Emerging Uses in the Treatment of Clostridium difficile Infection

Abstract

To provide new and / or alternative therapeutic solutions which may alleviate the symptoms associated with Clostridium difficile infection, preferably intestinal infections, and / or cure the physiopathological conditions related to / associated therewith.SOLUTION: The present invention relates to the use of specific strains of Lactobacillus paracasei or of a composition comprising the strains for the prevention and / or treatment of a physiopathological condition related to / associated with Clostridium difficile infection, preferably intestinal infections, or so-called CD-associated disease (CDAD) or CD infection (CDI).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of certain strains of Lactobacillus paracasei or compositions comprising said strains for the prevention and / or treatment of Clostridium difficile infections, preferably intestinal infections or physiopathological conditions related to / associated with so-called CD-associated disease (CDAD) or CD infection (CDI). [Background technology]

[0002] Clostridium difficile is a Gram-positive, anaerobic, spore-forming, rod-shaped bacterium that is widely distributed in nature both in the subsoil and in the intestinal tract of pets.

[0003] Contamination with this organism can also occur via the fecal-oral route, particularly as ingested spores survive the acid barrier of the stomach and germinate in the colon.

[0004] In humans, C. difficile is found in approximately 3% of healthy adults as a component of the saprophytic intestinal flora, and can be found at significantly higher rates (15–70%) in infants under 1 year of age.

[0005] Clostridium difficile generally does not cause any damage. However, under certain conditions, it can cause diarrhea of ​​varying severity, which in certain cases can be associated with fever and a significant overall impairment of the individual's health status in terms of physical, mental, and social aspects. In fact, diarrhea is often accompanied by other major symptoms such as nausea, vomiting, general malaise, pain, abdominal distension, and dehydration.

[0006] In clinical practice, C. difficile is known as a major cause of a severe form of colitis, defined as pseudomembranous colitis, characterized by widespread necrosis of the rectum and sigmoid colon, often accompanied by profuse diarrhea.

[0007] Of particular concern in this regard are several strains of Clostridium difficile, which are defined as enterotoxigenic because they are capable of producing enterotoxin A and / or cytotoxin B. These toxins are internalized through the intestinal mucosa and result in the death of enterocytes.

[0008] The histologic lesions range from type I morphology, characterized by isolated epithelial necrosis associated with an inflammatory infiltrate within the colonic lumen, to type III morphology, characterized by widespread epithelial necrosis and ulceration covered by a grayish pseudomembrane composed of mucin, neutrophils, fibrin, and cellular debris (hence the term pseudomembranous colitis).

[0009] The severity of C. difficile intestinal infection varies. Symptoms range from mild to profuse diarrhea (up to 10 liters of serous secretions per day) and may be accompanied by toxic megacolon, intestinal perforation, hypokalemia, intestinal bleeding, and sepsis. Diarrhea may be accompanied by fever, nausea, anorexia, general malaise, pain, abdominal distension, and dehydration. Diarrhea may be associated with mucus, blood, and fever. Infants and young children are often asymptomatic carriers. Indeed, while colonization appears favorable for the immature intestinal bacterial flora, the lack of pathological evolution is due to the inability of toxins to bind to receptors on enterocytes, which are also still immature.

[0010] Infections often appear after aggressive antibiotic therapy and are very commonly spread in hospital settings. Diarrhea is a common side effect of antibiotic therapy, but the disorder usually resolves once the drug is discontinued at the end of treatment. However, in the case of Clostridium, diarrhea, along with other symptoms, shows no signs of abating or improving.

[0011] Among the risk factors for Clostridium difficile infection, the following can be described: • The use of antibiotics, especially broad-spectrum antibiotics (i.e., those that can eliminate many different types of bacteria), and their use in conjunction with different antibiotics to cure long-standing or antibiotic-resistant infections. • The need for hospitalization or a stay in another treatment facility. ●Digestive system surgery. • Abdominal surgery involving the bowel. ●Staying in a kindergarten, nursery school or sanatorium. Colon problems, such as irritable bowel syndrome or colorectal cancer. ●Kidney problems. • A weakened immune system (caused by diabetes or medications). • Taking proton pump inhibitors or other medications that can reduce gastric acidity, which normally act as protection against infection. • Previous Clostridium difficile infection. ●Age over 65 years old.

[0012] In either case, treatment of the infection requires the use of antibiotics, albeit specifically targeted against this particular bacterium.

[0013] As previously indicated, the majority of C. difficile infections occur in settings such as hospitals and nursing homes, where many individuals are taking antibiotics and are in close contact with one another. The mortality rate of severe C. difficile infections is significant, leading to the adoption of essential preventative measures to limit the spread of disease in hospital settings.

[0014] In recent years, various cases have been successfully treated with fecal transplants.

[0015] Relying on probiotics has proven effective in the prevention / treatment of most types of diarrhea, including that induced by Clostridium difficile, through competitive inhibition of the pathogen, stabilization of the resident flora, and attenuation of increased intestinal permeability associated with, for example, rotavirus infection.

[0016] In view of the foregoing, there is a strong need for new and / or alternative therapeutic solutions that can alleviate symptoms associated with Clostridium difficile infections, preferably enteric infections, and / or cure physiopathological conditions related / associated therewith. Summary of the Invention [Problem to be solved by the invention]

[0017] The Applicant has found a solution to the above-mentioned needs through the use of probiotics based on bacteria and / or yeasts and / or other microorganisms. In particular, the solution of the present invention proposes the use of bacteria belonging to the genera Lactobacillus and / or Bifidobacterium, preferably the species Lactobacillus paracasei. [Means for solving the problem]

[0018] Indeed, as a result of using the probiotics described in detail below, an inhibition of C. difficile has been observed, evidenced above all by in vitro displacement tests.

[0019] The invention is described in detail below also by way of examples, which are not intended to be limiting in any way, with the aid of the following figures: In particular: [Brief explanation of the drawings]

[0020] [Figure 1]Figure 1A shows the results of a competitive adhesion test, i.e., the results of co-incubating eukaryotic cells with probiotics and with pathogens. More specifically, this figure illustrates CDIF adhesion in terms of the percentage of adherent live cells and viable cells after co-incubating HT29 cells with CDIF and with the various probiotics tested. Figure 1B shows the results of a test for the removal of adherent pathogens, i.e., the results of pre-treating eukaryotic cells with pathogens and then incubating with probiotics. More specifically, this figure illustrates CDIF adhesion in terms of the percentage of adherent live cells and viable cells after loading eukaryotic cells with CDIF and then incubating HT29 cells with the various probiotics tested. [Figure 2] Figure 2A shows the results of an adhesion exclusion test (eukaryotic cells were pretreated with the probiotic BIOK+ and then incubated with pathogens). More specifically, this figure illustrates CDIF adhesion in terms of the percentage of adherent live and viable cells after pre-stimulation by contact of HT29-MTX cells with the tested probiotic product, Bio-K+. Figure 2B shows the results of a competition in an adhesion test (co-incubation of eukaryotic cells with probiotics and with pathogens). More specifically, this figure illustrates CDIF adhesion in terms of the percentage of adherent live and viable cells after co-incubation of HT29-MTX cells with CDIF and with the tested probiotic product, Bio-K+. Figure 2C shows the results of an adhesion pathogen removal test (eukaryotic cells were pre-treated with pathogens and then incubated with probiotics). More specifically, this figure illustrates CDIF adhesion in terms of percentage of adherent viable cells and viable cells after loading eukaryotic cells with CDIF and then incubating HT29 cells with the different probiotics tested. [Figure 3] Figure 3 shows the results of a comparative analysis of the results of tests conducted with a particular example probiotic compared to a commercially available product known as Bio-K+ indicated for Clostridium difficile infection. DETAILED DESCRIPTION OF THE INVENTION

[0021] In this context, the definition of "probiotic" was devised by an expert group jointly convened by FAO and WHO in 2001 and is "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host." In particular, in Italy, the Ministry of Health defines probiotics as "microorganisms which, when taken in sufficient amounts, demonstrate the ability to exert a beneficial function on the body," essentially repeating the definitions of the two organizations mentioned above.

[0022] Clostridium difficile (hereafter CD) is a bacterium that is the clinical cause of various types of infections or diseases / conditions known as CD-associated diseases (CDAD) or CD infections (CDI) and can be characterized by different severity.

[0023] Indeed, the infection may manifest itself as mild diarrhea, but may also lead to pseudomembranous colitis, toxic megacolon and intestinal perforation. Severe clinical manifestations, associated above all with a risk of death, become more frequent when the infection is sustained by new, more virulent strains of bacteria.

[0024] CD infections are typically nosocomial and quite frequently present in epidemics.

[0025] A first aspect of the present invention relates to the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-01 or a composition comprising said strains for use in the prophylaxis and / or treatment of physiopathological conditions related to / associated with Clostridium difficile infections, preferably intestinal infections or so-called Clostridium difficile associated diseases (CDAD) or Clostridium difficile infections (CDI).

[0026] The disease associated with Clostridium difficile is preferably selected from pseudomembranous enterocolitis, pseudomembranous colitis, colitis without pseudomembranes, fulminant colitis, colitis with toxic megacolon and intestinal perforation, antibiotic-associated colitis, antibiotic-associated diarrhea, and diarrhea associated with C. difficile infection.

[0027] Both strains were isolated and deposited at SOFAR SpA. The bacterial strain L. casei DG® (Lactobacillus paracasei CNCM I-1572) was deposited at the National Collection of Cultures of Microorganisms of the Pasteur Institute of Paris under the accession number CNCM I-1572. This strain was originally designated Lactobacillus casei DG subcasei. The bacterial strain Lactobacillus paracasei LPC-S01 was deposited at the DSMZ under the following accession number: DSM26760.

[0028] The Clostridium difficile strains referred to in this context are preferably defined as enterotoxigenic, i.e., capable of producing an enterotoxin / cytotoxin, such as cytotoxin B and / or enterotoxin A. In a broader context, non-toxigenic, non-pathogenic, emerging, hypervirulent, antibiotic-resistant, and spore hyper-producer C. difficile strains are also considered.

[0029] Administration of the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 or said compositions is preferably effective in alleviating and / or treating and / or curing symptoms associated with said infections / CDAD / CDI and / or histological lesions caused by CD infections or CD-related diseases.

[0030] Indeed, infections / associated diseases caused by CD can be characterized by different degrees of severity. Infections may manifest themselves as mild diarrhea but may ultimately lead to abdominal conditions, such as protein-losing enteropathy and recurrent diarrhea, as well as extraintestinal conditions, such as bacteremia and splenic abscesses. Severe clinical manifestations, such as dehydration, hypokalemia, septic shock, pseudomembranous colitis, toxic megacolon, and intestinal perforation, are associated with a high risk of death and become more frequent as infections are sustained by new, more virulent bacterial strains.

[0031] Thus, according to a preferred embodiment of the present invention, said symptoms associated with said infection / CDAD / CDI are selected from diarrhea, dehydration, pseudomembranous colitis, enteropathy, toxic megacolon, intestinal perforation, sepsis, intestinal bleeding and hypokalemia.

[0032] The histological lesions are preferably type I and / or type III.

[0033] According to a preferred embodiment of the present invention, the composition further comprises a microorganism, preferably a bacterium, preferably a probiotic bacterium and / or a yeast and / or a fungus.

[0034] The bacterium preferably belongs to a genus selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus, Enterococcus and combinations thereof, more preferably the bacterium belongs to the genus Lactobacillus and / or Bifidobacterium.

[0035] According to a further preferred embodiment of the present invention, the bacteria of the genus Lactobacillus are selected from the group consisting of Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, and Lactobacillus cellobiosus. Lactobacillus cellobiosus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus johnsonii johnsonii, Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus mucosaeThe bacteria may belong to at least one of the following genus: Lactobacillus mucosae, Lactobacillus panis, Lactobacillus collinoides, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus salivarius, and Lactobacillus sanfranciscensis.

[0036] According to a further preferred embodiment, said bacterium belonging to the genus Lactobacillus is not a strain of Lactobacillus acidophilus CL1285 and / or Lactobacillus casei LBC80R and / or Lactobacillus rhamnosus CLR2.

[0037] According to a further embodiment of the present invention, the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 or the composition comprising said strains further comprises (and is further used in combination with) a bacterium belonging to the genus Lactobacillus which is not a strain of Lactobacillus acidophilus CL1285 and / or Lactobacillus casei LBC80R and / or Lactobacillus rhamnosus CLR2.

[0038] According to a further preferred embodiment of the present invention, the bacterium of the genus Bifidobacterium is selected from the group consisting of B. animalis, B. bifidum, B. breve, B. infantis, B. longum, B. adolescentis, B. catenulatum, B. angulatum, B. asteroides, B. boum, B. coerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. The bacterial strain is preferably selected from Bacillus inopinatum, B. lactis, B. magnum, B. merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B. pullorum, B. ruminantium, B. saeculare, B. subtile, B. thermocidophilum, B. thermophilum and B. tsurumiense, more preferably Bacillus clausii, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium The bacterium belongs to at least one species selected from Bacillus megaterium, Bacillus halodurans, Bacillus thuringiensis, Bacillus insolitus, and Bacillus marinus.

[0039] According to a further preferred embodiment of the present invention, the bacterium of the genus Propionibacterium belongs to at least one species selected from P. shermanii, P. acnes, P. australiense, P. avidum, P. cyclohexanicum, P. freudenreichii, P. granulosum, P. jensenii, P. microaerophilum, P. propionicum, and P. thoenii.

[0040] According to a further preferred embodiment of the present invention, the bacterium of the genus Streptococcus is selected from the group consisting of Streptococcus thermophilus, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus downei, Streptococcus dysgalactiae, and Streptococcus equinus. equinus, Streptococcus ferus, Streptococcus infantarius, Streptococcus iniae, Streptococcus intermedius, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis, Streptococcus peroris peroris, Streptococcus pneumoniae, Streptococcus pseudopneumoniaeThe bacterial strain may belong to at least one species selected from the group consisting of Streptococcus pseudopneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus tigurinus, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus uberis, Streptococcus vestibularis, Streptococcus viridans, and Streptococcus zooepidemicus.

[0041] According to a further preferred embodiment of the present invention, the bacterium of the genus Lactococcus belongs to at least one species selected from L. chungangensis, L. formosensis, L. fujiensis, L. garvieae, L. lactis, L. piscium, L. plantarum, L. raffinolactis, and L. taiwanensis.

[0042] According to a further preferred embodiment of the present invention, the bacterium of the genus Aerococcus belongs to at least one species selected from A. urinae, A. sanguinicola, A. christensenii, A. suis, A. urinaeequi, and A. urinaehominis.

[0043] According to a further preferred embodiment of the present invention, the bacterium of the genus Enterococcus is selected from the group consisting of Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus haemoperoxidus, Enterococcus hirae, Enterococcus malodoratus, Enterococcus moraviensis, Enterococcus mundtii, and Enterococcus pseudoavium. The bacteria belong to at least one species selected from Enterococcus pseudoavium, Enterococcus raffinosus, and Enterococcus solitarius.

[0044] According to a further preferred embodiment of the present invention, the yeast belongs to the genus Saccharomyces, more preferably to the species Saccharomyces cerevisiae and / or Saccharomyces boulardii.

[0045] The bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the further microorganisms are preferably live, in the latter case the resulting composition can also be defined as a probiotic.

[0046] Alternatively, the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the further microorganisms are dead or tyndallized.

[0047] In a further embodiment, said bacterial strain L. casei DG® and / or said bacterial strain Lactobacillus paracasei LPC-S01 and / or said further microorganism are in the form of a lysate and / or extract.

[0048] In the latter case, the resulting composition can also be defined as a paraprobiotic.

[0049] Alternatively, the bacterial strain L. casei DG (registered trademark) and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the further microorganisms may be present as individual components or some components may be present in the bacterial cell wall as exopolysaccharides.

[0050] In a further embodiment of the invention, the composition further comprises a metabolic bioproduct produced by said bacterial strain L. casei DG® and / or produced by said bacterial strain Lactobacillus paracasei LPC-S01 and / or produced by said further microorganism defined as a probiotic and / or produced by any other bacterial-derived product.

[0051] Thus, the compositions defined above may also be defined as probiotic or parabiotic or postbiotic, whether known or suspected, or are components of bacterial cell walls.

[0052] Generally, the microorganism further included in the composition of the present invention is a single microorganism or any combination of species identified in the EFSA QPS list.

[0053] The bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the further microorganisms are able to survive passage through the gastrointestinal tract and reach the colon alive.

[0054] The L. casei DG® and / or Lactobacillus paracasei LPC-S01 and / or the additional microorganisms, if present, are administered in an amount ranging from 1 to 300 billion cells (or even more if necessary), more preferably from 50 to 250 billion cells, even more preferably from 75 to 150 billion cells, preferably bacterial cells, per administration. In the case of application by enema and / or fecal transplant, the L. casei DG® and / or Lactobacillus paracasei LPC-S01 and / or the additional microorganisms, if present, are administered in an amount ranging from 100 to 300 billion cells, more preferably from 150 to 200 billion cells, preferably bacterial cells / probiotics / compositions, per administration / application.

[0055] According to a preferred embodiment, the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or, if present, the further microorganisms, preferably bacteria, are taken at least once or twice a day.

[0056] All routes of administration are contemplated in the present invention. The administration of the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the compositions described above is preferably oral, more preferably in the form of pills, capsules, tablets, granule powders, hard-shell capsules, orally dissolving granules, sachets, lozenges or drinkable vials.

[0057] Alternatively, the administration of the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the composition described above is in liquid form, preferably a syrup or drink.

[0058] Alternatively, the administration of the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the composition is carried out by adding them to food, preferably yogurt, cheese or fruit juice.

[0059] A further aspect of the present invention relates to a probiotic comprising a bacterium belonging to a genus selected from Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Aerococcus, Enterococcus and combinations thereof, or a composition comprising said probiotic, preferably for use in the prevention and / or treatment of physiopathological conditions related to / associated with Clostridium difficile infection, preferably intestinal infection or so-called Clostridium difficile associated disease (CDAD) or Clostridium difficile infection (CDI), wherein said probiotic or composition is formulated to act topically, preferably by rectal administration, preferably by enema, preferably by fecal microbiota transplantation.

[0060] The bacterium is preferably the bacterial strain L. casei DG® and / or the bacterial strain Lactobacillus paracasei LPC-S01 and / or the composition described above. The probiotic formulation or composition described above further comprises excipients generally acceptable for probiotic and / or pharmaceutical production.

[0061] According to a preferred embodiment of the present invention, the probiotic preparation or composition further comprises an anti-caking agent, preferably silicon dioxide and / or magnesium stearate.

[0062] According to a preferred embodiment of the present invention, the composition further comprises a coating agent, preferably gelatin.

[0063] In a further embodiment of the invention, the probiotic preparation or composition further comprises vitamins, trace elements, preferably zinc or selenium, enzymes and / or probiotic substances, preferably fructooligosaccharides (FOS), galactooligosaccharides (GOS), inulin, guar gum, or combinations thereof.

[0064] According to a preferred embodiment of the present invention, the probiotic preparation or composition may be associated or combined with further therapeutic approaches, preferably of the pharmacological or socio-behavioral type, preferably diet and / or a healthy lifestyle. [Example]

[0065] The pathogen used in the following examples was obtained from the ATCC American Type Culture Collection and was the following strain: Clostridium difficile ATCC 43255-VPI 10463.

[0066] The strains tested were: 1) L. casei DG (registered trademark) (Lactobacillus paracasei CNCM I-1572), 2) Lactobacillus paracasei LPC-S01 and 3) A combination (mixture) of L. casei DG® and L. paracasei LPC-S01 in a 1:1 ratio. 4) L. rhamnosus ATCC 53103 ("commercial" positive control)

[0067] Strains were cultured on Rogosa agar (L. casei and L. paracasei) and BHI Brain Heart Infusion (Clostridium difficile).

[0068] The aim was to verify the ability of Lactobacillus strains to exert inhibitory effects against CDIF.

[0069] Adhesion assay to verify the ability of exemplary probiotics to adhere to intestinal epithelial cells HT29-MTX-E12 (ECACC) was used as an example of a human intestinal epithelial cell line capable of producing mucus. Specifically, cells were cultured to form confluent monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with high glucose concentrations, 10% fetal bovine serum, 50 μg / ml gentamicin sulfate, and 2 mL of L-glutamine. Cells were maintained at 37°C in a 5% CO2 incubator.

[0070] Prior to the adhesion test, cells were washed with Hank's balanced salt solution, trypsinized, resuspended, and counted once using a hemocytometer so that they could be seeded into multiwell plates at known concentrations. Cells were allowed to grow for 2 days to form confluent monolayers before the adhesion test was performed.

[0071] On the day of the adhesion test, the spent medium was removed, the monolayer was washed with saline (Hank's Balanced Salt Solution), and DMEM medium without antibiotics or glutamine but supplemented with 1% fetal bovine serum was added to the cells. The cell monolayer was incubated at 37°C with 5% CO for 1 hour before contacting with bacteria. This procedure removes any residual antibiotics that may interfere with the test.

[0072] The cells were then "infected" to observe an MOI (multiplicity of infection) of 1:10 (cells:bacteria).

[0073] After 48 hours of confluence, cells were washed and incubated with DMEM medium containing exemplary bacterial strains (single or combined strains as described above) at a multiplicity of infection of 1:10 (MOI, epithelial cells:bacteria).

[0074] Incubation was carried out at 37°C for 60 minutes with slight shaking of the plate to favour cell contact.

[0075] At the end of the incubation, the culture medium was discarded, the cells were washed with sterile DMEM to remove non-adherent bacteria, and the adherent bacterial cells were quantified by viable count using serial dilution inoculation onto plates of agar MRS medium incubated at 37°C under anaerobiosis.

[0076] Adhesion Exclusion Test (Exclusion / Preincubation) After determining the number of HT29 cells per well, L. paracasei strains were added both individually and in combination (mixture) at a 1:1 ratio.

[0077] The bacteria were allowed to come into contact with the cells for 60 minutes, which were maintained at 37°C with slight shaking.

[0078] At the end of the incubation period, the medium was removed and the cells were washed with sterile medium to remove bacteria that had not adhered to the monolayer.

[0079] CDIF (MOI 1:10) was then added to the monolayer, and the cells were incubated at 37°C for 60 min.

[0080] At the end of the incubation period, the culture medium was removed and non-adherent bacteria were removed by washing with sterile medium.

[0081] To quantify CDIF adhesion to HT29 cells, cells were first released and then lysed, and bacteria were quantified by viable bacterial counts after inoculation onto Clostridium difficile Selective Agar under anaerobiosis at 37°C for 48–72 h.

[0082] HT29 cells not preincubated with any L. paracasei strain but inoculated with CDIF alone were used as a positive control, while cells not incubated with any bacteria were used as a negative control (sterility test).

[0083] Competition in adhesion test (competition / coincubation) After determining the number of HT29-MTX cells per well, the two L. paracasei strains (both individually and in combination at a 1:1 ratio) were added simultaneously with CDIF.

[0084] Bacteria were added at an overall MOI of 1:10 and a probiotic:pathogen ratio of 1:1. Bacteria were allowed to come into contact with the cells, which were maintained at 37°C with slight shaking, for 60 minutes.

[0085] At the end of the incubation period, the medium was removed and the cells were washed with sterile medium to remove bacteria that had not adhered to the monolayer.

[0086] To quantify CDIF adhesion to HT29 cells, cells were first released and then lysed, and bacteria were quantified by viable bacterial counts after inoculation onto Clostridium difficile Selective Agar under anaerobiosis at 37°C for 48–72 h.

[0087] HT29 cells inoculated with CDIF alone were used as a positive control, while cells not incubated with any bacteria were used as a negative control (sterility test).

[0088] Testing for removal of adherent pathogens (displacement / post-incubation) After determining the number of HT29-MTX cells per well, CDIF was added (MOI 1:10). The bacteria were allowed to come into contact with the cells for 60 minutes, which were maintained at 37°C with slight shaking.

[0089] At the end of the incubation, the medium was removed and bacteria that did not adhere to the monolayer were removed by repeated washing with sterile medium. Two L. paracasei strains (both individually and in a 1:1 ratio) were then added at a 10:1 ratio of probiotic to adherent pathogen.

[0090] The bacteria were allowed to come into contact with the cells, which were maintained with slight shaking at 37° C., for 60 minutes. At the end of the incubation period, the medium was removed and the cells were washed with sterile medium to remove bacteria that had not adhered to the monolayer.

[0091] To quantify CDIF adhesion to cells, cells were first released and then lysed, and bacteria were quantified by viable bacterial counts after inoculation onto Clostridium difficile Selective Agar under anaerobiosis at 37°C for 48–72 h.

[0092] HT29-MTX cells inoculated with CDIF alone were used as a positive control, while cells not incubated with any bacteria were used as a negative control (sterility test).

[0093] result The results are summarized in Figures 1A and 1B and demonstrate the following: 1) The L. casei DG® strain showed a mild ability to directly reduce CDIF adhesion by approximately 7% compared to conditions in which cells were incubated with the pathogen alone, while the L. paracasei LPC-S01 strain showed greater efficacy in competitively excluding CDIF (22% reduction in adhesion). A mixture of strains demonstrated enhanced inhibitory activity, resulting in a 31% reduction in CDIF adhesion (Figure 1A). 2) The L. casei DG® strain showed a moderate ability to reduce CDIF adhesion by approximately 27% compared to conditions in which cells were incubated with the pathogen alone (Figure 1B), while the L. paracasei LPC-S01 strain showed significant effectiveness in "removing" CDIF (75% reduction in adhesion).

[0094] Example 1 The exemplary strains were compared with commercially available products intended for the same purpose.

[0095] The pathogen used was the same strain as that used in the previous example.

[0096] The product Bio-K+ contains a mixture of Lactobacillus acidophilus CL1285, L. casei LBC80R and L. rhamnosus CLR2 at a published concentration of 50 billion CFU per 98g bottle, thus a published concentration of 5.0 x 10^10 CFU / 98g (5.1 x 10^8 CFU / g).

[0097] This product was subjected to decimal counting on selective media (ISTISAN method 08 / 36) to quantify the actual total lactobacillus concentration. The concentration of live and viable cells was 1.2 x 10^9 CFU / g, thus approximately twice the calculated theoretical concentration.

[0098] Adhesion Exclusion Test (Exclusion / Preincubation) After determining the number of HT29 cells per well, the products were added to the monolayer at a pathogen / probiotic MOI of 1:10. The bacteria were allowed to come into contact with the cells, which were maintained at 37°C with slight shaking, for 60 minutes. At the end of the incubation period, the medium was removed and the cells were washed with sterile medium to remove bacteria that had not adhered to the monolayer. CDIF was then added to the monolayer (MOI 1:10) and the cells were incubated at 37°C for 60 minutes.

[0099] At the end of the incubation period, the culture medium was removed, and nonadherent bacteria were removed by washing with sterile medium. To quantify CDIF adhesion to HT29 cells, cells were released and lysed, and bacteria were quantified by viable bacterial counts after inoculation onto Clostridium difficile Selective Agar under anaerobiosis at 37°C for 48–72 h.

[0100] Wells of HT29 cells not preincubated with the product but inoculated with CDIF alone were used as positive controls, while wells of cells not incubated with any bacteria were used as negative controls (sterility test).

[0101] Competition in adhesion test (competition / coincubation) After determining the number of HT29-MTX cells per well, the product was added simultaneously with CDIF. Bacteria were added at an overall MOI of 1:10. Bacteria were allowed to contact the cells, which were maintained at 37°C with slight shaking, for 60 minutes. At the end of the incubation, the medium was removed, and the cells were washed with sterile medium to remove bacteria that had not adhered to the monolayer. To quantify CDIF adhesion to cells, the cells were released and lysed, and the bacteria were quantified by viable bacterial count after inoculation onto Clostridium difficile Selective Agar at 37°C under anaerobiosis for 48-72 hours. Wells of HT29 cells inoculated with CDIF alone served as a positive control, while wells of cells not incubated with any bacteria served as a negative control (sterility test).

[0102] Testing for removal of adherent pathogens (displacement / post-incubation) After determining the number of HT29-MTX cells per well, CDIF was added (MOI 1:10). The bacteria were allowed to come into contact with the cells for 60 minutes, which were maintained at 37°C with slight shaking.

[0103] At the end of the incubation, the medium was removed, and bacteria that did not adhere to the monolayer were removed by repeated washing with sterile medium. The product was then added at a 10:1 bacteria / cell ratio as usual. The bacteria were allowed to come into contact with the cells, which were maintained at 37°C with slight shaking, for 60 minutes. At the end of the incubation, the medium was removed, and the cells were washed with sterile medium to remove bacteria that did not adhere to the monolayer. To quantify CDIF adhesion to cells, the cells were released and lysed, and the bacteria were quantified by viable bacterial count after inoculation onto Clostridium difficile Selective Agar at 37°C under anaerobiosis for 48-72 hours. Wells of HT29-MTX cells inoculated with CDIF alone served as a positive control, while wells of cells not incubated with any bacteria served as a negative control (sterility test).

[0104] result The results shown in Figure 2A consider CDIF adhesion as the percentage of viable and living cells adhering after pre-stimulation of HT29-MTX cells with the tested probiotic products. In other words, the CDIF adhesion in the absence of probiotic stimulation was assigned a value of 100, and the inhibitory potential of the test strains was expressed as the % reduction in CDIF adhesion compared to the positive control.

[0105] As shown, the test product showed the ability to enhance the inhibitory capacity, thus resulting in a very high reduction, equal to 90%, in the adhesion of CDIF.

[0106] Furthermore, after simultaneous treatment of cell lines with probiotics and pathogens, a possible reduction on CDIF adhesion to HT29-MTX cells was examined to assess the effect, if any, of competitive inhibition exerted by probiotics when co-incubated with CDIF.

[0107] Wells of HT29 cells inoculated with CDIF alone served as positive controls, while wells of cells not incubated with any bacteria served as negative controls (sterility test).

[0108] The results shown in Figure 2B consider CDIF adhesion as the percentage of adherent viable cells and viable cells after co-incubation of HT29-MTX cells with CDIF and the different probiotics tested. As before, CDIF adhesion in the absence of probiotic stimulation was assigned a value of 100, and the inhibitory potential of the test strains was expressed as the % reduction in CDIF adhesion compared to the positive control.

[0109] As shown, the test product demonstrated the ability to enhance the inhibitory capacity, thus resulting in a 30% reduction in the adhesion of CDIF.

[0110] Finally, we examined whether there was a reduction in CDIF adhesion to HT29-MTX cells after pretreatment of the cell line with pathogens followed by incubation with probiotics after removal of non-adherent pathogens.

[0111] Wells of HT29 cells inoculated with CDIF alone served as positive controls, while wells of cells not incubated with any bacteria served as negative controls (sterility test).

[0112] The results shown in Figure 2C consider CDIF adhesion as the percentage of adherent viable cells and viable cells after incubating HT29 cells with the various probiotics tested after loading eukaryotic cells with CDIF. As before, CDIF adhesion in the absence of probiotics was assigned a value of 100, and the inhibitory potential of the test strains was expressed as the % reduction in CDIF adhesion compared to the positive control.

[0113] As shown, the product exhibited very mild inhibitory potential, resulting in an 8% reduction in CDIF adhesion.

[0114] The table below summarizes the range of CDIF counts obtained in two consecutive tests, compared to an initial pathogen load of approximately 1.0 x 10^6 CFU, for the three different protocols tested.

[0115] [Table 1]

[0116] Comparing the data obtained in this study with data on the L. casei DG® strain, the L. paracasei LPC-S01 strain and a mixture of the two strains that were the subject of previous investigations, it was possible to derive the profile shown below.

[0117] Adhesion exclusion test (eukaryotic cells are pretreated with a probiotic and then incubated with a pathogen)

[0118] [Table 2]

[0119] Competition in adhesion assays (co-incubation of eukaryotic cells with probiotics and with pathogens)

[0120] [Table 3]

[0121] Adherent pathogen removal test (eukaryotic cells are pretreated with pathogens and then incubated with probiotics)

[0122] [Table 4]

[0123] The overall results shown by the three tablets demonstrate the following: 1) L. paracasei strain LPC-S01 was the most effective, demonstrating a very high inhibitory capacity (75%) against CDIF during the displacement test. In other words, unlike the commercial products tested, L. paracasei strain LPC-S01 acts after adhering to the pathogen and eliminates it, whereas the commercial products only perform a preventative function, preventing pathogen CDIF from adhering to cells. In this case, the L. paracasei strain LPC-S01 tested works even when the pathogen CDIF is already adhesive. 2) The 1:1 combination of L. casei DG® + L. paracasei LPC-S01 showed an interesting synergistic effect in terms of reducing the adhesive capacity of CDIF to cell lines. This combination proved advantageous compared to the individual strains in pretreatment and co-incubation protocols, while in substitution studies, the L. paracasei LPC-S01 strain proved more effective than the combination of the two probiotics. [Receipt of Trust] [Table 5-1] [Table 5-2] [Table 5-3] Table 5-4 Table 5-5 Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17

Claims

1. 1. A composition comprising as an active ingredient the bacterial strain Lactobacillus paracasei LPC-S01 deposited under accession number DSM 26760 for use in the prevention and / or treatment of a physiopathological condition caused by a Clostridium difficile infection and / or symptomatology associated with this physiopathological condition and / or histological lesions caused by a Clostridium difficile infection, the physiopathological condition caused by the Clostridium difficile infection is selected from pseudomembranous enterocolitis, pseudomembranous colitis, pseudomembranous colitis without pseudomembranes, fulminant colitis, colitis with toxic megacolon and intestinal perforation, antibiotic-associated colitis, antibiotic-associated diarrhea, and diarrhea associated with C. difficile infection; the symptomatology is selected from diarrhea, dehydration, pseudomembranous colitis, enteropathy, toxic megacolon, intestinal perforation, sepsis, intestinal bleeding, and hypokalemia; The composition, wherein the histological lesion is a type I morphology characterized by isolated epithelial necrosis associated with inflammatory infiltrates within the colonic lumen and / or a type III morphology characterized by diffuse epithelial necrosis and ulceration covered by a grayish pseudomembrane.

2. 2. The composition of claim 1, wherein the Clostridium difficile infection is an intestinal infection.

3. A composition described in claim 1 or 2, wherein the Lactobacillus paracasei LPC-S01 deposited under accession number DSM26760 is administered in an amount of 1 to 300 billion cells per intake.

4. 4. The composition of any one of claims 1 to 3, taken at least once or twice daily.

5. 5. The composition of any one of claims 1 to 4, formulated for oral administration.

6. 5. The composition of any one of claims 1 to 4, formulated for topical action.

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