to mimic clinical process of ETT washes. Different strains of P. aeruginosa were used to form biofilm in a sterile ETT. Three milliliters of a 1/100 dilution of an exponential phase culture in LB medium (0.01 optical density) were incubated in a sterile ETT, during 24 h, at 37°C, under shaking (200 rpm). After incubation, ETT were rinsed with PBS (pH 7.2) and was placed in a Falcon tube (15 mL) containing 5 mL solution of various concentrations of pLK diluted in LB medium (0, 10 and 100 μΜ), during 2 minutes, at room temperature. For each condition, segment of ETT was divided into two portions for both electron microscopy and bacteria numeration. Two bacteria enumerations were done to evaluate the pLK anti-biofilm activity. The first enumeration was determined after the following step (step 1): ETT section was placed in a Falcon tube containing 5 mL PBS, vortexed during 30 seconds, sonicated during 5 minutes and vortexed again during 30 seconds. Solution was removed and enumerated. Results obtained after 'step reflect live bacteria in the biofilm outer layer. Then, a second enumeration was determined after the following step (step 2): remaining ETT biofilm was removed with a 10 μί-ΐοορ and diluted in PBS. Obtained solution was enumerated. Results obtained after 'step 2' reflect live bacteria in the biofilm inner layer. For enumeration, 100 μΐ _^ of each dilution were spread on Cetrimide plates which were further incubated for 18 h at 37°C for isolation of colonies. All results were expressed on percentage (mean ± SEM) of CFU/100 μΐ,.

Same protocol was used on collected patient EET biofilms.

Scanning electron microscopy For scanning electron microscopy (SEM), the section of the ETT was fixed with 1 % (v/v) glutaraldehyde and 4 % (v/v) paraformaldehyde in 0.1 M PBS, pH 7.4, post-fixed in 2 % (v/v) osmium tetroxide, dehydrated in a graded acetone series, dried to the critical point using carbon dioxide, and sputter coated with platinum. Sections were examined with a Zeiss Gemini 982 scanning electron microscope.

Effect of sprayed pLK on Pseudomonas aeruginosa biofilm in endotracheal tubes

Same protocol as for 'Susceptibility assay of P. aeruginosa biofilm in ETT' was used. Only change was the administration step. EET were treated with 200 of sprayed LB, pLK 10 μΜ or pLK 100 μΜ during 2 minutes, at room temperature. Spray was done via an Aerosolizer Micro Sprayer® Model IA-1C (Penn Century). Following steps were identical to those described before.

Tolerance ofpLK in pigs

Animal experimentation were performed on four healthy piglets (Large White, 2-3 months of age, weight 30 ± 1 Kg; range 29 to 31 Kg) according to the guidelines of the Council Directive no. 86/609 of the European Economic Community of 24th November 1986, The protocol was approved by the "Comite d'Ethique en Experimentation Animale Val de Loire" (n° 00028.01). Animals were sedated and ventilated as previously described [18]. After tracheal intubation with a 7.0 mm internal diameter ETT, animals' lungs were mechanically ventilated with a Fabius Tiro Ventilator (Drager, Telford, PA, USA). We assessed the tolerance of pLK by mimicking the endotracheal suctioning procedure in the pig model and replacing sterile normal saline required for the suctioning by pLK 10 μΜ solution. The suctioning event was defined by the instillation of 3 mL of solution (sterile normal saline or pLK 10 μΜ solution) in the ETT; concomitantly, we placed a suction catheter through the artificial airway into the artificial airway into the trachea and applied a negative pressure as the catheter was being withdrawn. Instillation / suctioning events were realized every two hours during 6 hours (e.g. four instillation) either with normal saline or with pLK solution. At the end of the experiment, pigs were euthanized with an intravenous injection of sodium pentobarbital at 200 mg/kg (Dolethal, Vetoquinol, S.A., Lure, France). For each pig, bronchoalveolar lavage fluid (BAL) was obtained with instillation of 2 x 50 mL of cold sterile PBS into the lungs. BALs were centrifuged (2,000 rpm, 10 min, 4°C) and the supernatant was stored at -80°C for subsequent analysis. BAL cytokine levels interleukin-6 (IL-6) and IL-8, were assessed using commercially available immunoenzymatic assay (ELISA) kits containing pig-specific monoclonal antibodies, according to the manufacturers' instructions (R&D Systems, Minneapolis, MN, USA). The obtained concentrations were transformed into pg/ml values using a nonlinear regression curve. Histological studies were performed on eight samples were collected per pig: trachea, bronchial ramification, bronchus (right and left), different areas of the right lung (cranial and medial lobes) and the left lung (cranial and medial lobes). The samples were fixed in a 4% formaldehyde solution for subsequent histologic analysis. The tissue samples were embedded in paraffin, and 5 μιη histological sections were stained with hematoxylin and eosin. Pathologist who was blinded to the study groups performed the histological analyses. Examinations included testing for the presence of edema, intra-alveolar and interstitial hemorrhages and polymorphonuclear and mononuclear cell infiltration. Each assessed histological characteristic was attributed a score from 0 to 5 according to the level observed in the tissue.

Statistical analysis

Statistical analysis were performed only on in vitro studies using non-parametric test (Wilcoxon test) (GraphPad version Prism® 5). A p value of < 0.05 was considered as statistically significant. Results

pLK eliminate Pseudomonas aeruginosa (PAK-Lux) biofilm in 96-well microplate.

Biofilms were formed with a luminescent P. aeruginosa strain (PAK-Lux) in 96-well microplates and visualized by imager after treatment with either LB medium, or pLK at 10 or 100 μΜ. In absence of pLK, a dense and homogeneous luminescence was recorded, corresponding to the control condition. After a treatment with pLK 10 μΜ, we observed a decrease of luminescence, indicating a degradation of P. aeruginosa biofilm. Moreover, a total absence of luminescence was recorded corresponding to an elimination of the biofilm when treated with pLK 100 μΜ (Figure 1A).

Production of (PAK-Lux) biofilm in endotracheal tube and its susceptibility to pLK. Biofilm was formed with luminescent P. aeruginosa strain (PAK-Lux) in sterile ETT during 24 hours and controlled by luminescence visualization. To mimic clinical process of ETT instillation, we treated ETT experimental biofilm during 2 minutes, either with LB medium (control), pLK 10 μΜ or pLK 100 μΜ. Then ETT sections were washed and luminescence recorded. In absence of pLK, a dense and homogeneous luminescence was observed. On the opposite, after incubation with pLK 10 or 100 μΜ, the luminescence was strongly reduced, reflecting a destruction of PAK-Lux biofilm. These results were dose- dependent (Figure IB).

The biofilm structure was examined by SEM in the aim to visualize the bacteria morphology in the biofilm. We obtained high-resolution images of P. aeruginosa biofilm (Figure 2; Magnification x 5,000 and x 20,000). In absence of pLK, bacteria surface were smooth and bacteria were interconnected by fiber- like structures. After 2 minute incubation with pLK 10 μΜ or 100 μΜ, bacteria surface changed with apparition of micro-vesicles and the number of fiber- like structure was reduced (Figure 2).

pLK activity against P. aeruginosa biofilm was also evaluated by numeration of live bacteria after treatment. A first bacterial enumeration reflecting live bacteria in the biofilm outer layer, was determined by ETT wash with PBS following by vortex/sonication/vortex step (step 1). A second bacterial enumeration, reflecting live bacteria in the biofilm inner layer, was determined from the remaining ETT biofilm (step 2). Bacterial enumeration obtained without pLK treatment corresponded to 100 % of survival. After treatment, bacteria enumeration showed an almost complete killing with less than 1% of live bacteria with 10 μΜ and 100 μΜ pLK solution (Table 1, line 1). Altogether, these results suggested an antibiofilm effect of pLK characterized by a degradation of biofilm structure and an alteration of bacteria membrane.

Elimination of biofilm produced by a clinical strains of P. aeruginosa on EET

As for PAK-Lux strain, bio films were formed with different P. aeruginosa clinical strains in ETT. Without pLK treatment, images observed with SEM, were identical to those obtained with PAK-Lux biofilm, showing interconnected bacteria by fiber- like structures. After pLK treatment, a reduction of the fiber-like structures was noticed with presence of micro- vesicles at bacteria surface. Less than 0.5 % of live bacteria were enumerated when EET was treated with 10 μΜ and 100 μΜ pLK solution (Table 1, line 2).

pLK condensates the biofilm structure and unmasks bacteria of patient EET biofilm ETT were collected from mechanically ventilated patients, colonized by P. aeruginosa (Table 2). Sections of EET containing biofilm were treated either with LB, pLK 10 μΜ or pLK 100 μΜ. Without pLK treatment, an abundant biofilm was observed with complex matrices in the inner surface of the ETT and no bacteria were observed on this matrix surface. As expected, this biofilm was totally different from the artificial ones (Figures 2 and 3). This difference is most probably due to the presence of patient respiratory secretion which plays an important role in the biofilm formation. Another change was the P. aeruginosa concentration between artificial biofilm and ETT biofilm from patients, with about 1.10 ⁶ CFU/100 μΐ _^ and 5.10 ³ CFU/100 μΐ _^ , respectively. After pLK treatment, a compaction of biofilm matrix structure was observed with apparition of ETT wall and an unmasking of the bacteria (Figure 3). P. aeruginosa enumeration showed an elimination of more than 99 % of bacteria with 10 μΜ and 100 μΜ pLK solution (Table 1, line 3). If we observed pLK effect on individual ETT biofilm, we recorded less than 1 % of alive bacteria after step 1 and step 2, for 5 patient ETT biofilms on 7. These results also demonstrated the ability of pLK to eradicate multi-drug-resistant P. aeruginosa strains (Table 3). For the two others biofilms, about 90 % of P. aeruginosa were eliminated for the ETT bio film of patient 1, and only 50 % (after step 2) for the one of patient 4. However, in these two cases, and especially for patient 4, the biofilm were thicker than the others and it must be remembered that the mean of duration of mechanical ventilation was about 19 days (Table 2) and the results obtained with only one pLK administration. Altogether, these results put in evidence the double action of pLK with (a) its mucolytic property by condensing and degrading the biofilm matrix that unmasked bacteria and (b) its antibacterial effect.

Action of sprayed pLK on P. aeruginosa biofilm

We wanted to explore another way of instillation, while limiting the access to the lungs and avoiding ETT disconnection. Thus, we evaluated the efficiency of sprayed pLK (200 which is the maximum of delivery volume of Aerosolizer Micro Sprayer® Model IA-1C) on artificial biofilms formed in vitro (PAK-Lux and clinical strains) and on patient ETT biofilms. Previous works showed that sprayed pLK possesses the same activities that pLK in solution (Dubois et al, 2013). This method required a more precise movement, but we wanted to evaluate the eventual benefit of a spray, particularly for its action under pressure. Surprisingly, concerning artificial biofilms, we determined an elimination of about 96 % of bacteria when pLK was used at 100 μΜ, but only of 23 % after treatment with pLK 10 μΜ (Table 4, lane 1). However, for ETT biofilm from patients, pLK 10 μΜ and 100 μΜ were able to kill about 96.5 % and 92% of bacteria, respectively. When compared to the results obtained with pLK solution, we could hypothesize that the decrease of anti-bio film pLK activity, mostly for pLK 10 μΜ, could be due to (a) the difference of instilled pLK volume in the ETT (200 μΐ, when aerosolized compared to 3 mL when in solution), and (b) the difference of structure between artificial biofilm and ETT biofilm from patients.

Instillation of ETT by pLK solution induced no pulmonary lesions nor local inflammation in pigs

Altogether, our results showed that instillation of pLK in solution was the more efficient and simple way to eliminate P. aeruginosa biofilm. However, we had to demonstrate that repeated instillations of pLK solution have no side effects on trachea and lungs. To achieve this, four pigs were mechanically ventilated and ETT were instilled every 2 hours during 6 hours, as done in intensive care unit, either with physiological serum or pLK 10 μΜ solution. BAL analysis revealed no pro-inflammatory cytokine production. Indeed, mean concentration of IL- 6 was of 146 ± 26 pg/mL for the control group and of 155 ± 155 pg/mL for the treated group; and mean concentration of IL-8 was of 271 ± 11 pg/mL for the control group and of 85 ± 85 pg/mL for the treated group. For one treated pig, IL-6 and IL-8 concentrations were not detected with ELISA, explaining the calculated SEM. No tracheal or lung lesion and no local inflammation were observed by histological studies, in treated pigs compared to controls, showing that ETT repeated instillations with pLK 10 μΜ solution were well tolerated.

Conclusion

Herein, we demonstrated that pLK efficiently and rapidly disrupt P. aeruginosa biofilm existing on ETT from ICU ventilated-patients, without any issue of tolerance. Anti-biofilm properties of pLK were consistent for using this innovating anti-biofilm agent by a direct administration into the ETT during the endotracheal suctioning, a recommended and basic nurse-care of ventilated patients. Indeed, pLK could be proposed as an add-on therapy to enhance the effectiveness of systemic antibiotic during VAP treatment. Thus, while treating VAP with systemic antibiotic treatment, we propose that instillation with pLK could be integrated to the endotracheal suctioning. In this way, the combination of treatment - conventional antibiotics plus anti-biofilm therapeutic strategy - could efficiently sterilized both lungs and ETT from the responsive pathogen, and therefore may have a role in reducing relapse from persistent bacteria in ETT biofilm.

TABLES:

Table 1. pLK activity against P. aeruginosa biofilms. Biofilms formed in vitro in sterile ETT either by reference strain (PAK-Lux, line 1) or by clinical strains of P. aeruginosa (line 2) and biofilms from patient EET (collected during hospitalization, line 3) were treated with either PBS, pLK 10 μΜ or pLK 100 μΜ during 2 minutes. Percentage (mean ± SEM) of CFU/100 was determined. A first count corresponding to live bacteria in the biofilm outer layer was determined after vortex/sonication/vortex (step 1) and a second count corresponding to live bacteria in the biofilm inner layer was evaluated on the remaining ETT biofilm (step 2).

Table 2. Baseline characteristics of patients whom ETTs were collected

Patient characteristics at time of ETT collection N=7

Age (years), mean ± SEM 62 ± 5

Gender female, N(%) 5/7 (71%)

SAPS II, mean ± SEM 44 ± 7

Causes of intubation

Neurologic disorder 5/7 (71%)

Acute heart failure 2/7 (29%)

Duration of mechanical ventilation (days), mean ± SEM 19 ± 5

Mortality in ICU 2/7 (29%)

SAPS: Simplified Acute Physiologic Score, SEM: Standard Error of the Mean, ICU: Intensive Care Unit Table 3. P. aeruginosa data of patients whom ETTs were collected

Patient number Antibiotic P. aeruginosa

P. aeruginosa serotype treatment Susceptibility or MDR

1 yes susceptible Non-typable

2 no susceptible 06

3 yes MDR 03

4 yes susceptible O10

5 no susceptible 03

6 yes MDR 09

7 no susceptible Oi l

MDR: Multi-Drug-Resistant

Table 4. Sprayed pLK activity against P. aeruginosa biofilms. Biofilms formed in vitro in sterile ETT either by reference strain (PAK-Lux) or by clinical strains of P. aeruginosa and biofilms from patient EET (collected during hospitalization) were treated with either PBS, pLK 10 μΜ or pLK 100 μΜ during 2 minutes. Percentage (mean ± SEM) of CFU/100 μΕ was determined. A first count corresponding to live bacteria in the biofilm outer layer was determined after vortex/sonication/vortex (step 1) and a second count corresponding to live bacteria in the biofilm inner layer was evaluated on the remaining ETT biofilm (step 2).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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