Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Lacticaseibacillus rhamnosus DSM 33960 and its use in a method for the prevention and treatment of urogenital tract dysbiosis
• Lacticaseibacillus rhamnosus DSM 33960 bacterial strain and its use in a method for the prevention and treatment of conditions of dysbiosis of the urogenital tract, particularly the female urogenital tract, preferably vaginal dysbiosis.
• the vaginal microbiota although characterized by a few microbial species, is a dynamic ecosystem subject to change throughout a woman's life, from birth to pre- and post-menopausal phases.
• intrinsic factors such as race, immune system imbalance, genetic susceptibility, etc.
• extrinsic factors such as diet rich in vitamins and folic acid, use of oral contraceptives, sexual behaviors, hygiene habits, use of antibiotics or immunosuppressive therapies
• vaginal dysbiosis Alterations in the vaginal microbiota are associated with many diseases such as, for example, urogenital tract infections (e.g., by Candida) reproductive dysfunction, obstetric complications, and cancer.
• urogenital tract infections e.g., by Candida
• vaginal dysbiosis is treated with antimicrobial drugs (such as metronidazole and clindamycin).
• antimicrobial drugs have several undesirable effects, such as short-term recurrence and promotion of antibiotic resistance.
• alternative therapeutic strategies have been studied that can modulate the vaginal microbiota and correct its imbalances. In this context, the administration of probiotics, either alone or to complement antibiotic therapy, has gained increasing attention.
• probiotics currently known in the art frequently present host specificity problems and, consequently, poor efficacy in counteracting vaginal dysbiosis and related diseases.
• the probiotics currently known in the art are often found to be insufficiently safe and, therefore, unsuitable for use in medical field, for example, in the prevention or treatment of vaginal dysbiosis.
• probiotics currently known in the art are recognized as generally safe, both safety and probiotic characteristics are specific to each strain and cannot be considered as representative characteristics of the entire species to which the strain belongs.
• DSM 33500 for use in women with vagina! dysbiosis.
• bacterial strains particularly probiotic strains, that can prevent and/or counteract vaginal dysbiosis.
• probiotics capable of acting at the level of the vaginal microbiota in order to prevent and/or counteract imbalances in this microbiota, and related pathologies such as, for example, infections of the urogenital tract.
• Another purpose of the present invention is to make available an isolated bacterial strain for use in a method for the prevention and treatment of urogenital tract dysbiosis, preferably vaginal dysbiosis.
• Still purpose of the present invention is to make available an isolated bacterial strain that is effective in preventing and/or treating diseases or conditions related to dysbiosis of the urogenital tract, for example, infections of the urogenital tract , particularly vaginal infections.
• Further purpose of the present invention is to make available a probiotic bacterial strain that can be used, for example, in rebalancing the vaginal microbiota, effectively and safely, and that is host-specific.
• the object of the present invention which provides an isolated bacterial strain suitable for use in the prevention and/or treatment of dysbiosis of the urogenital tract, particularly vaginal dysbiosis.
• Lacticaseibacillus rhamnosus CA15 deposited by ProBioEtna Sri on July 28, 2021, at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), under deposit number DSM 33960.
• the microorganism (strain) according to the present invention i.e., Lacticaseibacillus rhamnosus CA15 DSM 33960 (also referred to in this description as “Lacticaseibacillus rhamnosus DSM 33960" or, more simply, “DSM 33960”) was isolated from the vaginal ecosystem of a healthy and asymptomatic Italian woman afferent to the Department of Obstetrics and Gynecology of the A.O.U. of the Policlinico - Vittorio Emanuele P.O. G. Rodolico of the University of Catania. To isolate the strain of the invention, vaginal exudate samples were processed as previously reported (Pino et al., 2021; 2019).
• Vaginal exudate taken using appropriate sterile pads provided with transport medium, was analyzed as follows. After removing the cells, employing sterile phosphate-buffered saline (PBS), serial dilutions were made and plated in de Man Rogosa and Sharp growth medium (MRS, Oxoid, Milan, Italy). The plates were incubated at 35-37°C for 40-48 h. In the context of the present invention "h” may mean hour or hours, intended as time, depending on the context.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain has a bacillary morphology and is Gram positive.
• the microorganism of the invention is catalase negative, that is, it does not have the enzyme catalase, which can hydrolyze hydrogen peroxide.
• DSM 33960 strain is, in addition, unable to produce spores and does not exhibit motility.
• the strain of the invention can be used as a medicament. Therefore, an object of the present invention is Lacticaseibacillus rhamnosus DSM 33960 strain for use as a medicament.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain could be observed toward a broad spectrum of potentially pathogenic bacteria and yeasts such as, for example, Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Candida tropicalis, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas monteilii, and Streptococcus agalactiae .
• Gardnerella vaginalis Escherichia coli
• Staphylococcus aureus Candida albicans
• Candida krusei Candida glabrata
• Candida parapsilosis Candida tropicalis
• Listeria monocytogenes Pseudomonas aeruginosa
• Pseudomonas monteilii Pseudomonas monteili
• the Applicant was able to observe additional advantageous features of the strain of the invention such as, for example, high anti-inflammatory and antioxidant activity, the ability to adhere to the intestinal mucosa, the ability to produce exopolysaccharides (EPS), the ability to produce biofilms, and the ability to antagonize biofilm formation by different Candida species as well as to inhibit the adhesion of a variety of pathogens and counteract the development of a variety of adherent pathogens.
• high anti-inflammatory and antioxidant activity the ability to adhere to the intestinal mucosa
• EPS exopolysaccharides
• biofilms the ability to produce biofilms
• antagonize biofilm formation by different Candida species as well as to inhibit the adhesion of a variety of pathogens and counteract the development of a variety of adherent pathogens.
• the strain of the invention particularly suitable for use in the prevention and/or treatment of dysbiosis of the microbiota of the urogenital tract, particularly of the female urogenital tract, preferably of the dysbiosis of the vaginal microbiota .
• the strain of the invention can be, advantageously, used in the prevention and/or treatment of alterations in the microbiota of the urogenital tract, preferably the vaginal microbiota.
• the Lacticaseibacillus rhamnosus CA15 DSM 33960 strain for use in the prevention and/or treatment of dysbiosis of the microbiota of the urogenital tract, preferably vaginal dysbiosis.
• the term “dysbiosis” refers to a condition of altered homeostasis of the microbiota associated with a specific microbial ecosystem of the human body.
• the terms “urogenital tract dysbiosis” and “vaginal dysbiosis” refer to an altered condition of the microbiota of the urogenital tract and vaginal microbiota, respectively, compared with their normal physiological conditions.
• the Lacticaseibacillus rhamnosus CA15 DSM 33960 strain for use in a method for the prevention and/or treatment of diseases related to dysbiosis of the urogenital tract microbiota, particularly the female urogenital tract microbiota, preferably the vaginal microbiota .
• vaginal infections examples include vaginal infections, vaginal inflammation, bacterial vaginosis (BV) and recurrences thereof, vulvovaginal candidiasis (WC), and sexually transmitted bacterial infections carried, for example, by Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.
• BV bacterial vaginosis
• WC vulvovaginal candidiasis
• sexually transmitted bacterial infections carried, for example, by Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.
• the Lacticaseibacillus rhamnosus CA15 DSM 33960 strain can be used for the prevention and/or treatment of urogenital tract infections, particularly female urogenital tract infections, preferably vaginal infections.
• the urogenital tract infections, particularly vaginal infections can be selected from infections due to one or more microorganisms such as Gardnerella vaginalis, Escherichia coli, Staphylococcus aureus, Candida spp., Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas monteilii, and Streptococcus agalactiae and combinations thereof.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain can be used for the prevention and/or treatment of urogenital tract infections, urogenital tract inflammation, bacterial vaginosis (BV) and recurrences thereof, vulvovaginal candidiasis (WC), mixed dysbiosis, and group B streptococcal infections.
• BV bacterial vaginosis
• WC vulvovaginal candidiasis
• mixed dysbiosis and group B streptococcal infections.
• the strain of the invention can be used to prevent and/or treat bacterial vaginosis (BV) and recurrences thereof, and/or vulvovaginal candidiasis (WC) , group B streptococci (GBS) infections, urinary tract infections caused by Pseudomonas aeruginosa and Escherichia coli.
• BV bacterial vaginosis
• WC vulvovaginal candidiasis
• GBS group B streptococci
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain can be, advantageously, used in the prevention and/or treatment of inflammation of the urogenital tract, particularly inflammation of the female urogenital tract, preferably vaginal inflammation, e.g., inflammation due to Gardnerella vaginalis, Escherichia coli, Candida spp., Pseudomonas aeruginosa, and Streptococcus agalactiae.
• vaginal inflammation e.g., inflammation due to Gardnerella vaginalis, Escherichia coli, Candida spp., Pseudomonas aeruginosa, and Streptococcus agalactiae.
• the strain of the invention can be administered orally (i.e., gastroenterally) and topically.
• the DSM 33960 strain of the invention is administered orally.
• the strain of the invention exhibits a high ability to survive stressful conditions in the gastrointestinal (Gl) tract.
• the strain of the invention exhibits high resistance to lysozyme (present, for example, in saliva), bile salts, gastric juice and acidic pH.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain can be administered as powders or granules, such as contained in a tablet, or as a swallowable capsule.
• the strain of the invention can be administered in dried or freeze-dried form.
• L. rhamnosus DSM 33960 strain does not show resistance to the antibiotics suggested by EFSA (European Food Safety Authority) , while resistance has been detected to metronidazole, clotrimazole, and boric acid, antimicrobials routinely used for the treatment of urogenital tract infections, particularly vaginal infections such as, for example, bacterial vaginosis (BV) and vulvovaginal candidiasis (WC).
• EFSA European Food Safety Authority
• L. rhamnosus DSM 33960 strain also showed resistance to fluconazole (an antifungal used to treat fungal infections that can also proliferate vagi nally).
• DSM 33960 strain can be advantageously used as an adjuvant to antimicrobial treatment, particularly antibiotic/antimycotic treatment, of urogenital tract infections, particularly female urogenital tract infections, preferably vaginal infections.
• adjuvant refers to the ability to enhance or complement the action of treatments that are the therapy of choice.
• the strain of the invention can be administered in combination with one or more antimicrobial agents.
• the strain of the invention can be administered in combination with one or more antimicrobial agents selected from metronidazole, clotrimazole, boric acid, fluconazole and mixtures thereof.
• the phrase "administered in combination with one or more antimicrobial agents” refers to the combined use of the strain of the invention with one or more antimicrobial agents, particularly for preventive and/or therapeutic purposes.
• the strain of the invention can be administered together with the antimicrobial agents or separately to the antimicrobial agents.
• the strain of the invention can be administered before or after or concurrently with the administration of antimicrobial agents.
• the strain of the invention has several advantages over probiotic microorganisms known in the art.
• the strain of the present invention can be used for the prevention and/or treatment of dysbiosis of the urogenital tract, particularly the female urogenital tract, preferably vaginal dysbiosis, effectively and safely for the host.
• the strain of the invention produces both H2O2 and lactic acid. This characteristic makes it particularly effective as an antagonist against many pathogenic microorganisms of the urogenital tract, particularly vaginal pathogens.
• DSM 33960 strain showed the ability to adhere to Caco-2 (intestinal epithelium model cells) HT29 (grade II colon adenocarcinoma cells) and VK2/E6E7 (vaginal epithelial cell lines) cell lines.
• the strain of the invention showed hydrophobicity, auto-aggregation ability and ability to co-aggregate with pathogens.
• microbial strains with probiotic potential should be able to adhere to and colonize the gastro-intestinal tract, interacting with resident bacteria.
• Several mechanisms are involved in the process of bacterial adhesion, which depends on intra- and extra-cellular properties.
• Autoaggregation intrinsic to specific microbial strains, is known to increase the potential for colonization while hydrophobicity, related to the presence of hydrophobic molecules on the cell surface of the strain, amplifies its ability to adhere to the host intestinal mucosa.
• the ability to co-aggregate with pathogens is considered a hallmark of specific probiotic strains, as it promotes the formation of a physical-chemical barrier that prevents colonization by pathogenic microorganisms.
• DSM 33960 strain produces exopolysaccharides (EPS) associated with a variety of health-promoting functions.
• EPS exopolysaccharides
• L. rhamnosus DSM 33960 strain exhibits important antioxidant and anti-inflammatory capacities to prevent tissue damage due to oxidative stress, and the development of related diseases, and to stimulate the immune system response in an anti-inflammatory manner as a response to pro-inflammatory stimuli.
• DSM 33960 strain also showed a high survival rate at low pH, in the presence of lysozyme, bile salts and during in vitro simulated gastrointestinal digestion. These characteristics make the strain of the invention particularly suitable for oral administration.
• Figure 1 Genetic fingerprint via PFGE ("Pulsed-field gel electrophoresis"). PFGE profiles of genomic DNA digested with Asci restriction enzyme of Lacticaseibacillus rhamnsous CA15 DSM 33960 strain. M: Lambda PFG marker (NEB, US).
• Figure 2 Results of an ABTS assay, designed to measure the antioxidant activity of Lacticaseibacillus rhamnsous CA15 DSM 33960 strain, using 2,2-azino di-(3-ethylbenzthiazoline sulfonate) (ABTS) substrate. Results are expressed as absorbance values at 500nm.
• Figure 3 Adhesion of Lacticasei bacillus rhamnsous CA15 DSM 33960 and Lacticaseibacillus rhamnsous GG strains to human intestinal mucus glycoproteins. The results are expressed as mean percent adhesion and standard deviation of three replicates each consisting of six technical replicates.
• Example 1 Isolation of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain, phenotypic and genotypic characterization
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was isolated from the vaginal ecosystem of a healthy patient, afferent at the Gynecologic Obstetric Clinic of the A.O.U. of Policlinico - Vittorio Emanuele P.O. G. Rodolico of the University of Catania, employing De Man, Rogosa and Sharpe (MRS) growth medium.
• the strain was stored, in pure culture, at -80 °C using a 20% glycerol (v/v) solution.
• the strain was subjected to genotypic characterization by: PCR species-specific tuf-gene , species-level characterization by 16S rDNA gene sequencing, and strain-level typing by Pulsed Field Gel Electrophoresis (PFGE) analysis, according to the requirements of the FAO/WHO 2002 guidelines.
• PFGE Pulsed Field Gel Electrophoresis
• the genomic DNA was subsequently amplified by conventional PCR using P1-P4 primers targeting the V1-V3 regions of the 16S rDNA gene.
• the resulting amplicon was sequenced and the species was identified through the BLAST database "Nucleotide collection (nt/nt)" (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Identification was considered unambiguous at >99% sequence homology.
• Strain-level typing was performed using pulsed field electrophoresis (PFGE).
• Plugs preparation, DNA extraction, and enzymatic restriction were performed using the CHEF Bacterial Genomic DNA Plug Kits (Bio-Rad, UK) according to the manufacturer's protocol indicated for Gram-positive bacteria. After digestion with Asci restriction enzyme at 37°C for 18 h, electrophoresis was performed using CHEF DR II (Bio- Rad, Milan, Italy) in 1 % Megabase agarose (Bio-Rad, Milan, Italy) in 0.5X TBE buffer. The gels were then stained with 1X Atlas ClearSight DNA Stain (BIOATLAS OU, Estonia) for one hour and visualized under UV light. The results of the PFGE analysis are shown in Figure 1 .
• Lacticaseibacillus rhamnsous CA15 DSM 33960 strain was revitalized in MRS broth medium and incubated at 37°C for 16-18 hours.
• the cell culture in exponential growth phase was transferred by smear to Blood Agar plates containing 5% defibrinated mutton blood (Biolife, Milan, Italy) and incubated at 37°C for 24-48 hours.
• Hemolytic activity was determined visually and distinguished as p-hemolysis, o-hemolysis, or y-hemolysis based on the presence of clear zones, green halos, or no zones around the colonies, respectively.
• the p-hemolytic strain Streptococcus pyogenes ATCC 19615 and the o-hemolytic strain Streptococcus pneumoniae ATCC 6303 were used as positive controls.
• the results, shown in Table 2, were expressed as "+” (presence of hemolytic activity) and "-" (absence of hemolytic activity).
• the tested strain showed no hemolytic activity.
• DNAse production was tested by transferring 5 pl of cell culture in exponential growth phase onto DNAse agar plates (Oxoid). After incubation at 37 °C for 48 h, the plates were treated with HC1 1 N for 5 min. The presence of clear areas around the colonies is considered as an indicator of DNAse production positivity.
• Gelatinase production was evaluated using gelatin agar plates (30 g/L gelatin, 5 g/L peptone, 3 g/L yeast extract, and 17 g/L agar). After incubation at 37 °C for 48 h, the surface of the plates was treated with saturated ammonium sulfate (Merck, Milan). The presence of clear areas around the colonies is considered as an indicator of positive gelatinase activity.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain to degrade mucin was evaluated using a suitable growth medium modified by the addition of 0.5% (w/v) of porcine stomach type III (Sigma-Aldrich, St. Louis, MO, USA).
• the cell culture in exponential growth phase was transferred into the aforementioned growth medium, and the plates were incubated at 37 °C for 72 h. Next, the plates were stained with 0.1% (w/v) starch black (Merck, Milan) in acetic acid 3.5 M for 30 min, then washed with acetic acid 1.2 M (Merck, Milan). The presence of halos around the colonies was considered as a positive result.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was reactivated by 2% inoculation in de Man Rogosa and Sharp broth medium (MRS, Oxoid, Milan, Italy) and incubated at 37°C overnight.
• the resulting biomass was subcultured five times in liquid medium containing 0.005% pyridoxal-5'-phosphate (Sigma Aldrich St. Louis, MO, USA) and biogenic amine production was stimulated by adding 0.1% histidine, 0.1% lysine, 0.1% tyrosine or 0.1% ornithine (Sigma Aldrich, St. Louis, MO, USA).
• Biogenic amine extraction and estimation protocols were performed as follows.
• the extracted and derivatized samples were separated by HPLC chromatography (Shimadzu Nexera XL UHPLC system equipped with an SPD M30A diode array detector) using a Discovery HS C18 250 mm x 46 mm x 5 pm column (Sigma Aldrich, St. Louis, MO, USA) applying a low pressure gradient of acetonitrile and water. Separation temperature was kept constant at 26 °C to allow better reproducibility of results. Six-point standard curves were generated and considered suitable if they had a correlation value R 2 >0.99. The samples were analyzed in duplicate, and the results are shown in Table 5 and expressed as mean and standard deviation.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in order to determine its resistance or susceptibility to certain antibiotics, in accordance with the European Food Safety Authority (EFSA) report, by minimum inhibitory concentration (MIC) assessment using EFSA's (2016) suggested cut-offs as a reference. For each antibiotic tested, the strain was classified as resistant (R) or susceptible (S). In addition to the antibiotics suggested by EFSA, the strain was tested for resistance/susceptibility to some antimicrobial agents used in common clinical practice (metronidazole, clotrimazole, and boric acid).
• EFSA European Food Safety Authority
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain is susceptible to all antibiotics suggested by EFSA.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed resistance against metronidazole, clotrimazole and boric acid. The strain also did not show genome-wide antibiotic resistance genes.
• the MIC and breakpoint values are shown.
• L. rhamnosus DSM 33960 strain In a survival test during gastrointestinal transit using the dynamic SHIME system, L. rhamnosus DSM 33960 strain also showed resistance to fluconazole (an antifungal used to treat fungal infections that can also proliferate vaginally). See, for more details, section 3.17-C below.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested for antagonistic activity against the potential pathogens Enterobacter cloaceae DSM 30054, Enterococcus faecalis DSM 2570, Escherichia coli ATCC 25922, Escherichia coli ATCC 700414, Escherichia coli DSM 105393, Candida albicans ATCC 10231, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida parapsilosis ATCC 90018, Candida tropicalis ATCC 13803, Gardnerella vaginalis ATCC 14019, Gardnerella vaginalis ATCC 14018, Listeria monocytogenes DSM 12464, Proteus mirabilis DSM 30116, Pseudomonas aeruginosa DSM 1117, Pseudomonas aeruginosa DSM 3227, Pseudomonas monteilii ATCC 700476,
• Antimicrobial activity was evaluated by employing both cells and metabolites produced during cell growth. The test with cells was performed by agar spot test. After incubation, antimicrobial activity was evaluated by measurement of the halo of inhibition obtained. The results, shown in Table 8, were expressed as (no activity); "+” (halo of inhibition ⁇ 10 mm in diameter); “++” (halo of inhibition between 11 and 20 mm in diameter); “+++” (halo of inhibition >20 mm in diameter).
• CFS Cell-free supernatant
• the strain under study showed a broad spectrum of antimicrobial activity. Based on the treatments, to which CFS was subjected, the antagonistic activity was, in all cases, attributed to the production of organic acids.
• Candida albicans ATCC 10231 Candida krusei ATCC 14243, Gardnerella vaginalis ATCC 14018, and Streptococcus agalactiae DSM 2134 (zone of inhibition larger than 20 mm). Zones of inhibition between 11 and 20 mm in diameter were observed against Candida glabrata ATCC 90030, Candida tropicalis ATCC 13803, Gardnerella vaginalis ATCC 14019, Listeria monocytogenes DSM 12464, Pseudomonas monteilii ATCC 700476, and Staphylococcus aureus ATCC 6538.
• Zones of inhibition having a diameter of less than 10 mm were found against: Escherichia coli ATCC 700414, Candida parapsilosis ATCC 90018, Pseudomonas aeruginosa DSM 1117, and Pseudomonas aeruginosa DSM 3227. 3.2. Production of hydrogen peroxide
• H2O2 The ability to produce hydrogen peroxide (H2O2) was evaluated by growing the strain, previously revitalized in broth medium, on MRS agar containing 0.25 mg/mL of 3,3',5,5'-tetramethylbenzidine and 0.01 mg/mL of horseradish peroxidase. After incubation for 72 h, the plates were exposed to air, and the production of H 2 O 2 was evaluated by the appearance of blue staining. The ability to produce hydrogen peroxide (H 2 O 2 ) was evaluated onthe basis of the time required for the appearance of blue staining as follows: absent (score 0, no production of blue staining); low (score 1, time > 20 min), medium (score 2, time 10-20 min) and high (score 3, time ⁇ 10 min). The results are shown in Table 9.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed ability to produce H2O2.
• the strain was classified as a high producer of H2O2 (score 3) because the time required for the appearance of blue staining was less than 10 minutes.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was quantitatively evaluated by HPLC chromatography following the protocol suggested by Chenoll et al. The test was performed in triplicate, and the results, expressed as mean (mmol/L) and standard deviation, are shown in Table 10.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed ability to produce acetic acid, butyric acid, lactic acid, propionic acid, and succinic acid in high amounts.
• U937 cell lines (human pro-monocytic cell line), were used as an in vitro model of inflammation.
• U937 cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L D-glucose (Gibco, Life Technologies, Milan, Italy) supplemented with 10% v/v bovine serum (FBS) (Invitrogen, Carlsbad, California, USA), 1% penicillin/streptomycin (Carlo Erba, Milan, Italy) and 60 mg/mL gentamicin (Gibco).
• DMEM Dulbecco's modified Eagle's medium
• FBS v/v bovine serum
• penicillin/streptomycin Carlo Erba, Milan, Italy
• 60 mg/mL gentamicin Gibco
• the cells were pretreated with lipopolysaccharide (LPS) at a concentration of 100 ng/mL for 2 h.
• LPS lipopolysaccharide
• the anti-inflammatory effect of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was evaluated by treating differentiated cells with the strain under study at a concentration of 10 pg/mL for 6 hours.
• cells were washed with PBS, harvested by trypsinization and then lysed for RNA extraction. Quantification of COX-1 (Cyclooxygenase-1), COX-2, IL-8 and IL-10 genes was performed by real-time qRT-PCR. Untreated cells were used as controls.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested for antioxidant activity by ABTS assay using the substrate 2,2-azino di-(3-ethylbenzthiazoline sulfonate).
• a standard curve was obtained using Trolox at different concentrations (20-1000 M). The results were expressed as molar concentration (M) of Trolox equivalent per liter (mol TE/L).
• antioxidant activity was estimated by testing the oxidation of oleic acid. In detail, 1 mL of cell-free supernatant was added to 1 mL of PBS (0.1 M, pH 7.0) and 1 mL of linoleic acid (50 mM) in ethanol (99.5%).
• Oxidation was measured by ferric thiocyanate determination. Butylated hydroxytoluene (BHT) and o-tocopherol (1 mg/mL) were used as positive controls. Uninoculated MRS broth without antioxidants was used as a negative control. Analyses were performed in triplicate.
• the tested strain showed antioxidant activity, counteracting linoleic acid peroxidation.
• the antioxidant activity of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was similar (P ⁇ 0.05) to that found for o-tocopherol.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested using intestinal cell lines, Caco-2 (ATCC HTB-37) and HT29 (Caucasian colon adenocarcinoma grade II, Merck, Milan, Italy) and human vaginal epithelial cell lines (VK2/E6E7 ATCC-CRL-2616). Each test was repeated three times in three separate experiments, and the results, shown in Table 12, are expressed as mean percent adhesion (%)and standard deviation. Adhesion ability was expressed as a percentage and was calculated by comparing the number of viable cells tested (8 log ufc/ml) and the number of adherent cells in the cell lines.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain demonstrates adhesion ability to all cell lines tested.
• the strain under study was labeled by addition to the growth medium (MRS broth) of 10 pil/ml tritiated thymidine (5-3Htymidine, 120 Ci mmol21; Amersham Biosciences, Little Chalfont, UK). After incubation at 37 °C for 18 h, cells were washed with PBS and standardized to an OD 6 oo of 0.25 ⁇ 0.05 corresponding to a cell number of about 8 log ufc/ml.
• MRS broth 10 pil/ml tritiated thymidine
• a solution of intestinal mucus was prepared by dissolving mucin (Mucin from porcine stomach type III, Merck, Milan, Italy) in NaOH 1M and diluting the solution in 1X PBS at a ratio of 1 :20.
• 150 l of the intestinal mucus solution was immobilized in wells of polystyrene microplates (Maxisorp, Nunc, Denmark) by overnight incubation at 4 °C. The plates were washed three times with PBS. 100 pl of the radiolabeled and standardized bacterial suspension was transferred to the wells and incubated for 1 h at 37 °C. Subsequently, the wells were washed three times with 200 pl of PBS to remove nonadherent cells.
• the adherent cells were released and lysed with 1% (w/v) sodium dodecyl sulfate (SDS) in NaOH (0.1 mol/liter) (200 pl per well) by incubation at 37°C overnight.
• SDS sodium dodecyl sulfate
• the contents of the wells were transferred to microcentrifuge tubes containing scintillation liquid (OptiPhase "HiSafe 3", Wallac, Turku, Finland), and radioactivity was measured by liquid scintillation.
• Adhesion was expressed as the percentage of radioactivity recovered after adhesion with respect to to the radioactivity of the bacterial suspension added to the immobilized mucus. Adhesion was determined in three independent experiments, and each test was performed in six replicates to calculate intra-test variation.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was used as the reference strain.
• adhesion percentage of 11.99 ⁇ 0.27 and 11.61 ⁇ 0.76 was obtained for CA15 and LGG strains, respectively.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested for the ability to produce biofilm according to the following.
• 96-well polystyrene microplates were used. Cell culture standardized to 9 log ufc/ml was inoculated in 5% (v/v) MRS broth and in MRS broth supplemented with TWEEN 80 (1% v/v) and incubated at 37°C for 24 and 72 hours. After incubation, the wells were washed three times with PBS (pH 7.0) and treated with 200 pl of crystal violet (0.1% w/v), prepared in isopropanol-methanol-PBS solution (1:1 :18 v/v), for 30 min.
• Biofilm production ability was reported as follows: no biofilm production (OD ⁇ ODc); weak biofilm production (ODc ⁇ OD ⁇ 2xODc); medium biofilm production (2x ODc ⁇ OD ⁇ 4xODc); high biofilm production (4xODc ⁇ OD ⁇ 8xODc); very high biofilm production (8xODc ⁇ OD). The test was carried out in triplicate and the results are reported in Table 13.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed high ability to produce biofilm in MRS after 24 and 72 h incubation. The ability to produce biofilm was found to be very high after growth for 72 hours in MRS with Tween 80.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was evaluated, using 96-well polystyrene microplates, by "pre-coating” and "co-incubation” methods.
• CFS cell-free supernatant
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was obtained by employing a standardized cell culture at 9 log ufc/ml subjected to centrifugation and filtration of the supernatant through 0.22 m filters.
• CFS 200 pL was transferred to wells of polystyrene microplates and incubated at 37 °C for 24 h. After incubation, CFS was removed and the wells were washed twice with 100 pL of PBS (pH 7.2). The cell suspension of each Candida species tested (150 pL), standardized to 7 log ufc/ml , was added, in separate wells, and incubated at 37°C for 24 hours. After incubation, the wells were washed gently with PBS (pH 7.2) then 99% methanol (100 pL) was added to fix the biofilm. After 15 minutes, the residual methanol was removed by washing then 2% crystal violet (100 pL) was added.
• Co-incubation 100 pL of CFS of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain and 150 pL of cell suspension of each Candida species tested, standardized to 7 log ufc/ml , were transferred to 96-well polystyrene microplates and incubated at 37 °C for 24 h. The washing and staining steps were performed as reported for the pre-coating test.
• Candida spp. suspension without CFS of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was used as a negative control. The optical density was measured at 650 nm.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain is found to inhibit the adhesion of all Candida species tested by exhibiting inhibition percentages above 50% in pre-coating assays and above 20% in co-incubation assays.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in polystyrene plates by transfer ring, to each well, 100 l of a cell suspension of the strain standardized to 8 log cfu/ml. After incubation for 1 hour at 37°C, unbound cells were removed by washing twice with PBS buffer. The tested pathogen strains were previously grown in liquid medium and radiolabeled. 100 l of each pathogen, standardized to 8 log cfu/ml, was added to the wells and incubated at 37°C for 1 hour.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed ability to inhibit adhesion of all tested pathogens with inhibition percentages above 50%.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was assayed as follows. In detail, radiolabeled and standardized pathogens, as reported previously, were added to the wells and incubated for 1 h at 37°C. Unbound pathogens were removed by washing three times with PBS buffer, and the nonradiolabeled Lacticaseibacillus rhamnosus CA15 DSM 33960 strain (100 l, 8 log cfu/ml) was added to each well. The plates were incubated for 1 h at 37°C. Then, the wells were washed again and the bound bacteria were released and lysed as described above.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain showed ability to inhibit all tested pathogens with percentages above 50%.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was evaluated using the phenol/sulfuric acid method, and the amount of EPS produced was assessed using glucose (50-500 mg/l) as a standard.
• the assay was performed in triplicate, and the results expressed in mg/L are shown in Table 17.
• Hydrophobicity was determined by mixing equal volume of xylene and cell suspension, of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain, in PBS (9 log cfu/ml ). After incubation at 37° C for 10 min, the suspension was kept at 37° C for 5 h to achieve phase separation. The aqueous phase was taken and subjected to absorbance determination at 600 nm. Hydrophobicity was calculated as the pertcentage reduction in absorbance (AAbs x 100) of the aqueous phase after incubation compared with the absorbance of the base suspension according to the following formula:
• H% Abs/ Absto x 100.
• Auto-aggregation was determined by using 1 ml of cell suspension (9 log ufc/ml ), of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain, in PBS buffer, vortexed for 10 seconds and incubated at room temperature for 5 hours. Then, an aliquot (0.1 ml) of the upper phase of the suspension was mixed with 500 l of PBS buffer and the absorbance at 600 nm was determined. Auto-aggregation (Auto-A%) was expressed as the percentage reduction in absorbance after 5 h (AAbs x 100) compared with that of the original suspension (AbstO) as follows:
• Co-aggregation (Co-A%) was evaluated using E. coli 555, G. vaginalis ATCC 14018, G. vaginalis ATCC 14019, C. albicans ATCC 10231, C. glabrata ATCC 90030, C. krusei ATCC 14243, C. parapsilosis ATCC 90018, and C. tropicalis ATCC 13803 strains as co-aggregation partners.
• equal volume (2 mL) of the cell suspension of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain and each of the pathogenic strains were mixed and vortexed for 30 seconds then kept at room temperature for 5 h.
• Co-aggregation was calculated as follows:
• Co-A% ((Abs C Ai5 + AbSp a t )/2 - Abs (C Ai5 + P at) / (Abs CA i5 + Abspat/2) x 100.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain exhibited good auto-aggregation, hydrophobicity, and coaggregation with all pathogens tested, indirectly evidencing the ability to adhere to the intestinal mucosa and prevent pathogen development.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was evaluated in MRS broth after 0, 30, and 120 min of incubation at 37°C. Lysozyme tolerance was determined by in-plate counting of viable cells. The ability to survive in the presence of lysozyme was assessed by determination of the survival rate (SR%), that is the survival percentage, which was calculated by taking into account the final (cfuF) and initial (cful) population density (cfuF/cful *100). The value 80% was considered as the minimum survival limit. Cell cultures, of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain, in the absence of lysozyme were used as control. The analysis was performed in triplicate.
• the tested strain showed ability to survive in the presence of lysozyme, presenting survival % of 95% and 93 % after incubation at 37°C for 30 and 120 min, respectively.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in MRS broth acidified to pH 2.0 and pH 3.0 by addition of HCI 1 M.
• MRS broth at pH 6.2 was used as a control.
• the strain under study was revitalized in MRS broth, and the cell suspension in exponential growth phase of growth (9 log ufc/ml ) was inoculated into the acidified culture medium. Aliquots were taken immediately after inoculation (0 h) and after 2 h and 4 h of incubation at 37°C. Tolerance to low pH values was determined by in-plate counting of viable cells. The percentage (%) of survival, calculated by taking into account the final (cfuF) and initial (cful) population density (cfuF/cful *100) was evaluated, and the value 80% was considered as the minimum survival limit.
• the tested strain showed ability to survive at pH 3.0 and 2.0, presenting survival % greater than 80% after incubation at 37°C for 2 hours and 4 hours (Table 20).
• the survival percentage at pH 3.0 after 4 hours was 86%.
• the survival percentage at pH 2.0 after 4 hours is 83%.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was evaluated as follows. The strain under study was revitalized in MRS broth, and the cell suspension in exponential growth phase (9 log ufc/ml ) was inoculated into MRS broth containing the above percentages of bile salts. MRS without bile salts was used as a control. Aliquots were taken immediately after inoculation (0 h) and after 2 h and 4 h of incubation at 37 °C, and viable cells were counted in plates.
• bile salts bovine bile salts, Oxgall; Sigma-Aldrich
• the tested strain showed ability to survive in the presence of bile salts at the concentrations of 0.5% and 1%.
• the survival rate to 0.5% bile salts after 4 hours of incubation is 87%.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was determined in vitro by using both a static system (SGJ/SIF) and a dynamic system (SHI ME), the latter capable of exactly reproducing the physiological, chemical and microbiological conditions of the Gl tract in vitro.
• SGJ/SIF static system
• SHI ME dynamic system
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain to survive during gastrointestinal (Gl) transit was determined in vitro using simulated gastric juice ("SGJ") and simulated intestinal fluid (“SIF").
• SGJ simulated gastric juice
• SIF simulated intestinal fluid
• SGJ pepsin, 0.5% NaCI, adjusted to pH 2 by adding HCI 1 M
• SIF pancreatin, 0.5% bile salt, 0.5% NaCI, 0.4% phenol, adjusted to pH 8 by adding NaOH 1 M
• All chemicals were obtained from Sigma Aldrich (St. Louis, MO).
• the strain exhibited excellent survival during simulated gastrointestinal transit under static conditions. During simulated gastrointestinal digestion, survival greater than 90% was observed. In detail SR% of 99.7% was found after treatment with simulated gastric juice (SGJ) and SR% of 95.7% was found after treatment with simulated intestinal fluid (SIF) (Table 22).
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was determined by using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®, ProDigest, Belgium). This system is capable of accurately simulating the entire human Gl tract, incorporating stomach, small intestine and different regions of the colon. Since it is a dynamic system, the physiological, chemical and microbiological conditions of the Gl tract are exactly reproduced.
• the dynamic SHIME system was set up by using a double-walled reactor, simulating the entire Gl tract, maintained at a temperature of 37°C, under constant stirring and anaerobic conditions by nitrogen insufflation. Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in capsule form with a population density of 10 log cfu/g and maintained in the SHIME system within a specific capsule holder.
• the stomach microenvironment was reproduced by employing gastric juice at pH 2 consisting of: KCI 0.66 g/L, NaCI 3.63 g/L, mucin 3.95 g/L, lecithin 3.4 g/L and pepsin 10 g/L. Digestion at the stomach level was simulated for 45 min. Next, the gastric digest was subjected to intestinal digestion then pancreatic juice consisting of: NaHCOs 2.6 g/L, bile salts 4.8 g/L, pancreatin 1.9 g/L, trypsin 10 g/L and chymotrypsin 10 g/L was added.
• duodenal 27 min incubation with controlled pH increase from 2.0 to 6.5
• jejunal 63 min incubation with controlled pH increase from 6.5 to 7.5
• ileal 90 min incubation at constant pH of 7.5 fractions were simulated.
• aliquots of NaHCOs (8.4 g/L) were added after 60, 90 and 120 min to determine the pH increase to the above values.
• the pH was continuously monitored using Senseline F410 pH meter (ProSense, Oosterhout, The Netherlands). Solutions of HCI (0.5 M) and NaOH (0.5 M) were dosed automatically, using a pump system, in order to determine a constant maintenance of pH within the above ranges.
• Table 23 shows the results for the survival of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain, expressed as log cfu/ml and survival % (SR%), as well as the capsule degradation rate.
• the stomach microenvironment was reproduced using gastric juice at pH 4.5 containing SHI ME® nutrient medium (PDNM001B 20.53 g/L, ProDigest, Ghent, Belgium), NaCI 3.63 g/L, KCI 0.65 g/L, lecithin 13.5 g/L and pepsin 40 g/L. Digestion at the stomach level was simulated for 120 min. Next, the gastric digest was subjected to intestinal digestion then pancreatic juice consisting of: NaHCO3 7.7 g/L, bile salts 15 g/L, pancreatin 10 g/L, trypsin 10 g/L and chymotrypsin 10 g/L was added.
• SHI ME® nutrient medium PDNM001B 20.53 g/L, ProDigest, Ghent, Belgium
• NaCI 3.63 g/L KCI 0.65 g/L
• lecithin 13.5 g/L lecithin 13.5 g
• duodenal 27 min incubation with controlled pH increase from 2.0 to 6.5
• jejunal 63 min incubation with controlled pH increase from 6.5 to 7.5
• ileal 90 min incubation at constant pH of 7.5 fractions were simulated.
• aliquots of NaHCOs (8.4 g/L) were added after 60, 90 and 120 min to determine the pH increase to the above values.
• the pH was continuously monitored using Senseline F410 pH meter (ProSense, Oosterhout, The Netherlands). Solutions of HCI (0.5 M) and NaOH (0.5 M) were dosed automatically, using a pump system, in order for the pH to be constantly maintained within the above ranges.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was determined by employing the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®, ProDigest, Belgium).
• SHIME® Simulator of the Human Intestinal Microbial Ecosystem
• the dynamic SHIME system was set up by employing a double-walled reactor, simulating the entire Gl tract, maintained at a temperature of 37°C, under constant stirring and anaerobic conditions by nitrogen insufflation. Lacticaseibacillus rhamnosus CA15 DSM 33960 strain and the antibiotic were introduced simultaneously inside the reactor.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in capsule form with a population density of 10 log cfu/g and maintained in the SHIME system within a specific capsule holder.
• the antibiotic fluconazole (200mg) previously dissolved in 50 ml of sterile water as per the dosage indication, was introduced sterilely through additional port.
• the stomach microenvironment was reproduced by employing gastric juice at pH 2 consisting of: KCI 0.66 g/L, NaCI 3.63 g/L, mucin 3.95 g/L, lecithin 3.4 g/L and pepsin 10 g/L. Digestion at the stomach level was simulated for 45 min.
• pancreatic juice consisting of: NaHCOs 2.6 g/L, bile salts 4.8 g/L, pancreatin 1.9 g/L, trypsin 10 g/L and chymotrypsin 10 g/L was added. Therefore, duodenal (27 min incubation with controlled pH increase from 2.0 to 6.5), jejunal (63 min incubation with controlled pH increase from 6.5 to 7.5) and ileal (90 min incubation at constant pH of 7.5) fractions were simulated. During the intestinal phase, after 60, 90 and 120 min, 8.4 g/L of NaHCOs were added to determine the pH increase to the above values.
• the acidifying capacity of Lacticaseibacillus rhamnosus CA15 DSM 33960 strain was tested in de Man Rogosa and Sharp broth medium (MRS, Oxoid, Milan, Italy) and monitored for 24 h by pH determination.
• MRS de Man Rogosa and Sharp broth medium
• the pH of the medium was determined at regular time intervals by pH meter (XS, PH 60 VIO LAB).
• Table 26 shows the pH values measured at different sampling times.
• Lacticaseibacillus rhamnosus CA15 DSM 33960 strain resulted in a gradual decrease in the pH of the medium used, reaching pH values of 3.82 after 24 hours of incubation at 37°C.

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