WO2020229521A1 - Methods for inhibiting or reducing bacterial biofilms on a surface - Google Patents

Abstract

The inventors have discovered that healthy human and mouse colon epithelium is a major source of active thrombin, released in the lumen. Using germ-free animals, they demonstrated that mucosal thrombin was directly regulated by the presence of commensal microbiota. Specific inhibition of lumenal thrombin activity caused macro-, microscopic damage and transcriptomic alterations of genes involved in host-microbiota interactions. Further, lumenal thrombin inhibition impaired the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin proteolyzed the biofilm matrix of reconstituted mucosa-associated human microbiota. Inventors demonstrated a previously unknown physiological role for epithelial thrombin that constrains biofilms at mucosal surfaces. Altogether these results provide new insights for treating biofilm on surface biological (but also artificial surface) such as mucosal surface, using thrombin of the invention as main active principle ingredient for inhibiting bacterial biofilm or in combination with antimicrobial agent and also new perspective related to the use of thrombin as a biomarker of dysbiosis associated diseases, especially mucosal dysbiosis associated diseases.

Description

METHODS FOR INHIBITING OR REDUCING BACTERIAL BIOFILMS ON A

SURFACE

FIELD OF THE INVENTION:

The present invention relates to methods for inhibiting or reducing bacterial biofilm formation or overgrowth. More specifically, the present invention relates to a method of inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying or administrating to the surface (biological or artificial) an amount of thrombin.

BACKGROUND OF THE INVENTION:

Epithelia can serve as a local source of proteases at the surface of mucosal organs such as in the skin ^{1 2}, the lung ³ and the digestive tract ⁴’ ⁵’ ⁶. Previous work indicates that bacterial infection ⁷, stress ⁵, low-grade ⁸ or high-grade inflammation ⁹’ ¹⁰ can trigger mucosal and the potentially epithelial release of active proteases by the host. Functional proteomic profiling has recently identified active proteases released by human intestinal mucosa in health and in inflammatory bowel diseases ^u. Among the identified active proteases, thrombin was found to be present both in healthy and inflamed human mucosa, although the cellular source of thrombin was not identified. Thrombin is a serine protease known to be synthesized in the liver. It plays a central role in hemostasis by converting plasma fibrinogen into fibrin and by promoting platelet aggregation via proteinase-activated receptor (PAR) activation. In addition to its coagulation pathway role, thrombin influences a number of pathophysiological processes, including inflammation, tissue repair, angiogenesis, and tumor invasion ⁴’ ¹². While the reported increased presence of thrombin in inflamed mucosa from inflammatory bowel disease patients could easily be explained by the bleeding associated with tissue damage ^u, it was more surprising to detect active thrombin in tissues from healthy individuals. Inventors thus wanted to investigate the cellular source of thrombin in the gut mucosa, hypothesizing that the intestinal epithelium can be a source of thrombin. Here, they identified the presence of thrombin in intestinal epithelial cell lines and in human and mouse healthy intestinal epithelium. Because active forms of thrombin were detected into the gut lumen, inventors postulated that it could play a role in host-microbiota interactions and overall in mucosa homeostasis. Intestinal microbiota naturally grows as a mucus-coated polymicrobial community, embedded as a biofilm organization, separated from the epithelial surface by sterile mucus layer ¹³’ ¹⁴’ ¹⁵’ ¹⁶’ ¹⁷. Nevertheless, biofilm encroachment at the surface of mucosal tissue is one of the most relevant drivers of persistent bacterial infections, thus constituting a major challenge for human and animal health ¹⁸. Not only have such biofilms been associated with diseases, they contribute directly to the foundation for inflammation-associated mucosal injuries ^{19, 20, 21}.

The development of bacterial biofilms is currently recognized as one of the most relevant drivers of persistent infections, and constitutes a major challenge for human and animal health. Biofilms are extremely resistant to medicinal treatment and immune system attacks, which leads to chronic reinfections. One of the most important characteristics of biofilms is their increased tolerance to antimicrobial agents due in part to their protective protein-matrix surrounding the bacteria. It has been proved that biofilms can tolerate up to 100 - 1000 times higher concentrations of antibiotics and disinfectants than planktonic cells22. Therefore, the prevention of biofilm formation and disruption of their matrix of already established biofilms is crucially important for clinical treatment of infectious diseases and biofilm-associated disease. One of the difficulties is to identify compounds that would inhibit biofilm formation, but that would also be safe for biological tissues.

Accordingly, there is a need to develop compounds that will be suitable for inhibiting bacterial biofilms formation and adhesion to tissues, 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 compounds for inhibiting bacterial biofilms adhesion and aggregation and for treatment or prevention of infection from a biofilm may be highly desirable.

Given the importance of spatial segregation between commensals and the epithelia, inventors postulated that the epithelium uses specific mechanisms to prevent the formation of a deleterious microbiota biofilm blanket in contact with host tissues. It is proposed that constitutively active thrombin, originating from the epithelium, maintains mucosal homeostasis via its ability to cleave microbiota biofilm-derived proteins, thereby preventing biofilm contact with tissues, and limiting bacterial invasion.

SUMMARY OF THE INVENTION:

Therefore, the present invention relates to a method of inhibiting or reducing bacterial biofilms formation comprising the step of administrating or applying to a surface an amount of thrombin (active or activated form).

The present invention also relates to a combination of thrombin and antimicrobial agent for use in preventing or treating infection from a bacterial biofilm.

The present invention also relates to a method of increase drug penetration at a mucosal surface comprising the step of administrating or applying to the surface an amount of thrombin.

The present invention also relates to a method for assessing a subject’s risk of having or developing dysbiosis associated diseases, said method comprising the step of measuring the level of thrombin in a biological sample obtained from said subject wherein the level of thrombin is positively correlated with the risk of said subject of having or developing a dysbiosis associated disease.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have now discovered that healthy human and mouse colon epithelium is a major source of active thrombin, released in the lumen. Using germ-free animals, they demonstrated that mucosal thrombin was directly regulated by the presence of commensal microbiota. Specific inhibition of lumenal thrombin activity caused macro-, microscopic damage and transcriptomic alterations of genes involved in host-microbiota interactions. Further, lumenal thrombin inhibition impaired the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin proteolyzed the biofilm matrix of reconstituted mucosa-associated human microbiota. Inventors demonstrated a previously unknown physiological role for epithelial thrombin that constrains biofilms at mucosal surfaces. Inventors report that lung, bladder and skin epithelia also expressed thrombin, suggesting that this role may be applicable to other host-microbiome surfaces. This discovery points route to new therapies targeting biofilms, important for a broad range of disorders, in the gut, and beyond.

Accordingly the inventors have found that thrombin has the following properties: i/ thrombin proteolyzed the matrix of complex polymicrobial biofilms of mucosa- associated human microbiota. Thrombin constrains biofilms at mucosal surfaces, acting as anti biofilm agents (Example 1) ii / thrombin in combination with antibiotics efficiently and synergistically eliminate bacteria from biofilms (Example 1). iii. 1/ that both thrombin activity and protein can be detected in human urine, intestinal biopsies, mucus and feces and 2/ that high thrombin activity and high protein level is correlated with urinary tract infection (i.e. inflamed bladder and dysbiosis), and inflammatory bowel disease (example 2)

Altogether these results provide new insights for treating biofilm on biological surface (and also artificial surface) such as mucosal surface, using thrombin of the invention as main active principle ingredient for inhibiting bacterial biofilm or in combination with antimicrobial agent. Furthermore these results also provide new perspective related to the the use of thrombin as a biomarker of dysbiosis associated diseases, especially mucosal dysbiosis associated diseases.

Methods and uses of the invention

A first aspect of the invention relates to a method of inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin.

In other words, the invention relates to thrombin for use for inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin.

As used herein, "thrombin" denotes the activated enzyme (also known as fibrinogenase, thrombase, thrombofort, topical, thrombin-C, tropostasin, activated blood-coagulation factor II, blood-coagulation factor Ila, factor Ila, E thrombin, beta-thrombin, gamma-thrombin, meizothrombin), which results from the proteolytic cleavage of prothrombin (factor II).

Thrombin is a serine protease (EC 3.4.21.5), an enzyme that, in humans, is encoded by the F2 gene (Gene ID: 2147). Prothrombin (coagulation factor II) is proteolytically cleaved by prothrombinase complex (serine protein, Factor Xa, and the protein cofactor, Factor Va) to form thrombin in the clotting process. The molecular weight of prothrombin is approximately 72 kDa. The catalytic domain is released from prothrombin fragment 1.2 to create several active enzyme thrombin, meizothrombin at 50,000 Da, thrombin alpha at 32,000 Da, thrombin beta 28,000 Da, thrombin gamma 15,000 Da.²³, ²⁴ , ²⁵)

The fully assembled prothrombinase complex (Factor Xa and Factor Va, phospholipid membranes, and Ca2+) catalyses the conversion of the zymogen prothrombin to the serine protease thrombin. Specifically, Factor Xa cleaves prothrombin in two locations, following Arg271 and Arg320 in human prothrombin. Because there are two cleavage events, prothrombin activation can proceed by two pathways. In one pathway, prothrombin is first cleaved at Arg271. This cleavage produces Fragment 1·2, comprising the first 271 residues, and the intermediate prethrombin 2, which is made up of residues 272-579. Fragment 1·2 is released as an activation peptide, and prethrombin 2 is cleaved at Arg320, yielding active thrombin. The two chains formed after the cleavage at Arg320, termed the A and B chains, are linked by a disulfide bond in active thrombin. In the alternate pathway for thrombin activation, prothrombin is first cleaved at Arg320, producing a catalytically active intermediate called meizothrombin. Meizothrombin contains fragment 1·2 A chain linked to the B chain by a disulfide bond. Subsequent cleavage of meizothrombin by Factor Xa at Arg271 gives Fragment 1*2 and active thrombin, consisting of the A and B chains linked by a disulfide bond. When thrombin is generated by Factor Xa alone, the first pathway predominates and prothrombin is first cleaved after Arg271, producing prethrombin 2, which is subsequently cleaved after Arg320. If Factor Xa acts as a component of the prothrombinase complex, however, the second pathway is favored, and prothrombin is first cleaved after Arg320, producing meizothrombin, which is cleaved after Arg271 to produce active thrombin. Thus, the formation of the prothrombinase complex alters the sequence of prothrombin bond cleavage.

Accordingly in a particular embodiment, the thrombin which can be used in the present invention are active or activated form of thrombin (Prothrombinase-mediated and autolytic- mediated degradation of prothrombin results in the formation of different forms of active thrombin):

Prothrombin (1-579 AA residues for Human form)

Prethrombin 2 (intermediate pathway 1 : residues 272-579 of Prothrombin)

Meizothrombin (intermediate pathway 1 : fragment 1*2 A chain (residues 1- 320 of Prothrombin) linked to the B chain (residues 321- 579 of Prothrombin) by a disulfide bond)

Active thrombin (or alpha thrombin) (formed by A chain (Light Chain: residues 272- 320 of Prothrombin) and B chain (Heavy chain or catalytic domain 321-579) linked by a disulfide bond of Prothrombin).

Beta- and gamma thrombin which are obtained after proteolysis of alpha thrombin and which are described in reference ^{23, 24 and 25} as active thrombin with an enzymatic activity (ie able to cleave Par4).

From reference²⁵ inventors evaluate the approximate size for different thrombin forms are: Prothrombin (~70,000Da), meizothrombin (several fragment at <50,000 Da), alpha- (~32,000Da), beta- (~28,000Da) and gamma (<15,000Da) thrombin.

Thrombin can be prepared by a variety of methods known in the art, and the term "thrombin" is not intended to imply a particular method of production. Both human and non human (e.g., bovine) thrombins can be used within the present invention.

Human and non-human thrombins are prepared according to methods known in the art. Purification of thrombin from plasma is disclosed by, for example, Bui-Khac et ah, U.S. Patent No.5,981 ,254. Purification of thrombin from plasma fractions, such as Cohn fraction III, is disclosed by Fenton et al.²⁶.

Alternatively, thrombin can be a recombinant thrombin prepared from a prethrombin precursor by activation with a snake venom activator as disclosed in US Patent.5,476,777. Other activators, such as factor Xa, can also be employed. Another method for producing recombinant human thrombin from recombinant prothrombin using recombinant ecarin is disclosed in US patent 8206967 and in Yonemura H. et al. ²¹.

Chemical synthesis of thrombin in particular in the case of the peptide derivatives (Solid Phase Peptide Synthesis techniques) can also be used.

A nucleic acid encoding thrombin can also be obtained from a genomic or cDNA library of a vertebrate, using suitable primers able to hybridize selectively with said nucleic acids. It can also be obtained by the classical techniques of polynucleotide synthesis.

The term“thrombin” should be understood broadly, it encompasses the mature forms of thrombin, variants and fragments thereof having same biological activity: reducing bacterial biofilm formation (through enzymatic activity of thrombin which cleaves the protein backbone of the biofilm matrix)

Typically a variant of thrombin has at least 80%, preferably, at least 85% more preferably at least 90%, more preferably at least 95% and even more preferably at least 99% identity with human mature form of thrombin. Typically, identity may be determined by BLAST or FASTA algorithms.

Precursors of said mature forms of thrombin, i.e. prothrombin and preprothrombin and nucleic acids encoding said precursors can also be used.

Other examples of polypeptides or nucleic acids suitable for use according to the invention are vertebrate, preferably mammalian, homologous of mature forms of human thrombin or precursors thereof, or nucleic acids encoding said polypeptides. Known vertebrate homologous of human thrombin include for instance bovine thrombin (already used in human therapy).

Preferably, if the patient is human, the thrombin is a mature form of human thrombin (UniProtKB - Q69EZ8). The protein sequence of said human thrombin, may be found in NCBI database with the following access numbers: mRNA NM_00506, and Protein : NP_000497 (prothrombin isoform 1 preproprotein : this variant (1) represents the longer transcript and encodes the longer isoform (1)) and mRNA NM_001311257, and protein_id : NP_001298186 (prothrombin isoform 2 preproprotein : this variant (2) uses an alternate in-frame splice site in the 5' coding region, compared to variant 1 and it encodes isoform 2, which is shorter than isoform 1).

The invention also encompasses the use of functional equivalents of the above-defined polypeptides. Functional equivalents are herein defined as peptide variants, having the same functional biological activity as the mature forms of thrombin (reducing bacterial biofilm formation through enzymatic activity of thrombin).

The effect of antibiofilm activity can be measured by monitoring condensation, dispersion and reduction of the bacterial biofilm present in a surface prior to and after application with the thrombin according to the invention, using in vitro assays (Susceptibility assay) adapted from different assays²⁸. Biological activity of mature form of thrombin (reducing bacterial biofilm formation through enzymatic activity of thrombin) can also be measured for example as described in example 1 (Gut microbiota biofilms/ Figure 5); the determination of biofilm biomass density variation by crystal violet (Reactifs RAL) assay as described in ¹³ and ¹⁹ and Biofilm rate of dispersal modulation can be assessed by measuring the optical density (600 nm) and assessing colony-forming unit (CFU) of biofilm-dispersed planktonic bacteria. Biofilm matrix-associated proteins (SYPRO biofilm matrix) and polysaccharides (wheat germ agglutinin) can also be stained after thrombin exposure. After extraction by disruption of electrostatic interactions using 1.5M NaCl buffer (pH 7.4), their total amount can be measured to evaluate overall disruptions of matrix-associated components by thrombin (see Figure 5, a, b, c).

By "amount" or "sufficient amount"“or dosage level” is intended to be an amount of thrombin of the invention, that, when applied brings about a positive response with respect to constrain /reduce the bacterial biofilm present in a surface and/or to act as anti- bacterial biofilm agents. Actual dosage levels of the thrombin of the present invention may be varied so as to obtain an amount of the thrombin which is effective to achieve the desired anti-biofilm response for a particular surface (biological or synthetic), mode of application (i.e. solution or by aerosolization) or administration.

Typically for a mature human biofilm (from human colon biopsies see example 1), the dosage when active thrombin of the invention applied in solution is between 1 to 1000 mU/mL, preferably between 10 to 500 m/UmL more preferably between 50 to 200 mU/mL, even more preferably 100 mU/mL. 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. Biofilm has also been described as well-organized microbial community enmeshed in a polymeric, carbohydrate-rich extracellular matrix (ECM) and adhering to an artificial or biological surface¹⁸.

For instance, intestinal microbiota naturally grows as a mucus-coated polymicrobial community, embedded as a biofilm organization, separated from the epithelial surface by sterile mucus layer. In the present invention, inventors demonstrated that thrombin is also expressed in epithelia from intestine, lung, bladder and skin, which also contains poly-microbials bacterial biofilms growing at these mucosal surfaces.

The term‘mucosal biofilnf denotes biofilms that grow on mucosal surfaces. Proposed clinical criteria for mucosal biofilm infections include: signs and symptoms of infection in otherwise culture-negative patients, chronicity or recurrence with periodic exacerbations and remissions, and minimal or no response to antimicrobials agents²⁹.

Many different bacteria may form biofilms, including gram-positive (e.g. Bacillus spp, Listeria monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum and Lactococcus lactis) and gram-negative species (e.g. Escherichia coli, or Pseudomonas aeruginosa) (see ³⁰).

Not only do bacteria form biofilm at the mucosal surface, they do so in a complex polymicrobial community. Such community from human colon mucosa can be cultured ex vivo using models like the Calgary Biofilm Device model using methods described in¹³’ ¹⁹.

Biofilms have been found to be involved in a wide variety of microbial infections in the body. Infectious processes in which biofilms have been implicated include several pathologies such as bacterial vaginosis, urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses, heart valves, and intervertebral disc (see³¹). More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds.

According a particular embodiment of the invention, the biofilm is produced by Cutibacterium bacteria. The term“ Cutibacterium bacteria” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in gut or skin of humans and other animals. The term“ Cutibacterium bacteria” refers to but it is not limited to gram-positive bacteria such as specoies C. acnes, C. avidum and C. granulosum.

In particular, the Cutibacterium biofilm according to the invention is Cutibacterium acnes biofilm. As used herein, the term“ Cutibacterium acnes” has its general meaning in the art and refers to gram-positive bacterium linked to the skin condition of acne. The species is largely commensal and part of the skin flora present on most healthy adult humans' skin. It may also be found throughout the gastrointestinal tract. It can also cause chronic blepharitis and endophthalmitis.

According a particular embodiment 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, gut or skin 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.

Pseudomonas aeruginosa represents a commonly used biofilm model organism since it is involved in different types of biofilm-associated infections ³². Examples of such infections include chronic wounds, chronic otitis media, chronic prostatitis and chronic lung infections in cystic fibrosis (CF) patients. About 80% of CF patients have chronic lung infection, caused mainly by P. aeruginosa growing in a non-surface attached biofilms surround by Polymorphonuclear neutrophil. According to another particular embodiment of the invention, the biofilm is produced by Staphylococcus bacteria. The term“Staphylococcus bacteria” has its general meaning in the art and refers to bacteria that occur normally or pathogenically in gut or skin of humans and other animals. The term“Staphylococcus bacteria” refers to but it is not limited to gram-positive bacteria Staphylococcus e.g; a bacterium of the S. aureus group, such as

S. aureus group - S. argenteus, S. aureus, S. schweitzeri, S. simiae

S. auricularis group - S. auricularis

S. carnosus group - S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans, S. simulans

S. epidermidis group - S. capitis, S. caprae, S. epidermidis, S. saccharolyticus

S. haemolyticus group - S. devriesei, S. haemolyticus, S. hominis

S. hyicus-intermedius group - S. agnetis, S. chromogenes, S. cornubiensis, S. felis, S. delphini, S. hyicus, S. intermedius, S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, S. schleiferi

S. lugdunensis group - S. lugdunensis

S. saprophyticus group - S. arlettae, S. caeli, S. cohnii, S. equorum, S. gallinarum, S. kloosii, S. leei, S. nepalensis, S. saprophyticus, S. succinus, S. xylosus

S. sciuri group - S. fleurettii, S. lentus, S. sciuri, S. stepanovicii, S. vitulinus

S. simulans group - S. simulans

S. warned group - S. pasteuri, S. warned,.

In padicular, the S. aureus biofilm according to the invention is Staphylococcus aureus biofilm.

Staphylococcus aureus is a common Gram-positive bacteria that can cause disease in animals, including humans. Staphylococcus aureus is, round-shaped bacterium that is a member of the Firmicutes, and it is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive for catalase and nitrate reduction and is a facultative anaerobe that can grow without the need for oxygen. Although S. aureus usually acts as a commensal of the human microbiota it can also become an oppodunistic pathogen, being a common cause of skin infections including abscesses, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. The emergence of antibiotic-resistant strains of S. aureus such as methicillin-resistant S. aureus (MRSA) is a worldwide problem in clinical medicine. Despite much research and development, no vaccine for S. aureus has been approved. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life- threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis. It is still one of the five most common causes of hospital- acquired infections and is often the cause of wound infections following surgery.

S. aureus is a significant cause of chronic biofilm infections on medical implants. S. aureus is often found in biofilms formed on medical devices implanted in the body or on human tissue. It is commonly found with another pathogen, Candida albicans, forming multispecies biofilms. The latter is suspected to help S. aureus penetrate human tissue. A higher mortality is linked with multispecies biofilms (Zago CE, et al. (2015). " PLOS One. 10 (4): e012320).

S. aureus biofilm has high resistance to antibiotic treatments and host immune response. S. aureus biofilms also have high resistance to host immune response. Though the exact mechanism of resistance is unknown, S. aureus biofilms have increased growth under the presence of cytokines produced by the host immune response (McLaughlin RA et al. (2006). Microbial Pathogenesis. 41 (2-3): 67-79). As used herein the term“surface” refers to any surface where bacteria (e.g. Pseudomonas bacteria or Staphiloccocus bacteria) are liable to grow on.

In some embodiments, the surface is an artificial surface or is a biological surface (such as mucosal surface).

Biological surfaces

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 thrombin 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 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 the present invention, inventors demonstrated that thrombin is expressed in epithelia from gut, lung, bladder and skin, which also contains poly-microbial bacterial biofilms growing at these mucosal surfaces. Several epithelia of biological tissues, such as vaginal tissue, gingival tissue and ocular tissue form a mucosal surface and a previous study discloses that thrombin is present at the eye epithelium ³³.

Accordingly, in a particular embodiment the surface is a mucosal surface of gut tissue lung tissue, bladder tissue, vaginal tissue, gingival tissue, ocular tissue and skin tissue.

In order to be directly administered to the skin or vaginal tissue, gingival tissue, ocular tissue, the thrombin according to the invention can be administered topically to the skin or to vaginal, gingival, ocular tissue.

In order to be directly administered to the lung, the thrombin according to the invention would be administered by any method allowing the specific targeting of the lungs such as inhaling or spray drying (intranasal administration or by aerosolisation)

In order to be administered specifically to the intestines (gut), the thrombin can be administered topically or rectally.

When administered orally, the compound according to the invention can be in the form of a sustained release composition so as to deliver the compound to the intestines.

In order to be administered specifically to the bladder, the thrombin can be administered systemically or intravesically.

In a preferred embodiment, the biological tissue is gut tissue, colon tissue and lung tissue.

Artificial surfaces

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 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 thrombin 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."

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)³⁴ . 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. Commercial 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 dacrocystalrhinostomy, 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.

Thus, the invention relates to a method of inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin, wherein the surface is an artificial surface or is a biological surface.

In other words, the invention relates to thrombin for use for inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin, wherein the surface is an artificial surface or is a biological surface.

In some embodiment, the biological surface is a mucosal surface of gut tissue, lung tissue, bladder tissue, vaginal tissue, gingival tissue, ocular tissue and skin tissue.

In some embodiment, the artificial surface is medical device. Thus, the invention relates to thrombin for use for inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin, wherein the surface is a mucosal surface of gut tissue, lung tissue, bladder tissue, vaginal tissue, gingival tissue, ocular tissue and skin tissue.

In some embodiment, the invention relates to thrombin for use for inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin, wherein the surface is a medical device.

In a particular embodiment, the present invention relates to a method of inhibiting or reducing Cutibacterium acnes biofilm formation on a surface comprising the step of applying to the artificial surface an amount of thrombin (active or activated form).

In other words, the present invention relates to thrombin (active or activated form) for use for inhibiting or reducing Cutibacterium acnes biofilm formation on a surface comprising the step of applying to the artificial surface an amount of said thrombin.

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 artificial surface an amount of thrombin (active or activated form).

In other words, the present invention relates to thrombin (active or activated form) for use for inhibiting or reducing Pseudomonas aeruginosa biofilm formation on a surface comprising the step of applying to the artificial surface an amount of said thrombin.

In a particular embodiment, the present invention relates to a method of inhibiting or reducing Staphylococcus aureus biofilm formation on a surface comprising the step of applying to the artificial surface an amount of thrombin (active or activated form).

In other words, the present invention relates to thrombin (active or activated form) for use for inhibiting or reducing Staphylococcus aureus biofilm formation on a surface comprising the step of applying to the artificial surface an amount of said thrombin

Combination of thrombin and antimicrobial agent

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

Accordingly, the present invention also relates to a method of inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin and an amount of at least one antimicrobial agent. The present invention also relates to a combination of thrombin and antimicrobial agent for simultaneous or sequential use in preventing or treating infection from a bacterial biofilm.

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.. 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, chloramphenicol and sulfisoxazole.

In some embodiments, the antibiotic agent of the present invention is ciprofloxacin and/or chloramphenicol.

Typically, the thrombin 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 thrombin 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. In preferred embodiment the Pseudomonas bacteria is a Pseudomonas aeruginosa bacteria.

In another embodiments, the method of the invention is particular suitable for preventing the development of Cutibacterium bacteria growth on the surface and thus for preventing any contamination or infection that can be driven by said bacteria. In preferred embodiment the Cutibacterium bacteria is a Cutibacterium acnes bacteria. In another embodiments, the method of the invention is particular suitable for preventing the development of Staphylococcus bacteria growth on the surface and thus for preventing any contamination or infection that can be driven by said bacteria. In preferred embodiment the Staphylococcus bacteria is Staphylococcus aureus bacteria

Another object of the invention is a method for treating infection from a bacterial biofilm comprising administering to a subject in need thereof a therapeutically effective amount of a thrombin as disclosed above and at least one antimicrobial agent.

In other words, the inventions relates to a thrombin as disclosed above and at least one antimicrobial agent for use for treating infection from a bacterial biofilm in a subject in need thereof.

By a "therapeutically effective amount" is meant a sufficient amount of compound to treat and/or to prevent the infection from a bacterial biofilm.

It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The thrombin according to the invention can be administered by any suitable route of administration. For example, thrombin according to the invention can be administered by oral (including buccal and sublingual), rectal, nasal, topical (intravesical), pulmonary, vaginal, or parenteral (including intramuscular, intra-arterial, intrathecal, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

The thrombin of the present invention and at least one antimicrobial agent, together with one or more conventional adjuvants, carriers, or diluents may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredients commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral uses. Formulations containing about one (1) milligram of active ingredient or, more broadly, about 0.01 to about one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms.

The thrombin of the present invention and at least one antimicrobial agent may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise compounds of the present invention or pharmaceutically acceptable salts thereof as the active component. The pharmaceutically acceptable carriers may be either solid or liquid. Solid form preparations include powders, tablets, pulls, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from about one (1) to about seventy (70) percent of the active compound. Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch gelatin, tragacanth, methylcellulose sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.

The term“preparation” is intended to include the formulation of the active compound with an encapsulating material as carrier, providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pulls, cachets, and lozenges may be as solid forms suitable for oral administration.

Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents, for example, such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizers, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

The thrombin of the present invention may be formulated for administration as suppositories. Typically, a low melting wax, such as a mixture of fatty-acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously.

The thrombin of the present invention and at least one antimicrobial agent may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non- aqueous carriers, diluents solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil, and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

The thrombin of the present invention and at least one antimicrobial agent may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Formulations suitable for topical administration to the eye include eye drops wherein the active ingredient is suspended or dissolved in a suitable carrier, preferably an aqueous solvent.

Regarding delivery of thrombin within a biocompatible surgical sealant, Thrombin is already delivered as such in gelatin matrix/thrombin sealants^{35, 36, 37}.

The thrombin according to the present invention may be formulated for intrapulmonary administration. Suitable formulations for intrapulmonary applications have a particle size typically in the range of 0.1 to 500 microns and are administered by inhalation through the nasal tract or through the mouth in the form of a dry powder or via an aerosol.

Method of increase drug penetration

Delivery of drugs topically in the skin, and/or at mucosal surfaces, including the airways, the gastro-intestinal tract, the oral cavity, the genito-urinary tract, represents a desirable alternative to invasive delivery by injection, and could avoid hepatic first-pass metabolism. However, tissue surface and mucosa poor permeability constitute important challenges. For many drugs and drug delivery systems, the penetration into and permeation across biological surfaces such as the skin and the mucosa, is a bottleneck to be overcome for sufficient drug bioavailability. For successful topical or mucosal drug administration, several barriers have to be crossed, and among them, tissue biofilm is the very first one. It is both a physical and metabolic barrier, which contribution to drug passage and metabolism has been under-estimated (Nok et al Arch Pharm Res. 2017). The resident microbiota uses various mechanisms to alter the disposition, efficacy and toxicity of drugs and xenobiotics: such reactions involve reduction, hydrolysis, dihydroxylation, acetylation, deacetylation, proteolysis, deconjugation, and deglycosylation processes (³⁸, ³⁹and ⁴⁰). For instance, commensal gut microbiota biofilm may express enzymes that either metabolically activate or inactivate drugs (ex in ⁴¹). This concept is not uniquely applicable for gut microbiome, as it is a concern as well for other mucosa : ⁴². The present inventors demonstrate that the epithelium uses specific mechanisms to prevent the overgrowth of microbiota biofilm blanket in contact with host tissues. In their results, the inventors have demonstrated that thrombin, originating from the epithelium, maintains mucosal homeostasis via its ability to cleave microbiota biofilm- derived proteins, thereby preventing biofilm contact with tissues, and limiting bacterial invasion. Therefore, it is logical to propose that the use of thrombin could be offered to facilitate a more efficient and rapid passage of topically-delivered drugs across mucosal or skin barriers. In view of the above, a local administration of thrombin would represent a very promising way to increase drug penetration, particularly at mucosal surface wherein bacterial biofilm.

Regarding the fact that:

the thrombin possesses the ability to cleave microbiota biofilm-derived proteins, and

thrombin is expressed in epithelia from gut, lung, bladder and skin, which also contains poly-microbial bacterial biofilms growing at these mucosal surfaces;

it is herein proposed that administration of thrombin would be beneficial to increase drug penetration at a mucosal surface.

The present invention also relates to a method of increase drug penetration at a mucosal surface comprising the step of administrating to the surface an amount of thrombin.

In other words, the invention relates to thrombin for use for increasing drug penetration at a mucosal surface comprising the step of administrating to the surface an amount of thrombin.

By "amount" or "sufficient amount"“or dosage level” is intended to be an amount of thrombin of the invention, that, when applied brings about a positive response with respect to constrain /reduce the bacterial biofilm present in a surface and to increase drug penetration. Actual dosage levels of the thrombin of the present invention may be varied so as to obtain an amount of the thrombin which is effective to achieve the desired anti-biofilm response for a particular mucosal surface, mode of administration (i.e. topic, enema, suppository, intravesical or by aerosolization, within a surgical glue or biocompatible hydrogel sealant) .

As used herein, the term “increase” means a statistically significant increase compared to a control value (without thrombin), preferably, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 90%, or at least 100% increase of the control value.

The quantification may be relative (by comparing the amount of the drug penetration to a control with known amount of drug penetration without thrombin for example and detecting “higher” or“lower” amount compared to that control) or more precise (i.e. : quantitative), at least to determine the specific amount relative to a known control amount (i.e.: to determine the difference between the concentration value and the control value).

These quantification assays of a drug penetration can be conducted in a variety of ways. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art.

The level of the drug penetration may be determined by using cell culture in vitro models used for studying mucosal drug delivery such as Caco-2 cell model (Monolayer, standard model for oral epithelial absorption) HT29 or Calu-3 cell models (Monolayer, mucus producing) TR146 cell model (Multilayered squamous epithelium) ⁴³ or organoid models such as colon organoids as described in ⁴¹.

For example, determination of the drug penetration level at the mucosal surface can be performed by a variety of techniques and method any well-known method in the art: Diffusion of drug in the mucus is most often assessed by studies applying fluorescent nanoparticles, which are detected by using multiple particle tracking⁴⁴ and/or fluorescence recovery after photobleaching⁴⁵. Other promising techniques include capillary penetration using magnetic beads and a magnetic field⁴⁶ and nuclear magnetic resonance (NMR) with either pulsed-field or pulsed-gradient spin-echo⁴⁷. Quantitative analysis of drug distribution in complex bacterial biofilms could also be measured using the laser interferometry method as described in ⁴⁸. This method could calculate the amount (mol) of drug accumulated in the biofilm formed on nucleopore membrane. This technique could also be adapted to biofilms grown at mucosal surfaces.

Diagnostic methods according to the invention:

In an additional study (Example 2) inventors demonstrate 1) that the thrombin expression and the level of activity can be detected in biological samples (such as urine or feces (see also example 1) or biopsy tissue sample) 2) that high thrombin activity and high protein level in those samples are correlated with urinary tract infection (i.e. inflamed bladder and dysbiosis see figure 11) or with condition associated to dysbiotic mucosa: Crohn’s disease (CD) or Ulcerative Colitis (UC) (see figure 12).

Accordingly, another aspect of the invention consists of a method for assessing a subject’s risk of having or developing dysbiosis associated diseases (DAD), said method comprising the step of measuring the level of thrombin in a sample obtained from said subject wherein the level of thrombin is positively correlated with the risk of said subject of having or developing a dysbiosis associated disease.

A high level of thrombin is predictive of a high risk of having or developing a dysbiosis- associated disease.

A low level of thrombin is predictive of a low risk of having or developing a dysbiosis associated disease.

As used herein, the term "biological sample" as used herein refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy. In a particular embodiment regarding the method for assessing a subject’s risk according to the invention, the biological sample is a body fluid (such urina or feces) or tissue biopsy of said subject.

More particularly the body fluid sample, is urina or feces sample. Indeed, the inventors have surprisingly demonstrated that thrombin, known until now to be a protein expressed by the liver, is also expressed by human epithelial cells, and is a circulating protein present in urine and feces, including in health conditions.

As defined herein the term“thrombin” refers to active form thrombin disclosed above. This active form of thrombin are selected from the list consisting ofMeizothrombin or thrombin (alpha, beta or gamma thrombin).

In one embodiment of the method defined above, one or more biological markers are quantified together with thrombin.

As used herein, a "biological marker" encompasses any detectable product that is synthesized upon the expression of a specific gene, and thus includes gene-specific mRNA, cDNA and protein.

The various biological markers names specified herein correspond to their internationally recognized acronyms that are usable to get access to their complete amino acid and nucleic acid sequences, including their complementary DNA (cDNA) and genomic DNA sequences. Illustratively, the corresponding amino acid and nucleic acid sequences of each of the biological markers specified herein may be retrieved, on the basis of their acronym names, that are also termed herein "gene symbols", in the GenBank or EMBL sequence databases. All gene symbols listed in the present specification correspond to the GenBank nomenclature. Their DNA (cDNA and gDNA) sequences, as well as their amino acid sequences are thus fully available to the one skilled in the art from the GenBank database, notably at the following Website address : "http://www.ncbi.nlm.nih.gov/".

Of course variant sequences of the biological markers may be employed in the context of the present invention, those including but not limited to functional homologues, paralogues or orthologues of such sequences.

As used herein, the term“subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably, a subject according to the invention is a human.

The term "dysbiosis associated diseases " refers to or describes diseases reflecting mishandling of the microbiota by the host immune system, to altered metabolism of commensal microbiota (e.g. reduced production of short chain fatty acids, increased incorporation of iron) and to clinical situations in which restoration of a normal microbiota is likely to be better achieved by strengthening natural homeostatic mechanisms than antibiotics.

Disruptions in the microbiome can allow outside factors or even pathogenic members of the commensal microbiome to take hold in the gut environment. Dysbiosis has been reported to be associated with various diseases, such as periodontal disease, (Nath SG, et al (2013). Journal of Indian Society of Periodontology. 17 (4): 543-5) inflammatory bowel disease (Lepage P, et al (2013). Gut. 62 (1): 146-58), chronic fatigue syndrome (Lakhan SE, et al (October 2010). Nutrition & Metabolism. 7: 79.) obesity (Tumbaugh PJ, et al (2006). Nature. 444 (7122): 1027-31) colorectal cancer (Castellarin M, et al (2012). Genome Research. 22 (2): 299-306) bacterial vaginosis (Africa CW et al (2014). International Journal of Environmental Research and Public Health. 11 (7): 6979-7000) and colitis (Mazmanian SK (2008). Journal of Pediatric Gastroenterology and Nutrition. 46 Suppl 1 : El l-2.).

In preferred embodiment“dysbiosis associated diseases” are associated with mucosal dysbiosis (« mucosal dysbiosis associated diseases”) that occurs in intestinal, colon, lung skin or bladder disease. Such“mucosal dysbiosis associated diseases” notably include but are not limited to :

- intestinal diseases selected from the list consisting of inflammatory bowel disease (IBD such as Crohn's disease), irritable bowel syndrome (IBS), and coeliac disease (see Carding S et al Microb Ecol Health Dis. 2015; 26: 10.3402),

- colon disease such as colitis (especially Ulcerative Colitis), colorectal cancer or celiac disease.

- bladder disease such as urinary tract infection or pyelonephritis.

- skin disease such acne, chronic wound ulcers

- lung disease such as cystic fibrosis or chronic obstructive pulmonary disease.

The level of the thrombin may be determined by using standard enzymatic, electrophoretic and immunodiagnostic techniques, including immunoassays such as chromogenic substrate cleavage, competition, direct reaction such as immunohistochemistry, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. For example, determination of the thrombin level can be performed by a variety of techniques and method any well known method in the art: RIA kits (DiaSorin; IDS, Diasource) Elisa kits (Thermo Fisher, EHTGFBI, R&D DY2935, IDS (manual) IDS (adapted on open analyzers) Immunochemiluminescent automated methods (DiaSorin Liaison, Roche Elecsys family, IDS iSYS) (Janssen et ak, 2012).

In a particular embodiment, the methods of the invention comprise contacting the biological sample with a binding partner.

As used therein, binding partner refers to a molecule capable of selectively interacting with thrombin.

The binding partner may be generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. Polyclonal antibodies directed against thrombin can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against thrombin can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler et al. Nature. 1975; 256(5517):495-7 ; the human B-cell hybridoma technique (Cote et al Proc Natl Acad Sci U S A. 1983;80(7):2026- 30); and the EBV-hybridoma technique (Cole et al, 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti- thrombin, single chain antibodies. Antibodies useful in practicing the present invention also include anti- thrombin including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to thrombin. For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

In another embodiment, the binding partner may be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk et al. (1990) Science, 249, 505-510. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al. (1996) Nature, 380, 548-50).

The binding partners of the invention such as antibodies or aptamers, may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labeled", with regard to the binding partner, is intended to encompass direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labeled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Ini 11, Rel86, Rel88.

The aforementioned assays generally involve the bounding of the binding partner (ie. antibody or aptamer) in a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against thrombin. A body fluid sample containing or suspected of containing thrombin is then added to the coated wells. After a period of incubation sufficient to allow the formation of binding partner- thrombin complexes, the plate(s) can be washed to remove unbound material and a labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

As the binding partner, the secondary binding molecule may be labeled.

Different immunoassays, such as radioimmunoassay or ELISA, have been described in the art.

Measuring the level of thrombin with or without immunoassay-based methods may also include separation of the proteins: centrifugation based on the protein's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the protein's affinity for the particular solid-phase that is use. Once separated, thrombin may be identified based on the known "separation profile" e. g., retention time, for that protein and measured using standard techniques. Alternatively, the separated proteins may be detected and measured by, for example, a mass spectrometer.

In a preferred embodiment, the method for measuring the level of thrombin comprises the step of contacting the biological sample with a binding partner capable of selectively interacting with thrombin to allow formation of a binding partner- thrombin complex.

In more preferred embodiment, the method according to the invention comprises further the steps of separating any unbound material of the blood sample from the binding partner- thrombin complex, contacting the binding partner- thrombin complex with a labelled secondary binding molecule, separating any unbound secondary binding molecule from secondary binding molecule- thrombin complexes and measuring the level of the secondary binding molecule of the secondary binding molecule- thrombin complexes.

Measuring the expression (and notably the expression level) of the transcript of thrombin can be performed by a variety of techniques well known in the art.

Typically, the expression of a transcript (and notably the expression level) may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT- PCR).

Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A“detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labelled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labelled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and / or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook— A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene-1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDaminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al, Science 281 :20132016, 1998; Chan et ah, Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors hased on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.). Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hyhridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670, 113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labelled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques. For example, a biotinylated probe can be detected using fluorescein-labelled avidin or avi din-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et ak, Proc. Natl. Acad. Sci. 83 :2934-2938, 1986; Pinkel et ak, Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et ak, Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et ak, Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labelled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labelled with a nonfluorescent molecule, such as a hapten (such as the following non limiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, coumarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labelled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labelled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labelled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labelled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labelled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are“specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et ah, Hepatology 2014, 60: 1674-1685 or Ueno A et al, Journal of hepatology 2014, 61 : 1080-1087).

Typically, a high or a low level of thrombin is intended by comparison to a control reference value.

Said reference control values may be determined in regard to the level of thrombin present in biological samples taken from one or more healthy subject or to the thrombin distribution in a control population.

In one embodiment, the method according to the present invention comprises the step of comparing said level of thrombin to a control reference value wherein a high level of thrombin compared to said control reference value is predictive of a high risk of having a dysbiosis associated diseases and a low level of thrombin compared to said control reference value is predictive of a low risk of having a dysbiosis associated diseases. The control reference value may depend on various parameters such as the method used to measure the level of thrombin or the gender of the subject.

Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of thrombin in blood samples previously collected from the patient under testing.

A“control reference value” can be a“threshold value” or a“cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the thrombin levels (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the thrombin level (or ratio, or score) determined in a biological sample derived from one or more subjects who are responders to dysbiosis associated diseases treatment. In one embodiment of the present invention, the threshold value may also be derived from thrombin level (or ratio, or score) determined in a biological sample derived from one or more subjects who are not affected with dysbiosis associated diseases. Furthermore, retrospective measurement of the thrombin levels (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.

"Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to dysbiosis associated diseases (DAD), and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to AMC and/or congenital peripheral neuropathy disease conversion and therapeutic AMC and/or congenital peripheral neuropathy disease conversion risk reduction ratios.

"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to a dysbiosis associated diseases condition or to one at risk of developing a dysbiosis associated diseases. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of dysbiosis associated diseases, such as cellular population determination in peripheral tissues, in urine or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to dysbiosis associated diseases, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a dysbiosis associated diseases. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for dysbiosis associated diseases. In other embodiments, the present invention may be used so as to help to discriminate those having dysbiosis associated diseases from normal.

The invention also relates to the use of thrombin as a biomarker of dysbiosis associated diseases, especially mucosal dysbiosis associated diseases.

Monitoring of treatments

Monitoring the influence of agents (e.g., drug compounds) on the level of expression of one or more tissue-specific biological markers of the invention can be applied for monitoring the malignant potency of the treated dysbiosis associated diseases of the patient with time. For example, the effectiveness of an agent to affect thrombin expression can be monitored during treatments of subjects receiving anti-dysbiosis treatment.

Accordingly, a second object of the invention also relates to method for monitoring the effect of a therapy for treating dysbiosis associated diseases in a subject comprising the step of measuring the level of thrombin in a first fluid sample (urine or feces) obtained from said subject at tl and measuring the level of thrombin in a second fluid sample obtained from said subject at t2 wherein when tl is prior to therapy, t2 is during or following therapy, and when tl is during therapy, t2 is later during therapy or following therapy, and wherein a decrease in the level of thrombin in the second sample as compared to the level of thrombin in the first sample is indicative of a positive effect of the therapy on dysbiosis associated diseases in the treated subject.

In another embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of (i) obtaining a pre- administration biological sample from a subject prior to administration of the agent; (ii) detecting the thrombin level; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting thrombin level in the post administration samples; (v) comparing thrombin level in the pre-administration sample with the level of expression in the post-administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased thrombin level during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage, or indicative to the necessity to change the treatment. Conversely, decreased thrombin fluid may indicate efficacious treatment and no need to change dosage.

Because repeated collection of biological samples from the dysbiosis associated diseases -bearing patient are needed for performing the monitoring method described above, then preferred biological samples is fluid or tissue samples susceptible to contain (i) cells originating from the patient's dysbiosis associated diseases tissue, or (ii) specific marker expression products synthesized by cells originating from the patients dysbiosis associated diseases tissue, including nucleic acids and proteins.

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. Intestinal epithelium releases active thrombin in gut lumen. A. F2 mRNA transcripts (Factor 2, thrombin) were detected in colonic crypt epithelium from three different healthy human (left), and three different human cancer cell lines (Caco-2, SW480 and HT-29, right). Representative blots from 3 independent experiments. B. Immunostaining of Epithelial cell adhesion molecule (EPCAM, epithelial cell marker) and human thrombin in human colon biopsy display thrombin-expressing epithelial cells (stars) and secreted thrombin in the lumen (arrows). Scale bar corresponds to 20 pm. C. Immunostaining of thrombin and nuclei confirmed the specific staining of thrombin in Caco-2 cells. Scale bar corresponds to 50 pm. Representative images of three human donors, with at least 3 independent fields are presented in B and C. D. Western blots revealed the presence of prothrombin (72 kDa) and its active isoforms (50, 32, 28 and 15 kDa) in Caco-2 cell protein extract (left blot). Released thrombin quantity was higher in apical (middle blot) compared to basolateral media (right blot). Representative blots from 3 independent experiments. E. Western-blot confirmed the presence of human prothrombin and its active isoforms in colonic biopsy supernatant from three healthy donors. Representative blot from 3 independent experiments F. Caco-2 cultured for 24 hours in serum- free medium releases a trypsin-like activity (boc-VPR-AMC fluorescent substrate) that is dose-dependently inhibited by the thrombin inhibitor (lepirudin). Activity assay was reproduced in 3 independent experiments.

Figure 2. Epithelial thrombin is activated by epithelial-prothrombinase complex and is directly regulated by the presence of commensal microbiota. A. mRNA transcripts for the two genes of the prothrombinase complex (gene F5 and gene F10) were detected in two different human cancer cell lines (Caco-2 and HT-29) and isolated human colonic crypts. The same samples used in Figure 1A to detect F2 transcripts were used to detect F5 and F10 transcripts in Caco-2 and HT-29. Representative blots from 3 independent experiments. B. Western blots revealed the presence of Factor 10 (inactive, 74 kDa and active form 54 kDa, left blot) as well as Factor 5 (inactive, 300 kDa ; active heavy chain 94 kDa and active light chain 74 kDa, right blot) in Caco-2 cell protein extract (left blot) . Representative blots from 2 independent experiments. C. Epithelial thrombin activity produced by Caco-2 was dose- dependently inhibited by the presence of the F10 inhibitor Apixaban during 24 hours (0.01 mM, 0.1 pM and 1 pM). Activity assay was reproduced in 3 independent experiments. D. Germfree mice (GF, n=5), germfree recolonized with specific-pathogen free (SPF) cecal content (GF>SPF, n=5) and conventionally-bred mice (CONV, n=8) were sacrificed at 10-14 weeks old. Relative expression of F2 mRNA transcript (thrombin) was unchanged in germ-free mouse liver compared to conventionally-bred mouse liver and GF>SPF. In germ- free mouse, F2 mRNA was significantly reduced in colon mucosa compared to conventionally-bred mouse colon mucosa. GF recolonized with SPF microbiota had identical transcription of epithelial thrombin compared to conventional mice. One-way ANOVA with Fisher’s LSD test ** P < 0.01 versus GF group.

Figure 3. Intestinal homeostasis is compromised after inhibition of basal thrombin activity in colon lumen. C57B1/6 mice were treated daily with either vehicle (n=5) or lepirudin (n=10, lOpg/day) via intracolonic route, euthanized after 10 days and used for further analysis (A to D). A. Macroscopic (Wallace scoring endpoint ⁴⁹) and B. Microscopic damage scores (⁵⁰) in lepirudin-treated group were significantly greater than the scores measured in vehicle controls. C. Hematoxylin-eosin images revealed a normal histology in vehicle-treated group, while in lepirudin-treated group we observed crypt elongation (lower left panel), areas with goblet cell depletion (upper right panel), as well as neutrophils transmigration in the lumen (lower right panel, arrows). Scale bars represent 50 pm. Representative images of vehicle (n=5) and lepirudin-treated (n=10) group are reported. D. Transcriptome analysis oi Muc2 , Camp , Tff3 , Deft>4 , Reg3g , Reg3b , Cox 2, Nos 2, Tnf Iftig, Cxcll , IL17a , Adgrel , ILlb , Cldnl , Cldn2 , Cldn5 , Tjpl Zol and Ocln genes was performed by qPCR. Principal coordinate analysis with Bray-Curtis dissimilarity matrix illustrates distances between each mouse transcriptome (each dot represents an individual mouse transcriptome), and demonstrate a significant separation between vehicle and lepirudin-treated group (Permanova P = 0.0367).

Figure 4. Suppression of basal thrombin activity modifies spatial organization of microbiota biofilm. C57B1/6 mice were treated daily with either vehicle (n=5) or lepirudin (n=10, 10pg/day) via intracolonic route, euthanized after 10 days. A. and B. Distal colon sections were Camoy’s fixed. All bacterial cells were labeled with the universal probe Eub338 for fluorescent in situ hybridization. Wheat germ agglutinin was used to stain the polysaccharides-rich mucus layer on these samples. All images were counterstained with the nuclear stain, 4’,6-diamidino-2-phenylindole DAPI. A. In vehicle-treated colon, double-arrows highlight the presence of sterile mucus layer separating the colon epithelium (dashed line) from the dense microbiota biofilm. B In contrast, lepirudin-treated animals were characterized by a complete disorganization of microbial biofilms and mucus layer in distal colon. Adherent and isolated microcolonies were evident (arrows) and rods were visible in submucosal tissue (asterisks). Images are representative of n = 5 mice. Scale bars represent 20 pm. C. Abnormal alterations to gut microbiota biofilm organization were blindly recorded and demonstrated an increased damage score in animals treated with lepirudin compared to vehicle control groups (See Table I Biofilm Damage Score). Unpaired Mann- Whitney test * P < 0.001 D. Bacterial translocation, of aerobes and anaerobes, into mesenteric lymph nodes (MLN) was significantly greater in animals treated with lepirudin compared to vehicle-treated animals. Unpaired Mann- Whitney test * P < 0.05

Figure 5. Thrombin destroys multispecies microbiota anaerobic biofilms cultured ex vivo from human colon biopsy. Multispecies anaerobic biofilms were generated from 5 different human healthy donors. Mature biofilms were then exposed to various concentrations of human active thrombin or inactive boiled thrombin for 24 hours. A. Active thrombin, but not boiled thrombin, dose-dependently reduced total biofilm biomass (crystal violet assay), B. as well as reduced the total content of matrix-associated proteins (FilmTracer Ruby matrix biofilm stain). One-way ANOVA with Fisher's LSD test versus control group without thrombin * P < 0.05 **P < 0.01, ***p < 0.001. Each concentration with > 12 biofilms per donor, N=3 independent experiments. C. Representative confocal 3D-surfaces reconstructions from human biofilms confirmed a strong effect of thrombin (100 mU/ml, equivalent to 1 nM, 24 hours) on matrix-associated proteins (lower panels), while total adherent bacteria staining (propidium iodide, in upper panels) was marginally affected. Scale bars represent 20 pm. Representative images from at least 3 independent experiments. D. Scanning electron microscopy was performed on the same human biofilm treated or not with thrombin (100 mU/ml, equivalent to 1 nM, 24 hours). Biofilms from vehicle-treated group were fully covered with a thick matrix slime with unnoticeable bacteria (upper panel). Biofilm treated with thrombin had an altered matrix structure, bacteria cells becomes clearly visible (lower panel). Representative images from n=3 healthy donor's biofilm. Scale bars represent 1 pm.

Figure 6. Thrombin is produced in all major epithelia under healthy conditions. F2 mRNA transcript (thrombin) was detected by reverse transcription PCR in A. human and B. C57B1/6 mouse epithelia. A. F2 mRNA is present in lane RT + corresponding to human hepatocyte cell line (Hep G2), liver tissue, intestinal epithelial cell line (Caco-2), lung epithelial cell line (HTB-29), healthy skin epidermis, human isolated epithelial cells from colon crypts and primary culture of urothelium. Lanes RT- are negative control for amplification of genomic contamination. Small gaps represent the same gel from which irrelevant lanes were cut out. Larger gaps correspond to three different gels, PCR conditions were 40 cycles at 60°C for HepG2, Caco-2, HTB-39 and 35 cycles at 62°C for epidermis, colon crypts and urothelium. Amplicon at 297 bp (arrow) was sequenced and confirmed to be human F2 mRNA. B. F2 mRNA is present in lane +RT corresponding to liver tissue, colon tissue, colon mucosa, skin tissue, lung tissue, bladder tissue, and ileum tissue from mouse. Lanes -RT are negative control for amplification of genomic contamination. Small gaps represent the same gel from which irrelevant lanes were cut out. Larger gaps correspond to two different gels, PCR conditions were 40 cycles at 60°C for liver, colon tissue, colon mucosa, skin and 35 cycles at 62°C for lung, bladder and ileum tissue. Amplicon at 180 bp (arrow) was sequenced and confirmed to be mouse F2 mRNA. RT-PCR experiments have been reproduced at least twice independently.

Figure 7. Active thrombin increases bactericide effect of chloramphenicol in Staphylococcus aureus biofilms. Biofilms of Staphylococcus aureus (strain ATCC 2913) were generated for 48 hours on the Calgary Biofilm Device (brain-heart infusion broth) and under anaerobic conditions (anaeropack jar system). Mature biofilms were exposed to various concentrations of human active thrombin (0.1 and 1 U/ml) with various concentrations of chloramphenicol (0 to 2500 pg/ml) in minimal M63/ 1% glucose media for additional 24 hours. Mortality of bacteria was evaluated by measuring the remaining metabolically alive bacteria (resazurin conversion assay) within biofilms compared to the mortality measured in vehicle- treated controls. Total biofilm biomass (cell + matrix) was measured using the crystal violet stain and compared to the percentage of biomass in vehicle-treated controls. A. Active thrombin dose-dependently reduced total biofilm biomass (0.1 and lU/ml). Mortality was increased when biofilms were exposed to 1 U/ml of thrombin, but not at 0.1 U/ml. * p/0.05 for mortality and ### p < 0.001 for biomass, one-way ANOVA with Fisher's LSD test. B-C. At concentrations above 313 pg/ml, chloramphenicol reduced B. total biofilm biomass and C. mortality. Co incubation of chloramphenicol with thrombin (0.1 and 1 U/ml) allowed the reduction of biofilm biomass and mortality at lower concentrations of chloramphenicol (10 to 313 pg/ml for biofilm biomass; 10 to 78 pg/ml for mortality). Experiments have been reproduced in two independent experiments, with a total of n = 9-29 biofilms per concentrations. * p<0.05, ** p<0.01, *** p <0.001, one-way ANOVA with Fisher's LSD test.

Figure 8. The thrombin inhibitor lepirudin prevents thrombin-induced human multispecies microbiota biofilm biomass reduction. Multispecies anaerobic biofilms were generated from one human healthy donor. Mature biofilms were then exposed to various concentrations of human active thrombin with or without addition of lepirudin (thrombin irreversible inhibitor). Thrombin, without lepirudin, dose- dependently reduced total biofilm biomass (crystal violet assay). Thrombin-induced effect on biomass was totally abolished when biofilms were incubated with thrombin and lepirudin (1.4 nM and 14 nM). Two-way ANOVA analysis with Fisher’s LSD test versus control group no-F2 * P < 0.05, ***P < 0.001. Each concentration with 4-16 individual biofilms, N=2 independent experiments.

Figure 9. Gastrointestinal damage induced by oral anticoagulant treatments Dabigatran (thrombin inhibitor) and warfarin (vitamin K antagonist). C57B1/6 mice were treated daily with either vehicle (n=12), warfarin (n=7, 10 mg/L in drinking water ad libitum ), or dabigatran etexilate (50 mg/kg/day oral gavage, n=12) for 7 days and euthanized for further analysis (A to D). A. Change in body weight and B. fecal score (consistency and presence of blood) were recorded daily. C. Macroscopic damage score (purpura/petechia, tissue hematoma and edema) in both warfarin and dabigatran etexilate-treated group were significantly greater than the scores measured in vehicle controls. D. Bacterial translocation of aerobes (warfarin) and anaerobes (warfarin and dabigatran-etexilate), into mesenteric lymph nodes (MLNs) was significantly greater in animals treated with oral anticoagulants compared to vehicle-treated animals. One-way ANOVA test with Fisher’s LSD * P < 0.05 versus CTR group. Figure 10. Anti-biofilm effects of thrombin against antibiotic-sensitive clinical strains of Pseudomonas aeruginosa. Four clinical strains were isolated from human pulmonary catheters at Tours Hospital between 10/2016 and 01/2017. A. Biofilms of each Pseudomonas aeruginosa isolates were generated during 48 hours on the Calgary Biofilm Device (Luria broth) and under anaerobic conditions (anaeropack jar system). Mature biofilms were then exposed to various concentrations of human active thrombin (1, 10, 100 and 1000 mU/ml) in minimal M63/ 1% glucose media for additional 24 hours. Total biofilm biomass (cell + matrix) was measured using the crystal violet stain and compared to the percentage of biomass in vehicle-treated controls. Active thrombin dose-dependently reduced total biofilm biomass (at 100 and lOOOmU/ml) of clinical strain #1 and #3. Under these conditions, human thrombin had no effect on biofilms biomass of Pseudomonas aeruginosa isolate #2 and isolate #4. * p<0.05, ** p<0.01, one-way ANOVA with Fisher's LSD test. Experiments have been reproduced in two independent experiments, with a total of n= 6-12 biofilms per concentrations. B. Antibiogram has been performed on each isolate by disc diffusion method (Kirby-Bauer method). Antibiotic susceptibility threshold has been set up at 15 mm (<15 mm = sensitive; >15 mm = resistance). As indicated in panel A, thrombin was effective against biofilm generated from strains #1 and #3, which were resistant to all (strains #1) or several antibiotics (strain #2 was resistant to Piperacilllin+tazobactam, piperacilline, colistin and ciprofloxacine).

Figure 11. A. Western-blot analysis for protein expression in urine. Western blots revealed the presence of active forms of human thrombin (50-60 kDa meizothrombin; 30 kD alpha-thrombin; 15 kDa gamma-thrombin) in human urine. Lane 1 is recombinant human prothrombin (70 kDa), lane 2 is recombinant active thrombin (Sigma Aldrich T6884); lane 3 is molecular weight marker, lane 4 is urine from healthy donor #1, lane 5 is urine from healthy donor #2, lane 6 is urine from patient with urinary tract infection #1, lane 7 is urine from patient with urinary tract infection #2.B. Enzymatic assay for thrombin activity in urines. Specific thrombin proteolytic activity (against Boc-VPR-amc substrate) inhibited with thrombin inhibitor (dabigatran) was measured in 50 mΐ of human urine from 4 healthy donors and 3 urinary tract infection patients. Only female were included in the study

Figure 12. Thrombin expression (protein and mRNA) in human colon biopsy, mucus or fecal samples from healthy controls or patients with dysbiotic mucosa: Crohn’s disease (CD) or Ulcerative Colitis (UC). Thrombin was detected (A) by western-blot in human colon mucosa (lanes 3, 4), mucus (lane 5) and feces (lanes 6, 7), (B) by immunohistochemistry and further quantification of immunofluorescence associated with intestinal epithelium in controls and inflammatory bowel disease patients (Crohn’s disease: CD and Ulcerative Colitis: UC) tissue samples, and (C) by qRT-PCR in tissue samples from inflammatory bowel disease patients (Crohn’s disease: CD and Ulcerative Colitis: UC).

Figure 13. Thrombin effect on biofilm formed by two clinical strain of Propionibacterium acnei. After maturation (72 hours), biofilms were exposed to various concentrations of human thrombin (human recombinant thrombin, Sigma, vehicle was PBS) at 37 degrees, under anaerobic conditions. Additional control condition has been performed, where the highest concentration of human thrombin has been heat-inactivated (95 degrees, 15 minutes). After 24 hours exposure, biofilm total biomass (i.e. bacteria cells and their surrounding biopolymeric matrix) was quantified using the crystal violet assay (see DOI: 10.1046/j.1365-2958.1998.01062.x). Data are represented as a percentage of biomass compared to vehicle-treated biofilms. Significant differences versus vehicle-treated biofilms is depicted with P values ** <0.01, *** <0.001 using two-way ANOVA and Dunnetfs comparison test.

Table 1. Biofilm damage score of the distal colon microbiota

EXAMPLE 1:

Material & Methods

Human tissue collection

Colon cancer resections from human donors were provided by the Centre Hospitalier de Toulouse (France). Biopsies used in the study were collected from macroscopically healthy area at distance from cancer tissue and when necessary transferred in sterile tubes for anaerobic transport (BBL, BD Bioscience). Tissue was manipulated under aseptic conditions and maintained throughout on ice and on PBS sterile buffer. Written and verbal informed consent was obtained before enrollment in the study, and the Ethics Committee approved the human research protocol (ClinicalTrials.gov Identifier: NCT01990716). Human crypts were isolated, cultured, and colon organoids were generated as described in ⁵¹, the absence of inflammatory cells in organoid cultures was confirmed (lack of CD45-positive staining).

Animals.

All animal procedures were approved by the local Laboratory Animal Ethics Committee, Toulouse. C57BL/6 mice were kept in ventilated cages and acclimatized to the study conditions for two weeks before entering experiments. Mice used for experiments were 8 weeks old. Germ-free C57BL/6 mice (10-12 weeks old) generated by 2-stage embryo transfer and housed in flexifilm gnotobiotic isolators were obtained from McMaster University’s Axenic Gnotobiotic Facility. Samples were shipped to INSERM.

In conventionally-bred mice, thrombin inhibition was induced by daily intracolonic instillation of Lepirudin (10 pg, Bachem, Ki=0.2 pM) under 3% isoflurane anesthesia. Control animals were treated with an equal volume of vehicle (0.9 % NaCl). After instillation, the animals were kept upside-down for 2 min. Animals were euthanized by cervical dislocation after 10 days of experimentation. The cumulative macroscopic damage was obtained by measuring colon thickness edema (in mm) and blind Wallace scoring endpoints ⁵². Histological damage scoring was performed on formalin-fixed, paraffin-embedded sections stained with hematoxylin-eosin, and according to a previously published scoring system ⁵⁰, on 5 fields per tissue section. Macroscopic and histologic scoring was blindly performed by skilled experimenters (one experimenter for macroscopic and two experimenters for histological scoring). Mesenteric lymph nodes were collected aseptically, weighed, homogenized and plated on Columbia blood agar (BD Biosciences) for 24 hours for aerobes and 48 hours for anaerobes (anaerobic jar) at 37°C.

To investigate gastrointestinal damage associated with oral anticoagulants, one group of mice (n=12) was treated orally with the direct thrombin inhibitor Dabigatran etexilate (50 mg/kg/day in mice corresponding to a human dose of ~4 mg/kg/day, SelleckChem, Euromedex, France). Another group (n=7) was given warfarin in drinking water ad libitum (10 mg/L, Bristol-Myers Squibb, France). The dose based on water consumption intake was 2mg/kg/day, which is equivalent to a human dose of 10 mg daily. Daily, a fecal score was recorded based on i) fecal consistency (score 0 for normal feces, 1 for soft feces, 2 for diarrhea) and ii) the presence of blood in the feces (Hemoccult tests, score 0 for negative, score 1 for positive, score 2 for gross bleeding). We also investigated potential macroscopic lesions in the stomach (purpura/petechia, tissue hematoma and edema), as well as bacterial translocation in mesenteric lymph nodes (for aerobe and anaerobe presence).

Cell culture.

Human intestinal epithelial cell lines (Caco-2, HT-29, SW480; ATCC, USA), human hepatocyte (Hep G2), human lung epithelial cells (HTB-29) were grown in DMEM high glucose GlutaMAX Suplemented with lx non-essential aminoacids, lx penicillin/streptomycin and 10% FBS (Gibco). Briefly, 3xl0⁵ IECs were plated on flat-bottom 6-well plates and grown for 7 days at 37°C 5% CO2 with culture medium replacement three times a week. Additionally, lxlO⁵ Caco2 cells were plated on polycarbonate 12-transwells plates and grown for 21 days, as described above. For harvesting of cellular supernatants, cells were washed twice with PBS Ca²⁺/Mg²⁺-free and kept for 24 h in the cell culture medium described above, but without FBS.

Gut microbiota biofilms.

For mucosa-associated biofilm reconstitution, human colonic biopsies were transferred in sterile tubes for anaerobic transport (BBL, BD Bioscience) just after collection at the endoscopy. Colon biopsies were homogenized in a microtube pestle and mucosa-associated microbiota was first cultured overnight in rich anaerobe media (Wilkins-Chalgren broth, ThermoFisher Scientific) Supplemented with L-cysteine (5%, Sigma- Aldrich). Biofilms were generated on the Calgary Biofilm Device (Innovotech, Edmonton, Canada) as previously described ^{13, 19, 53, 54}. All steps described herein were performed in anaerobic conditions in jar (Anaeropack, ThermoFisher Scientific). Mature biofilms (48 hours) were transferred onto a new plate of minimal M63 media Supplemented with glucose (2%), L-cysteine (0.5 %) and containing various concentration of human active thrombin (thrombin from human plasma, 2000 NIH units /mg of proteins, Sigma T6884), thrombin inhibitor lepirudin (Bachem) or its vehicle (phosphate buffered saline, pH 7.4 with 0.1 % bovine serum albumin) for 24 hours. The biofilm biomass density and bacterial viability were determined respectively by crystal violet (Reactifs RAL) and rezasurin (Sigma-Aldrich) assay as previously described in ¹³ and ¹⁹. Matrix-associated proteins and matrix-associated polysaccharides (N-acetlyglycosamines and sialic acid) were stained respectively with FilmTracer Ruby matrix biofilm stain, ThermoFisher Scientific) and wheat germ agglutinin (ThermoFisher Scientific) for 2 hours. Matrix was then extracted after incubation for 30 minutes in 1.5 M Nacl buffer based on previously described method ⁵⁵, and specific fluorescence was measured on fluorescent spectrophotometer (Tecan). Biofilm rate of dispersal was assessed by measuring the optical density (600 nm) and assessing colony-forming unit (CFU) of biofilm-dispersed planktonic bacteria recovered in the challenge plate. All results were expressed as percentages change of that of the means in the vehicle- treated group set as 100 %.

Imaging of biofilms.

Biofilms were stained for 1 hour, without fixation, with FilmTracer Ruby matrix biofilm stain (specific matrix-associated protein stain), fluorescein-labeled wheat germ agglutinin (matrix-associated polysaccharides stain) and propidium iodide (DNA-RNA stain, Invitrogen) and visualized on a confocal microscope (Zeiss LSM 710). Three-dimension surface rendering of stained biofilm was performed on Imaris Bitplane (v8, Concord, MA, USA). Alternatively, pegs containing treated biofilms were broken with needle nose pliers, and fixed in 2 % glutaraldehyde (Sigma-Aldrich) in 0.1 M Sorensen phosphate buffer (pH 7.4). Biofilms were dehydrated, dried by critical point drying (Leica EM CPD 300), and coated with 6 nm Platinium on a Leica EM Med 020 before being examined on a FEI Quanta 250 FEG scanning electron microscope, at accelerating voltages of 5 and 10 kV. FIJI freeware was used for final image mounting (v.1.51).

Bacteria growth rate.

Human mucosa-associated microbiota were grown for 24 hours in rich anaerobe media (Wilkins-Chalgren). Saturated cultures were then diluted in 96-well microplate to optical density of 0.1 (OD₆oo_nm) in minimal M63 media Supplemented with glucose (2%), L-cysteine (0.5 %) and containing various concentration of human active thrombin (0 to 1250 mU/ml). The growth curves of each inoculum were generated from continuous Oϋboohh_i reading every 20 minutes for 15 hours. Each value was expressed using means of duplicate experiment for each microbiota.

Tissue Imaging. For fluorescent in situ hybridization (FISH), Camoy's-fixed mice colon tissues were paraffin-embedded. Slides were hybridized with 1 mM of a universal bacterial 16S fluorescent rRNA probe (EUB338-Cy3, Eurofins) and counterstained for DNA by 4’,6-diamidino-2- phenylindole (DAPI, Sigma-Aldrich) and polysaccharides content (wheat germ agglutinin labeled with fluorescein, ThermoFisher). Human colonic biopsies were cryopreserved in Optimal Cutting Temperature (OCT, Dako) and sectioned at 6 pm of thickness. Slides were thawed at room temperature for 20 minutes and blocked for 1 hour (1% BSA, 0.3% Triton X- 100, PBS IX). Tissue were immunostained with goat polyclonal anti-thrombin (Santa Cruz, sc-16972) overnight, and double labeled with secondary antibody (anti-goat alexa-555, Life Technologies). Slides were counterstained with DAPI and fluorescein-labeled wheat germ agglutinin. Epithelial cells were highlighted with anti-Epcam staining (1/200 dilution; Abeam). To reveal unspecific staining of thrombin antibody, isotype control was used and incubated at the same concentration and under the same experimental conditions (data not shown). Representative images were obtained from blind acquisition of 4 different fields per animals. We acquired all images on Leica LSM 710 confocal microscope, and FIJI freeware was used for final image mounting (v.1.51).

16S rDNA Sequencing.

Total DNA was extracted from faeces at both baseline and after the treatment with lepirudin as previously described 47. The 16S bacterial rDNA V3-V4 regions were targeted by the 357wf-785R primers and analyzed by MiSeq at RTLGenomics (Texas, USA). An average of 11,000 (between 6896 and 15901) sequences was generated per sample. A complete description of the applied bioinformatic filters is available at www.rtlgenomics.com. Cladograms were drawn by the Huttenhower Galaxy web application (huttenhower.sph.harvard.edu/galaxy/) via the LEfSe (Linear Discriminant Analysis Effect Size) algorithm 48.

Transcription assays.

mRNA from intestinal epithelial cells, crypts and organoids were extracted by using the Nucleospin RNA/Protein kit (Macherey-Nagel). mRNAs from other human and animal tissues were extracted using the Qiagen RNeasy kit according to the manufacturer’s instructions (Qiagen) and reversely transcribed into cDNA (iScript cDNA synthesis kit, Biorad). The PCR was performed on 384-well plates and on LightCycler 480 (Roche). The expression levels of genes were normalized to both Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransf erase (HPRT) as reference genes. Fold changes in the mRNA levels were calculated with the comparative 2-DD(2ΐ method. For RT-PCR blot analysis, 1 pg mRNAs were reverse transcripted using Maxima First Strand cDNA kit (Thermo Fisher). Subsequent PCR was performed on 50 ng of cDNA at 60°C for 40 cycles or 62°C for 35 cycles. The PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Gel images were captured using Quantum ST4 1000/26MX (Fisher Scientific). Specific band were sequenced and were blasted on National Center for biotechnology information (blastn NCBI), and aligned using Clustal Omega program. All the sequences correspond to thrombin (99% homology). Representative blots were selected from at least three independent experiments.

Measurement of thrombin activity.

Proteolytic activity was measured in Caco-2 cell supernatant samples with BOC-Val- Pro-Arg-amino-4-methylcoumarin hydrochloride (0.5-150 mM) as substrate in 50 mM Tris, 10 mM CaC12, 150 mM NaCl, pH=8.3 (Sigma- Aldrich). Thrombin activity was identified from overall Arg-cleaving enzymes by pre-incubating supernatants with increasing concentrations of the specific thrombin inhibitor lepirudin for 30 min at 37°C (15.6-1000 pM; Bachem, GmbH). Velocity (reaction rate per min) was calculated by the change in fluorescence (excitation: 355 nm, emission: 460 nm), measured over 15 min at 37°C on a Varioskan Flash microplate reader (Thermo Fisher Scientific). No thrombin activity was detected using this assay in pure FBS used for cell culture.

Western-blots.

Total protein extract of Caco-2 cells was prepared by using the Nucleospin RNA/Protein kit (Macherey-Nagel, GmbH). Protein from Caco-2 cell supernatant was precipitated in 15% trichloroacetic acid at 4°C during 90 min. The pellet was washed twice in cold acetone (-20°C) and solubilized in 20 pL of protein solving buffer with tris-(2-carboxyethyl)-phosphine hydrochloride (PSB-TCEP; Macherey-Nagel). Samples were then heated at 95°C for 5 min, clarified by centrifugation at 12000 x g for 5 min and the solubilized sample was loaded into 4- 20% Mini-Protean TGX precast gels (Bio-Rad, GmbH). Human feces from 3 donors (1 g) were suspended in 1 mL PBS buffer, homogenized, centrifuged and supernatant was stored in -20°C. The mucus was collected in buffer (Tris 40 mM, NaCl 150 mM, EDTA 20 mM, pH 8 and protease inhibitor cocktail, Sigma) by scrapping the colon mucosa of human resection. Lysed samples from human and mouse tissues and feces were diluted in Laemmli buffer 4X (Biorad), Supplemented with 2-mercaptoethanol and heated at 95°C. Samples were run with at least 20 pg of total protein on Precast Gel 4-15% (Biorad) and transferred to nitrocellulose membrane (Biorad). The membrane was blocked 1 hour (PBS, 5% milk and 1% bovine serum albumine) and incubated overnight with anti-thrombin antibody (Santa Cruz sc- 16972, 1/200 dilution), anti-Factor 10 (Abeam, Ab79929, 1/200 dilution), anti-Factor 5 (Abeam, Abl08614, 1/200 dilution), in blocking buffer. Detection was achieved using secondary antibody coupled to horseradish peroxidase (donkey anti-goat IgG, Promega) during 1 hour and a chemiluminescent substrate (ECL from Amersham, Chemidoc XRS, Biorad). Pro-thrombin and active forms of thrombin were used as control (70 and 60 ng of proteins respectively, Sigma-Aldrich). Representative blots were selected from at least three independent experiments.

N-terminomics/TAILS workflow.

Human microbiota biofilms from 3 different healthy donors have been generated on the Calgary Biofilm Device as describe above. Biofilms were treated with either vehicle (PBS) or recombinant human thrombin (100 mU/ml, equivalent to 1 nM) for 24 hours at 37°C. Biofilm- associated proteins were extracted in 6M Urea, 4% SDS buffer, precipitated in trichloroacetic acid (15% final) and were further processed to N-terminomics/TAILS and shotgun proteomics analysis (data not shown). Briefly, samples were alkylated with iodoacetamide, peptide were then labelled with isotopically heavy [40 mM ¹³CD₂0 +20 mM NaB¾CN (sodium cyanoborohydride)] or light labels [40 mM light formaldehyde (CH2O) + 20 mM NaB¾CN]. Samples were then processed for N-terminal enrichment ⁵⁶’ ⁵⁷ and processed to high performance liquid chromatography (HPLC) and mass spectrometry (MS) at the Southern Alberta Mass Spectrometry (SAMS) core facility at the University of Calgary, Canada. Spectral data were matched to peptide sequences in the human UniProt protein database of twelve common bacterial species using the Andromeda algorithm as implemented in the MaxQuant software package v.1.6.0.1, at a peptide-spectrum match false discovery rate (FDR) of < 0.05. The cleavage site specificity was set to semi-ArgC (free N-terminus), with up to two missed cleavages allowed. Significant outlier cut-off values were determined after log(2) transformation by boxplot-and-whiskers analysis using the BoxPlotR tool (data not shown).

Statistical analyses.

Graphic representation and statistical analyses were performed using GraphPad Prism (v6, La Jolla, USA). Mann- Whitney's non-parametric t tests were used accordingly after D'Agostino-Pearson normality test. For multiple variables, we used two-way ANOVA followed with Fisher's LSD test. For multiple variables, we used two-way ANOVA followed with Dunnetf s test. An associated P value less than 5% was considered significant. All central values are means for dot-plots and histograms. Error bars represent standard error of the mean. To represent visual distances in multivariate dataset (metagenomic and transcriptomic) we used principal co-ordinate analysis (PCoA) using Past 3 software 49. Permutational multivariate analysis of variance (PERMANOVA Bonferroni corrected) with Bray-Curtis dissimilarity was used for comparing transcriptomic dataset (Past 3). Images for western-blots, RT-PCR and microscopy were obtained from at least 3 independent experiments and/or at least 3 independent human donors.

Results

Intestinal epithelium expresses and releases constitutively active thrombin into the lumen.

Reverse transcription analysis showed the presence of thrombin mRNA in intestinal epithelium from healthy human colon crypts (HC) as well as in three different human intestinal epithelial cell lines (Caco-2, HT-29, SW480, Figure 1A). Immunofluorescence detection of thrombin in healthy human colonic biopsies illustrated the expression of thrombin protein in the intestinal epithelium (stars), and in the lumen (arrows, Figure IB). Thrombin protein expression was confirmed in the human epithelial cell line (Caco-2) by immunohistochemistry (Figure 1C). Western-blot analysis of cell extracts and supernatants from Caco-2 cells grown on transwells confirmed the presence of released thrombin, both in its inactive proform (72 kDa band) and in its active forms (meizothrombin at 50 kDa, thrombin alpha at 32 kDa, thrombin beta 28 kDa, thrombin gamma 15 kDa, Figure ID). Most of the thrombin protein was released on the apical side of Caco-2 monolayers grown in transwells, only discrete bands were detected in Caco-2 basolateral supernatants (Figure ID). Western-blot analysis also confirmed the presence of thrombin in supernatants from incubated human colon biopsies harvested from healthy donors (Figure IE). Both the proform and active forms of thrombin were found in human colon biopsy supernatants, mucus scraped at the surface of colon mucosa, and fecal samples from healthy human volunteer and naive mice (). Proteolytic activity released by unstimulated Caco-2 cells (24 hours) was concentration-dependently inhibited by lepirudin, a specific inhibitor of thrombin further demonstrating that epithelial cells release an active thrombolytic activity in the range of 50 mU/ml (Figure IF). Overall, these data demonstrate that intestinal epithelial cells are a local source of active thrombin, released and active mostly on the luminal side of intestinal mucosa.

In addition, we demonstrated that thrombin protein can be detected in colon mucosa, mucus and feces of both naive mice, and healthy humans (data not shown). This suggests that the presence and quantification of thrombin in such samples could be used as a marker of biofilm invasiveness or mucosa health.

Thrombin regulation in intestinal epithelium

Prothrombin activation is known to be tightly regulated by the prothrombinase complex, composed of heterodimers of coagulation factors Xa and Va. Because active thrombin was detected in supernatants from intestinal epithelial cell line (Caco-2, Figure IF), we investigated the possible presence of transcripts from the prothrombinase complex F5 and F 10 genes in two human intestinal epithelial cell lines (Caco-2 and HT-29) as well as in isolated human colonic crypts. In all cases, transcripts from F5 and F 10 genes were detected (Figure 2A), showing that intestinal epithelial cells possess all the machinery required for the production of active thrombin. Further, we confirmed the presence of the F 10 and F5 proteins in cell lysates of Caco- 2 (Figure 2B). Finally, in the presence of the prothrombinase FI 0-specific inhibitor Apixaban (0.01 mM, 0.1 mM and 1 pM), thrombin activity produced by intestinal epithelial cells was significantly inhibited, proving that prothrombinase complex is present in intestinal epithelial cell cultures and is involved in active thrombin generation (Figure 2C).

Because most of the thrombin protein produced by unstimulated intestinal epithelial cells was released at the apical side, we postulated that thrombin expression might be regulated by luminal factors, and potentially the presence of microbiota at the epithelial surface. We therefore investigated the mucosal expression of thrombin in the colons of germ-free mice. Average threshold cycle was 16 for the liver, and 28 for colon mucosa. We observed that thrombin mRNA was significantly reduced (by 70%) in germ-free mice compared to the levels detected in conventionally-bred mice (Figure 2D). Liver expression of thrombin was not different in germ-free compared to conventional mice (Figure 2D). Interestingly, mucosal transcription of thrombin mRNA was completely restored in germ-free mice 3 weeks after recolonization with pathogen-free standard microbiota (cecal content) (Figure 2D). These results confirmed the constitutive production of active thrombin in intestinal epithelium, and its control by the presence of microbiota.

Constitutive thrombin activity preserves mucosal homeostasis.

To determine the physiological role of thrombin at mucosal surfaces, we inhibited its activity, by administering the irreversible thrombin-specific inhibitor, lepirudin, intracolonically to mice 22. After ten daily administrations ( 10 pg per day per mouse), we observed a significant increase of macroscopic damage compared to animals receiving intracolonic administration of vehicle (NaCl 0.9%) (Figure 3A). Histological analysis also revealed significant damage in the distal colon of animals treated with lepirudin (Figure 3B). The histological damage consisted of crypt elongation, area of goblet cell depletion, as well as neutrophil transmigration into the lumen (Figure 3C). We performed a transcriptomic qPCR analyses of genes involved in host-microbiota related functions (Defb4, Tff3, Reg3g Reg4b, Camp, Muc2), tight junctions (Zol, Ocln, Cldnl, Cldn2, Cldn5) as well as inflammatory markers (Cox2, Nos2, TNFa, IFNg, IL17A, Adgrel, Cxcll, ILlb) in the distal colon of vehicle- and lepirudin-treated animals. We used a principal coordinate analysis (PCoA) with Bray- Curtis dissimilarity matrix to show overall distance in each mouse transcriptome (Figure 3D). As illustrated by PcoA, but also by hierarchical clustering dendrogram (), there was a significant shift between the transcriptome of vehicle- and lepirudin-treated groups (Permanova P value = 0.0379). The strongest discriminants in this shift were group of genes involved in host- microbiota interactions (Camp, Muc2 and Tff3;) as well as the inducible nitric oxide synthase (Nos2). This separation caused by lepirudin failed to reach significance for inflammatory genes (Permanova P value = 0.0949,) or tight junction genes (Permanova P value = 0.553,). Together, these results demonstrate that inhibition of epithelial thrombin activity in the lumen is sufficient to cause intestinal injuries and to cause alterations in host-microbiota related transcriptome.

Impact of constitutive thrombin activity on gut microbiota

To determine whether constitutive thrombin activity would change the composition and relative abundance of gut microbiota, we sequenced the 16S rDNA V3-V4 regions from fecal samples of mice at day 0 and after 10 days treatment with lepirudin. Principal component analysis revealed that the shift induced by the intracolonic administration of the thrombin inhibitor lepirudin was modest as group's convex hulls were largely superimposed. However, the cladogram obtained by LEfSe (Linear discriminant analysis effect size) analysis showed a lepirudin-specific microbial signature characterized by a higher abundance of the genus Bamesiella (belonging to the phylum Bacteroidetes and family Porphyromonadaceae) compared to the vehicle-treated group.

As the physiology of complex microbial communities is strongly dependent on the immediate surroundings of each microbe, we then asked if thrombin would alter the microbial microenvironment. Fluorescent in situ hybridization was used to visualize spatial

organization of microbiota biofilm. In animals treated with vehicle, no mucosa-adherent bacteria were observed, and no bacteria were detected deep in the crypts, within the epithelial cells or in the submucosa. Microbiota in the distal colonic mucosa was clearly separated from the epithelia surface by a dense mucus layer, free of bacteria, although high density microbial biofilms were obvious in areas where mucus contacted fecal material (Figure 4A). Intestinal biofilms in animals treated with lepirudin had a strikingly different spatial organization from that of vehicle-treated mice (Figure 4B and 4C). Short and long rod bacteria could be seen forming microcolonies or partially segregated from each other at the outer edge of the mucus layer (when present) and feces. These microcolonies were largely encased in a loose polysaccharide-rich matrix (Figure 4B, 4C). Lepirudin allowed isolated planktonic bacteria to colonize the submucosal tissues (star, Figure 4B, 4C). As this result indicated a possible breach of the mucosal barrier, we investigated whether commensals were able to translocate across the mucosa to distant organs such as mesenteric lymph nodes. The results show that the numbers of both aerobic and anaerobic bacteria were significantly increased in the mesenteric lymph nodes of mice treated with intracolonic administration of the thrombin inhibitor, lepirudin, compared to animals given vehicle (Figure 4D). Overall, the findings indicate that while ablation of mucosal thrombin activity modestly alters microbiota community composition, it does create opportunities for invasive behavior. These results suggest that constitutive thrombin activity at the surface of intestinal mucosa exerts a constraining role on mucosal biofilms, that prevent its contact with the epithelial surface.

Thrombin alters the protein matrix of human-derived microbiota biofilms.

To further explore if and how thrombin could contribute further to the spatial segregation of commensal biofilms, we first cultured mucosa-associated microbiota, from 4 healthy human colon biopsies, in the presence of active human thrombin in liquid media. After 15 hours exposure under these specific conditions of incubation, no direct bactericidal or bacteriostatic property was detected upon thrombin exposure. We then reconstituted mucosa- associated human microbiota under its natural biofilm phenotype in anaerobic conditions. Mature human biofilms were generated for 48 hours and were then exposed for an additional 24 hours to active or inactivated (boiled) human thrombin. Biofilm biomass (cells and total matrix-associated content) was significantly reduced dose-dependently, starting from 10 mU/ml of active thrombin (Figure 5A). Boiled thrombin (500 mU/ml) did not cause such an effect (Figure 5A), nor did thrombin in the presence of its specific inhibitor Lepirudin (Figure 8). At concentrations lower than 100 mU/ml, thrombin had no effect on bacterial viability within biofilms. Starting at concentrations of 50 mU/ml, active human thrombin increased the dispersion of live biofilm-derived bacteria. To further understand the origin of biofilm biomass reduction further, we stained matrix-associated proteins (SYPRO biofilm matrix) and polysaccharides (wheat germ agglutinin). We then extracted the biofilm matrices by disruption of electrostatic interactions using 1.5M NaCl buffer (pH 7.4), and measured the amount of fluorescently labelled matrix-associated proteins and polysaccharides. We found that thrombin, starting at 1 mU/ml, significantly reduced total content of matrix-associated proteins (Figure 5C). At higher concentrations (> 100 mU/ml), thrombin reduced the total polysaccharides content in biofilms. Scanning electron microscopy revealed bacterial cells hidden beneath the dense and fully covering matrix slime in untreated human biofilms (Figure 5D upper panel). When a biofilm from the same patient was treated with human thrombin (100 mU/ml for 24 hours), individual bacteria became visible as the matrix was severely damaged (Figure 5D lower panel). Further, we have investigated the molecular mechanisms by which active thrombin (100 mU/ml for 24 hours) cleaves matrix-associated proteins by using N-terminomics/TAILS and shotgun proteomics analysis (Data not shown), together with the putative protein corresponding to the cleaved peptides and the microbial species known to express such peptides. Overall, these data provide evidence that human thrombin can damage mature human multispecies biofilms, through enzymatic processing of selective protein constituent of the biofilm matrix backbone.

Thrombin alters biofilms of pathogenic bacteria and potentiates the effects of antibiotics.

We investigated the effects of thrombin on biofilms of four different clinical isolates of pathogenic Pseudomonas aeruginosa. We observed that thrombin was efficient at reducing the total biofilm biomass of 2 clinical strains (strain #1 and #3), in a dose-dependent manner (at 100 and lOOOmU/ml) (Figure 10). On the two other strains (clinical isolate #2 and #4), thrombin had no effect on total biofilm biomass (Figure 10). Interestingly, the effects of thrombin did not correlate with the sensitivity of the strains to antibiotics. As shown in Figure 10B, clinical isolate #1 was resistant to all antibiotics, but its biofilms was greatly reduced by thrombin exposure (Figure 10 A). We also demonstrated that thrombin exposure induced mortality and decreased the total biofilm biomass of cultured pathogenic Staphylococcus aureus strain (Figure 7). These results demonstrate that thrombin can alter the biofilm of some pathogenic bacteria such as P. aeruginosa or S. aureus , but is not active against all biofilms. Further, it demonstrates that thrombin can alter the biofilm of pathogenic strains that are otherwise resistant to antibiotics.

Next, we investigated whether thrombin could modify the efficacy of antibiotics on biofilms. At concentrations above 313 pg/ml, chloramphenicol reduced the total biofilm biomass and mortality of Staphylococcus aureus biofilms (Figure 7). Co-incubation of chloramphenicol with thrombin (0.1 and 1 U/ml) allowed the reduction of biofilm biomass and mortality at lower concentrations of chloramphenicol (10 to 313 pg/ml for biofilm biomass; 10 to 78 pg/ml for mortality) (Figure 7). Importantly, we demonstrated here that thrombin enhanced the effects of the antibiotic Chloramphenicol against S. aureus biofilms, allowing the use of a lower concentration of this antibiotic.

Thrombin is expressed in skin, lung, bladder and small intestine

As thrombin (F2 gene) appeared to be of significant importance for intestinal mucosa homeostasis by constraining microbial biofilms, we hypothesized that epithelia from all major host-microbiota surfaces can also produce thrombin for the same purpose. We detected F2 mRNA in epithelial cell line derived from intestine, skin and lung as well as in intestinal crypt epithelium, and bladder urothelium derived from healthy human tissues (Figure 6A) as well as from C57B1/6 mice (Figure 6B). We sequenced the specific F2 amplicon (297 bp for human, 180 bp for mouse), and performed a blast alignment revealing a > 99% homology with the respective human and mouse F2 genes, confirming an active transcription of the thrombin gene. These data thus further expand our knowledge on the presence of active thrombin in epithelial organs, which at least in the intestine is under the direct regulation of microbiota. Our data thus point to a previously unknown role for thrombin in epithelia-biofilms interactions.

DISCUSSION

The present findings demonstrate that thrombin, a serine protease classically involved in the coagulation cascade and known to be produced in the liver, can originate from intestinal epithelium, where it can in principle, contribute to mucosal protection by maintaining healthy spatial segregation between the host and the microbiota.

Adding to our current report, ectopic expression of thrombin (both mRNA and protein) has been suspected in the epithelium from benign and malignant prostate tumors ²³, in brain endothelial cells ²⁴, as well as in epithelial-like cells that form ovarian follicle, granulosa cells ²⁵. In our study, we detected unequivocal expression of thrombin in non-tumoral intestinal, lung, skin and bladder epithelium. Furthermore, we demonstrated that epithelial production of thrombin mRNA is directly controlled by microbiota colonization. Interestingly, thrombin mRNA production in liver remained unchanged in germ- free mice, suggesting that the intestinal epithelium possesses specific regulatory mechanisms for the local production of thrombin in the digestive tract.

Although anticoagulants and direct thrombin inhibitors administered orally are increasingly used for treatment of chronic cardiovascular pathologies 26, their safety remains a concern. Patients commonly suffer from gastrointestinal adverse effects that may be severe and even fatal in the case of gastrointestinal bleeding ²²’ ²⁷. Our results demonstrated a protective role for constitutively produced epithelial thrombin. It thus can be hypothesized that chronic inhibition of epithelial thrombin activity by oral anticoagulant treatments could possibly lead to uncontrolled and invasive biofilm phenotypes, initiating tissue damage. Indeed, in Figure 10, mice were orally treated with dabigatran etexilate, a direct thrombin inhibitor used in humans for cardiovascular pathologies, or with warfarin, a vitamin K antagonist. We observed in these mice gastrointestinal bleeding (presence of blood in feces), tissue damage in stomach (petechia, purpura, edema), and bacterial translocation to mesenteric lymph nodes. In addition to its transcriptional regulation, inactive pro-thrombin has to be cleaved into active thrombin. This activation can be achieved not only by autoproteolysis, but also by the activity of many other proteases of the coagulation cascade. Among the many different possible ways to activate thrombin, we investigated the concurrent presence along with thrombin, of the prothrombinase complex composed of Factors 5a and 10a. Both factors are present (both mRNA and protein) and biologically active in the intestinal epithelium to transform inactive to active thrombin. Further, we found that thrombin activity is under the control of the prothrombinase complex, as Apixaban, an inhibitor of this complex, was able to inhibit the release of thrombin activity from intestinal epithelial cells. Pro-thrombin activation also requires the vitamin K-dependent carboxylase (specifically the VKORC1 subunit)⁵⁸ known to be produced by commensal microbiota. Germ-free mice reach an euthanasia endpoint when fed an irradiated AIN-76A diet if not supplemented with vitamin K ²⁹. Of note, vitamin K deficiency in humans resulting from broad-spectrum parenteral antibiotic treatment, is associated with severe gastrointestinal damage, such as bleeding and perforated gastric ulcers ³⁰. Here, we propose that thrombin inhibition in the lumen of distal colon might directly predispose to mucosal injuries, at least in part due to microbiota biofilm adhering to mucosal surfaces. In addition to this previously unknown role for thrombin in microbiota organization, other pathophysiological roles for epithelial thrombin are plausible, but yet unexplored. More research is needed to determine whether constitutive expression of epithelial thrombin might also regulate epithelial biology, potentially by protease-activated receptors activation.

Based on our results, a role for thrombin activity in the modulation of Barnesiella abundance is suspected, although thrombin’s effect on shaping overall microbiota composition is likely to be minor. Under inflammatory conditions, human neutrophil elastase is able to cleave out small C-terminal highly cationic fragments from thrombin, highly cationic, which in turn exert bactericidal effects on isolated pathogens ³¹. Adding to these reports, our data suggest that full-length active thrombin, exerts a specific biofilm-disrupting activity that depends on its proteolytic function. Together, thrombin seems to have a dual action on microbes: to segregate host mucosa from bacterial biofilms for the full-length active thrombin, and bactericidal effects on planktonic bacteria for truncated, proteolytically inactive thrombin. Interestingly, western- blot analysis demonstrated that different forms of thrombin (pro-form, active form, truncated forms) are present at the epithelial surface. Each form might exert diverse effects on bacteria in vivo , adding to the impact of proteolytically active thrombin on bacterial biofilm biomass.

Biofil bacteria are embedded in a protective matrix having a complex composition (e.g. RNA/DNA, proteins, polysaccharides). The development of this biofilm organization constitutes a major challenge for human and animal health 18. Therefore, the prevention of biofilm overgrowth and disruption of already established deleterious biofilms is crucially important. Destroying the biofilm matrix backbone, for example via enzymatic lysis, seems to be an interesting approach for biofilm eradication. Several microbially-derived enzymes have indeed been reported to degrade components of bacterial biofilm matrix, although these reports relied exclusively on monospecies biofilms 32, 33, 34, 35, 36. Our study adds a previously unsuspected component of biofilm matrix regulation: active thrombin produced by the host epithelial mucosa. Epithelial-derived thrombin can thus be added to the list of matrix-degrading antibiofilm agents, with a strong effect on biofilms originating from a complex multispecies human microbiota. Our study suggests that epithelial-derived proteolytic factors are produced as a physiological response towards microbiota biofilms growing at the mucosal surface of the digestive tract. Our demonstration that thrombin is also expressed by epithelia from lung, bladder and skin, where the same role for thrombin could be expected, further suggests that this novel mechanism may be a target for new therapies to modify pathogenic biofilm encroachment at these host-microbial surfaces.

We determined that the concentration of thrombin released by monolayers of intestinal epithelial cells was in the range of 50 mU/ml. We used a similar thrombin concentration for in vitro experiments on biofilms. Interestingly, this thrombin concentration range is very low compared to the systemic blood thrombin activity required to induce coagulation (in the range of 10 U/ml) 37. Although we report a protective function for basal thrombin activity, where low concentrations of epithelial thrombin could be beneficial to control mucosal biofilms and avoid their direct contacts with tissue, increased thrombin activity is also associated with chronic inflammation of the gut in cases of inflammatory bowel disease patients 11. In that case, one can hypothesize that these relatively high concentrations of thrombin at mucosal surfaces might be detrimental, fragmenting and dispersing biofilms to cause tissue damage. Indeed, an inflamed area of the colon is associated with an overall destruction of normal biofilm organization, with heterogeneous morphology, ranging from isolated cells to clusters of epithelia-adherent biofilms ^{13, l6· 20}’ ^{21, 38}. Further studies related to the role of epithelial-derived thrombin in pathologies, and to the control of thrombin expression and activity, are thus warranted.

The biofilm phenotype of intestinal microbiota has been well established in the healthy digestive tract and is conserved throughout the animal kingdom ¹³’ ¹⁵’ ¹⁷’ ¹⁹’ ³⁹. The ability to maintain such biofilms that distances the microbiota from host tissue is likely of evolutionary significance for digestive health as well as for host survival. We propose that epithelial thrombin helps maintain a spatial segregation between the microbiota biofilm and the host. The present findings can have a direct impact on human health in view of the link between biofilms abnormal overgrowth on mucosal surface, in the gut, and beyond (e.g. inflammatory bowel diseases, colorectal cancer, cystic fibrosis, urinary tract infections and chronic wound ulcers)

18 40

EXAMPLE 2 (Thrombin as Biomarker of dysbiosis associated diseases)

Methods:

Collection of human urine

Human urinary samples were collected between July and October 2017. Samples were collected from women hospitalized in the Adult Emergency Department of Toulouse University Hospital, France. Samples were classified as two groups: patients with urinary clinical symptoms and with positive bacteriuria were classified as urinary tract infection group (UTI), those without urinary clinical symptoms and negative for bacteria in the urine were classified as the healthy group. According to French regulation on observational database analyses, the study did not need specific informed consent requirements.

Collection of human intestinal tissues, mucus and feces

Colon cancer resections from human donors were provided by the Centre Hospitalier de Toulouse (France), and only non-cancerous margins were used. Mucus was collected at resection surface. Tissues were collected, manipulated under aseptic conditions and maintained throughout on ice and on PBS sterile buffer. Written and verbal informed consent was obtained before enrollment in the study, and the Ethics Committee approved the human research protocol (ClinicalTrials.gov Identifier: NCT01990716).

Thrombin-activity dosage in human urine

Proteolytic activity was measured in 50 mΐ of urine with BOC-Val-Pro-Arg-amino-4- methylcoumarin hydrochloride (150 mM) as substrate in 50 mM Tris, 10 mM CaC12, 150 mM NaCl, pH=8.3 (Sigma- Aldrich). Samples were pre-incubated for 30 min at 37°C to activate proteolytic activity in the buffer. Velocity (reaction rate per min) was calculated by the change in fluorescence (excitation: 355 nm, emission: 460 nm), measured over 15 min at 37°C on a Varioskan Flash microplate reader (Thermo Fisher Scientific). Specific thrombin activity was identified from overall Arg-cleaving enzymes by pre-incubating supernatants with 1 mM concentration of the specific thrombin inhibitor dabigatran BIBR953 for 30 min at 37°C (Sigma-Aldrich, France). Thrombin activity in mU/ml was calculated after the slope velocity of known concentration of human active thrombin (Sigma Aldrich T8664, 0 to 500 mU/ml).

Detection of thrombin by western blots.

Protein from 400 mΐ of urine was precipitated in Protein Precipitator PP kit according to the manufacturer's protocol (from Total DNA, RNA, and protein isolation kit, Macherey- Nagel). Protein pellet was solubilized in 60 pL of protein solving buffer (2% final SDS) and resuspended in Laemmli buffer. Samples were then heated at 95°C for 5 min, clarified by centrifugation at 12000 x g for 5 min. Samples were run with 20 pg of total protein on Precast Gel 4-20% Mini-Protean TGX precast gels (Biorad) and transferred to nitrocellulose membrane (Biorad). The membrane was blocked 1 hour (PBS, 5% milk and 1% bovine serum albumin) and incubated overnight with anti-thrombin antibody (Santa Cruz sc- 16972, 1/200 dilution) in blocking buffer. Detection was achieved using secondary antibody coupled to horseradish peroxidase (donkey anti-goat IgG, Promega) for 1 hour and a chemiluminescent substrate (ECL from Amersham, Chemidoc XRS, Biorad). Pro-thrombin and active forms of thrombin were used as control (70 and 60 ng of proteins respectively, Sigma-Aldrich).

Human feces from 3 donors (1 g) were suspended in 1 mL PBS buffer, homogenized, centrifuged and supernatant was stored in -20°C. The mucus was collected in buffer (Tris 40 mM, NaCl 150 mM, EDTA 20 mM, pH 8 and protease inhibitor cocktail, Sigma) by scrapping the colon mucosa of human resection. Lysed samples from human and mouse tissues and feces were diluted in Laemmli buffer 4X (Biorad), Supplemented with 2-mercaptoethanol and heated at 95°C. Samples were run with at least 20 pg of total protein on Precast Gel 4-15% (Biorad) and transferred to nitrocellulose membrane (Biorad), and incubations with antibodies were performed as described above.

Immunohistochemistry in tissue samples

Human colonic biopsies were cryopreserved in Optimal Cutting Temperature (OCT, Dako) and sectioned at 6 pm of thickness. Slides were thawed at room temperature for 20 minutes and blocked for 1 hour (1% BSA, 0.3% Triton X-100, PBS IX). Tissue were immunostained with goat polyclonal anti-thrombin (Santa Cruz, sc- 16972) overnight, and then with secondary antibody (anti-goat alexa-555, Life Technologies). Slides were counterstained with DAPI and fluorescein-labeled wheat germ agglutinin. Epithelial cells were highlighted with anti-Epcam staining (1/200 dilution; Abeam). We acquired all images on Leica LSM 710 confocal microscope, and FIJI freeware was used for final image mounting (v.1.51).

Results: An Arg-cleaving proteolytic activity was detected in human urine (figure 11). This activity was inhibited by pre-treatment with thrombin-specific inhibitor (dabigatran BIBR953), confirming that the nature of this proteolytic activity was thrombin. Healthy donors (4 female donors) had an average thrombin-specific activity of 0.73± 0.38 mU/ml versus 18.1± 14.8 mU/ml for patients with urinary tract infection (3 female patients, bacteriuria was confirmed in vitro).

Thrombin protein expression was confirmed in urine from two donors and two UTI patients (400 mΐ of urine) with western-blot and specific thrombin immunostaining. Specific bands were unnoticeable in urine samples from healthy donors, although several active forms of thrombin (meizothrombin, alpha-thrombin and gamma thrombin) were observed in urine from UTI patients.

Results also demonstrated that thrombin protein can be detected in human intestinal mucosa biopsies (lanes 3 and 4 of Western-blot shown in Figure 12 A), mucus harvested at intestinal surface (lane 5) and in human feces (lanes 6 and 7). Further, it was found that thrombin protein expression associated with epithelium was significantly increased in biopsies of Inflammatory Bowel Disease patients, particularly in ulcerative colitis patients (see Figure 12B). Thrombin mRNA expression was detected in mucosal biopsies of controls and inflammatory bowel disease (Crohn’s disease: CD and ulcerative colitis: UC) patients (Figure 12C). A trend towards increased thrombin mRNA expression in inflammatory bowel disease tissues was noticed, but did not reach significance due to the low number of samples.

Overall, these data demonstrated that 1/ that both thrombin activity and protein can be detected in human urine, intestinal biopsies, mucus and feces and 2/ that high thrombin activity and high protein level is correlated with urinary tract infection (i.e. inflamed bladder and dysbiosis), and inflammatory bowel disease.

EXAMPLE 3

Methods:

Two clinical strains of Propionibacterium acnei (other names Cutibacterium acnes, Corynebacterium acnes , strain 6919, strain 11827, American Tissue Type Culture ) were cultured under anaerobic condition to form a biofilm using the MBEC device (minimal biofilm eradication device, or Calgary Biofilm Device, Innovotech, Canada). After maturation (72 hours), biofilms were exposed to various concentrations of human thrombin (human recombinant thrombin, Sigma, vehicle was PBS) at 37 degrees, under anaerobic conditions. Additional control condition has been performed, where the highest concentration of human thrombin has been heat-inactivated (95 degrees, 15 minutes). After 24 hours exposure, biofilm total biomass (i.e. bacteria cells and their surrounding biopolymeric matrix) was quantified using the crystal violet assay (see DOI: 10.1046/j.1365-2958.1998.01062.x).

Results:

As shown in figure 13, the total biofilms of cultured Propionibacterium acnei clinical strain (strain 6919) was greatly reduced by thrombin exposure.

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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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Claims

CLAIMS:

1. A method of inhibiting or reducing bacterial biofilm formation on a surface comprising the step of applying to the surface an amount of thrombin.

2. The method according to claim 1 wherein thrombin is active or activated form of thrombin selected from the list consisting of Prothrombin, Prethrombin 2, Meizothrombin and active thrombin (alpha, beta or gamma thrombin).

3. The method according to claim 2 wherein thrombin is a mature form of active human thrombin.

4. The method according to claim 1 to 3 wherein bacterial biofilm is Pseudomonas biofilm or Staphylococcus biofilm.

5. The method according to claim 1 to 4 wherein the surface is an artificial surface or is a biological surface.

6. The method according to claim 5 wherein the biological surface is a mucosal surface of gut tissue, lung tissue, bladder tissue, vaginal tissue, gingival tissue, ocular tissue and skin tissue.

7. The method according to claim 5 wherein the artificial surface is a medical device.

8. The method according to claim 1, wherein the method further comprises the step of applying to the surface at least one antimicrobial agent.

9. A combination of thrombin and antimicrobial agent for simultaneous or sequential use in preventing or treating infection from a bacterial biofilm.

10. The method according to claim 8 or a combination for use according to claim 9 wherein thrombin is active or activated form of thrombin selected from the list consisting of Prothrombin, Prethrombin 2, Meizothrombin and active thrombin (alpha , beta or gamma thrombin).

11. The method or a combination for use according to claim 10 wherein infection from bacterial biofilm is infection from Pseudomonas biofilm or infection from Staphylococcus biofilm.

12. The method according to claim 8 or a combination for use according to claim 9 wherein antimicrobial agent is an antibiotic antimicrobial agent.

13. A method for assessing a subject’s risk of having or developing dysbiosis associated diseases, said method comprising the step of measuring the level of thrombin in a biological sample obtained from said subject wherein the level of thrombin is positively correlated with the risk of said subject of having or developing a dysbiosis associated disease.

14. The method according to claim 13 comprising the step of comparing said level of thrombin to a control reference value wherein: - a high level of thrombin compared to said control reference value is predictive of a high risk of having or developing a dysbiosis associated diseases and

- a low level of thrombin compared to said control reference value is predictive of a low risk of having or developing a dysbiosis associated diseases