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
• • 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
  • A61K35/745—Bifidobacteria
• • 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
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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, e.g. protozoa; 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
• • 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, e.g. protozoa; 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• • 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
• • 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

Definitions

• the present invention is directed to the field of probiotics delivery.
• a healthy microbiota requires bacterial colonization which provides the host multiple benefits including resistance to a broad spectrum of pathogens, essential nutrient biosynthesis and absorption, and immune stimulation that maintains a healthy gut epithelium and an appropriately controlled systemic immunity.
• microbiota functions can be lost or deranged, resulting in increased susceptibility to pathogens, altered metabolic profiles, or induction of proinflammatory signals that can result in local or systemic inflammation or autoimmunity.
• Urogenital infections such as yeast vaginitis, bacterial vaginosis, and urinary tract infection remain a major medical problem in terms of the number of women afflicted each year. These diseases affect the organs and tissues related to the reproductive system.
• microbiota is mainly represented by lactobacilli
• the microbial composition of the biocoenosis is characterized by a decrease in the number of lactobacilli and their replacement by pathogenic anaerobic microorganisms.
• a change in the vaginal flora characterized by the decrease of lactobacilli appears to be the major factor causing the syndrome bacterial vaginosis.
• antimicrobial therapy is generally effective at eradicating these infections, there is still a high incidence of recurrence.
• the patient’s quality of life is affected, and many women become frustrated by the cycle of repeated antimicrobial agents whose effectiveness is diminishing due to increasing development of microbial resistance.
• Lactobacillus iners The most common Lactobacillus sp. in the lower genital tract are Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri.
• lactobacilli have in maintaining vaginal health, while preventing genital tract infections.
• the beneficial effect of Lactobacillus crispatus on colitis has also been reported.
• vaginal suppositories formulation in which the probiotics are viable under the vaginal conditions, are able to adhere to the vaginal epithelial cells for a successful colonization. Moreover, it is important that such formulations are resistant to the common antibiotics used in the treatment.
• composition comprising a first lipophilic carrier, a co-cultured probiotic bacteria in the form of a dried biofilm comprising Lactobacillus crispatus and at least one additional bacterial species selected from the group consisting of: L. gasseri, L. jensenii, and L. rhamnosus, and a first agent comprising: an antibiotic agent, a pH adjusting agent, or both.
• composition comprising: a co cultured probiotic bacteria in the form of a dried biofilm comprising: (i) Faecalibacterium prausnitzii and at least one additional bacterial species selected from the group consisting of: Blautia obeum, Bl. coccoides, Bacteroides vulgatus, and Dorea (Eubacterium) formicigenerans, (ii) Lactobacillus crispatus and at least one additional bacterial species selected from the group consisting of: L. gasseri, L. jensenii, and L.
• a method for modulating the flora in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the herein disclosed composition, thereby modulating the flora in the subject.
• a method for preparing the herein disclosed composition comprises the steps of: (a) inoculating a growth medium comprising a particle with L. crispatus, (b) incubating the particle with L. crispatus from step (a) under conditions suitable for allowing L. crispatus to attach to the particle; (c) inoculating the particle of step (b) with at least one additional bacterial species; and (d) culturing the inoculated particle of step (c) under conditions suitable for forming a biofilm comprising L. crispatus and at least one additional bacterial species.
• inoculating a growth medium comprising a particle with a first bacteria selected from: (i) L. crispatus ; (ii) Bif. adolescentis, or (iii) F. prausnitzii; incubating the particle with the first bacteria under conditions suitable for allowing the first bacteria to attach to the particle; inoculating the particle with at least one additional bacterial species; and culturing the inoculated particle under conditions suitable for forming a biofilm comprising: (i) L. crispatus ; (ii) Bif. adolescentis, or (iii) F. prausnitzii, and at least one additional bacterial species.
• the co-cultured probiotic bacteria in the form of a dried biofilm and the first agent comprising an antibiotic agent are homogeneously dispersed within the composition.
• the co-cultured probiotic bacteria in the form of dried biofilm is 10% to 50% (w/w) of the total composition.
• the co-cultured probiotic bacteria in the form of dried biofilm is attached to a particle.
• the particle is selected from the group consisting of: seeds, MCC, dicalcium phosphate, a polysaccharide, and any combination thereof.
• the composition further comprises a second layer.
• the second layer comprises a second lipophilic carrier, a second agent or both.
• the release of the co-cultured probiotic bacteria in the form of dried biofilm is slower than the release of the second agent.
• any one of the first agent and the second agent is an antibiotic.
• the composition is for use in the treatment of bacterial vaginosis.
• the composition further comprises a stabilizer, a preservative, a lubricant, a viscosity modifying agent, a buffering agent, fatty acids, and combinations thereof.
• the composition is formulated for a delivery route selected from the group consisting of: oral, vaginal, rectal, and topical.
• the composition is in the form of a suppository.
• the flora is a vaginal flora, a gut flora, or a skin flora.
• the method is for preventing or treating a dysbiosis related condition or an intestinal or metabolic disease in a subject in need thereof.
• the dysbiosis related condition or an intestinal and metabolic disease is selected from the group consisting of: bacterial vaginosis, a urogenital infection, ulcerative colitis, inflammatory bowel disease (IBD), Crohn's disease, colorectal cancer, obesity, and celiac disease.
• the subject is afflicted with or at risk of developing bacterial vaginosis, a urogenital infection, dysbiosis, ulcerative colitis, an inflammatory bowel disease (IBD), Crohn's disease, or any combination thereof.
• IBD inflammatory bowel disease
• the at least one additional bacterial species is selected from the group consisting of: L. jensenii, L. gasseri, and L. rhamnosus.
• the at least one additional bacterial species is selected from the group consisting of: Bif. Longum sub.longum, and Bif. breve.
• the at least one additional bacterial species is selected from the group consisting of: Bl. obeum, Bl. coccoides. Bac. vulgatus, and Dorea (Eubacterium) formicigenerans.
• the first bacteria is F. prausnitzii and the inoculating steps are performed simultaneously.
• Figures 1A-1G present pictures of the different suppository formulations presented in table 1 ( Figures 1A-1F), and a diagram of the experimental design and assays that were performed to optimize growth of bacteria in biofilm using bacteria in form of biofilm as well as to evaluate bacteria in form of biofilm developmental phase.
• the use of pH 3.5 in the pH resistance assay was determined to have a pH value close to the pH that prevails in woman vagina (pH 4-5).
• Susceptibility of planktonic bacteria to the resistance assays was also determined and results were subsequently compared to bacteria in form of biofilm results.
• CFU colonies forming units ( Figure 1G).
• Figure 2 presents a bar graph of acid resistance of planktonic bacteria of L. iners.
• Figure 3 presents a bar graph of acid resistance of L. iners Bacteria in form of biofilm following growth in a small-scale set-up. Aerobic and anaerobic conditions were examined as well as aeration condition (static vs. stir).
• Figure 4 presents a bar graph of acid resistance of L. iners Bacteria in form of biofilm following growth in a medium scale set-up.
• Figure 5 presents a bar graph of antibiotics resistance of L. iners Bacteria in form of biofilm following growth in a medium-scale set-up.‘Control’ refers to Bacteria in form of biofilm that was not exposed to antibiotics. Numbers in the x-axis are antibiotics concertation in pg/mL.
• Figure 6 presents a bar graph of acid resistance of planktonic bacteria of L. jensenii.
• Figure 7 presents a bar graph of acid resistance of L. jensenii Bacteria in form of biofilm following growth in a small-scale set-up while examining two agitation speeds (70 and 130 rpm).
• Figure 8 presents a bar graph of acid resistance of L. jensenii Bacteria in form of biofilm following growth in a medium-scale set-up while examining two agitation speeds (70 and 130 rpm).
• Figure 9 presents a bar graph of antibiotics resistance of L. jensenii Bacteria in form of biofilm following growth in a medium-scale set-up.‘Ctrl’ refers to Bacteria in form of biofilm that was not exposed to antibiotics. Numbers in the x-axis are antibiotics concertation in pg/mL.
• Figure 10 presents a bar graph of acid resistance of planktonic bacteria of L. crispatus.
• Figure 11 presents a bar graph of acid resistance of L. crispatus Bacteria in form of biofilm following growth in a small-scale set-up while examining agitation and non agitation conditions.
• Figure 12 presents a bar graph of acid resistance of L. crispatus Bacteria in form of biofilm following growth in a medium scale set-up.
• Figure 13 presents a bar graph of antibiotics resistance of L. crispatus Bacteria in form of biofilm following growth in a medium-scale set-up.‘Ctrl’ refers to Bacteria in form of biofilm that was not exposed to antibiotics. Numbers in the x-axis are antibiotics concertation in pg/mL.
• Figure 14 presents a bar graph of acid resistance of planktonic bacteria of L. gasseri.
• Figure 15 presents a bar graph of acid resistance of L. gasseri Bacteria in form of biofilm following growth in a small-scale set-up while examining two agitation speeds (70 and 130 rpm).
• Figure 16 presents a bar graph of acid resistance of L. gasseri Bacteria in form of biofilm following growth in a medium scale set-up.
• Figure 17 presents a bar graph of antibiotics resistance of L. iners Bacteria in form of biofilm following growth in a medium-scale set-up.‘Ctrl’ refers to Bacteria in form of biofilm that was not exposed to antibiotics. Numbers in the x-axis are antibiotics concertation in pg/mL.
• Figure 18 presents a bar graph of acid resistance of planktonic bacteria of L.rhamnosus.
• Figure 19 presents a bar graph of acid resistance of L. rhamnosus Bacteria in form of biofilm following growth in a small-scale set-up while examining two agitation speeds (70 and 130 rpm).
• Figure 20 presents a bar graph of acid resistance of L. rhamnosus Bacteria in form of biofilm following growth in a medium scale set-up.
• Figure 21 presents a bar graph of antibiotics resistance of L. rhamnosus Bacteria in form of biofilm following growth in a medium-scale set-up.‘Ctrl’ refers to Bacteria in form of biofilm that was not exposed to antibiotics. Numbers in the x-axis are antibiotics concertation in pg/mL.
• Figure 22 presents a bar graph of the effect of cranberries on Bacteria in form of biofilm survival in suppositories for two months ‘cran’ refers to cranberries;‘supp’ refers to suppositories.
• Figure 23 presents a bar graph of survival of wet- and dry- Bacteria in form of biofilm after 1 and 3 months in suppositories.
• Figure 24 presents a bar graph of survival of L. plantarum Bacteria in form of biofilm in suppositories containing different ratio of Bacteria in form of biofihmexcipients, 1:5 or 1:10, respectively.
• Excipients comprised of two oil-based carriers, vegetable butter and cocoa butter‘supp’ refers to suppositories.
• Figure 25 presents a bar graph of the effect of different particles on the growth of L. gasseri Bacteria in form of biofilm.
• Figure 26 presents a bar graph comparing the stability of a suppository formulation comprising a combination of Pentasa with Bacteria in form of biofilm ‘supp A’ refers to L. gasseri and L. rhamnosus biofilm ‘supp B’ refers to L. gasseri and L. rhamnosus biofilm with Pentasa.
• Figure 27 presents a picture of colonies morphologies of L. rhamnosus (LRh), L. jensenii (LJ) and L. gasseri (LG) as obtained from the co-culture experiment. Bacteria cells were released from the biofilm by vortex before plating on MRS agar plate for CFU counting.
• Figures 28A-28B present bar graphs of co-culture of bacterial strains. Bacterial population growth and biofilm development (pH resistance) was compared between the strain growth alone and together. Dashed line refers to the threshold for bacterial resistance; Bacterial population that survived above this point is considered resistant to the tested treatment.
• (28A) present a bar graph of co-culture of strain L. jensenii (LJ) with L. rhamnosus (LRh)
• (28B) present a bar graph of co-culture of strain L. gasseri (LG) with L. rhamnosus (LRh).
• Figures 29 presents a picture of colonies morphologies of L. rhamnosus (LRh), L. jensenii (LJ), L. gasseri (LG) and L. crispatus (LCr) as obtained from the co-culture experiment. Bacteria cells were released from the biofilm by vortex before plating on MRS agar plate for CFU counting. Images of the bacteria colonies were taken from the agar plate.
• Figures 30A-30B present bar graphs of co-culture of three or four bacterial strains. Bacterial population growth and biofilm development (pH resistance) of each strain in the co-culture (the current experiment) was compared to their growth alone (former experiments). Dashed line refers to the threshold for bacterial resistance; Bacterial population that survived above this point is considered resistant to the tested treatment.
• (30A) presents a bar graph of Co-culture of L. rhamnosus (LRh), L. jensenii (LJ) and L. gasseri (LG)
• (30B) present a bar graph of Co-culture of L. rhamnosus (LRh), L. jensenii (LJ), L. gasseri (LG), and L. crispatus (LC).
• Figure 31 presents a bar graph showing affinity of the L. gasseri (LG) Bacterial population to various particle size.
• Figures 32A-32C present bar graphs of the effect of different pH levels and animal-based and non-animal-based growth mediums on the growth and developmental state of LCr (32A), LG (32B), and LJ (32C) cultures.
• Figures 33A-33C present bar graphs of the effect of animal-based medium and nonanimal 1 based medium on growth and development (pH and antibiotics resistance) of LG (33A), LRh (33B), and LCr (33C) Bacterial population. Dashed line refers to the threshold for bacterial resistance; bacterial population that survived above this point is considered resistant to the tested treatment.
• Figure 34 presents a vertical bar graph showing that Bifidobacterium adolescentis ( BfA ) has an improved growth after 72 h when cocultured with Bifidobacterium longum sub.longum ( BLL ) and B. breve (BfBr) compare to its growth alone.
• BfA Bifidobacterium adolescentis
• Figure 35 presents a vertical bar graph showing that BfA has an improved growth when cocultured with BfBr compare to its growth alone after 24 h and 72 h of growth.
• Figure 36 presents a vertical bar graph showing that BfA has higher relative abundance (RA) when it is added 24 h prior to the addition of BLL , B. animalis (BB-12), and BfBr. BfA was added either in parallel with BLL and BfBr (‘control’) or 24 h before the addition of BLL and BfBr (‘Advantage to BfA’).
• Figure 37A-37B present vertical bar graphs showing improved growth of BfA ( ⁇ 1 log difference) when it is added 24 h prior to the addition of BLL , BB-12 and BfBr ('Advantage to BfA') compare to its addition in parallel to the other Bifidobacterium species ('Control').
• the relative abundance values are normalized to bacteria count (log scale).
• Figure 38A-38B present vertical bar graphs showing improved growth of BfA ( ⁇ 1 log difference) when it is added 24 h prior to the addition of BLL , BB-12 and BfBr ('Advantage to BfA') compare to its addition in parallel to the other Bifidobacterium species ('Control').
• the relative abundance values are normalized to bacteria count (CFU/mL).
• Figure 39 presents a vertical bar graph showing that Faecalibatcerim prausnitzii ( FaP ) show an improved growth when cocultured with either Blautia obeum ⁇ BIO), Bl. coccoides ( BIC ) or both, compare to its growth alone.
• Figure 40 presents a vertical bar graph showing that FaP show an improved growth when cocultured with either Bacteroides vulgatus ( BaV) or both BaV and Dorea (Eubacterium) formicigenerans (DoF) compare to its growth alone.
• BaV Bacteroides vulgatus
• Dorea Eubacterium formicigenerans
• Figure 41 presents a vertical bar graph showing that FaP shows the improved growth when coculture with either Bifidobacterium adolescentis (BfA) or Bacteroides thetaiotaomicron ( BaT ).
• BfA Bifidobacterium adolescentis
• BaT Bacteroides thetaiotaomicron
• Figure 42 presents a vertical bar graph showing that FaP is cocultured with Bacteroides spp. (right column), a clear advantage is observed for FaP growth in comparison to its growth in coculture with only Clostridiales spp. (left column).
• Figure 43A-43B present vertical bar graphs showing that FaP has a higher relative abundance (RA) in biofilm (43B) compared to the planktonic part (43A) during 72 h of growth.
• the present invention provides a composition comprising at least one bacteria in the form of bio film.
• the biofilm is in the form of dried biofilm or the form of a powder biofilm.
• the biofilm is biofilm particles.
• biofilm particles refers to bacteria (e.g., probiotic bacteria) in the form of biofilm and in a form of particles.
• the composition comprises a plurality of bacteria.
• a plurality is any integer greater than 1, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, etc., or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
• At least one bacterium within the composition produces biofilm. In some embodiments, all bacteria within the composition produce biofilm.
• the composition comprises planktonic bacteria.
• the planktonic bacteria do not produce biofilm.
• the planktonic bacteria produce a low amount of biofilm compared to the biofilm producing bacteria.
• the composition comprises planktonic bacteria and biofilm producing bacteria, wherein the planktonic bacteria are stuck, entrapped, merged, or embedded, under, onto, or within the biofilm produced by the biofilm producing bacteria.
• the herein disclosed probiotic bacteria in the form of biofilm comprises a mixture of planktonic bacteria and bacteria in the form of biofilm.
• composition comprising a first lipophilic carrier, a co-cultured probiotic bacteria in the form of a dried biofilm comprising Lactobacillus crispatus and at least one additional bacterial species selected from the group consisting of: L. gasseri, L. jensenii, and L. rhamnosus, and a first agent comprising: an antibiotic agent, a pH adjusting agent, or both.
• co-cultured probiotic bacteria in the form of a dried biofilm and the first agent comprising an antibiotic agent are homogeneously dispersed within the composition.
• the composition comprises a bacteria in the form of biofilm comprising: Faecalibacterium prausnitzii and at least one additional bacterial species selected from: Blautia obeum, Bl. coccoides, Bacteroides vulgatus, Dorea (Eubacterium) formicigenerans, or any combination thereof.
• the composition comprises a bacteria in the form of biofilm comprising: Lactobacillus crispatus and at least one additional bacterial species selected from: L. gasseri, L. iners, L. jensenii, L. rhamnosus, or any combination thereof.
• the composition comprises a bacteria in the form of biofilm comprising: Bifidobacterium adolescentis and at least one additional bacterial species selected from: Bif. Longum sub.longum, Bif breve and a combination thereof.
• the term“probiotic” refers to a beneficial or required bacterial strain that can also stimulate the growth of other microorganisms, especially those with beneficial properties (such as those of the vaginal flora and gut flora).
• the biofilm comprises at least one bacterial strain derived from vaginal microflora.
• the at least one bacterial strain derived from vaginal microflora is a probiotic bacterium.
• the biofilm comprises at least one bacterial strain derived from gut microflora.
• the at least one bacterial strain derived from gut microflora is a probiotic bacterium.
• the biofilm comprises at least one bacterial strain derived from the colon. In some embodiments, the biofilm comprises at least one bacterial strain derived from the stomach. In some embodiments, the biofilm comprises at least one bacterial strain derived from the small intestine.
• the at least one probiotic bacteria is selected from the genera Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Faecalibacterium, Pediococcus, Leuconostoc, Bacillus, Escherichia coli, or any combination thereof.
• Non-limiting examples of gut-derived strains include Lactobacillus rhamnosus GG (LGG), Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus rhamnosus, Propionibacterium freudenreichii, Bifidobacterium breve, Lactobacillus reuteri, Lactobacillus salivarius, Bifidobacterium infantis, Streptococcus thermophiles, and Faecalibacterium prausnitzii.
• LGG Lactobacillus rhamnosus GG
• Streptococcus thermophilus Lactobacillus acidophilus
• Bifidobacterium lactis Bifidobacterium breve, B
• the biofilm comprises at least one Lactobacillus bacterial strain.
• Lactobacillus include Lactobacillus crispatus, Lactobacillus Gasseri, Lactobacillus iners, Lactobacillus Jensenii, Lactobacillus rhamnosus, Lactobacillus Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii ssp. Bulgaricus.
• the probiotic bacteria can colonize a vaginal tissue.
• the probiotic bacteria are more proficient in colonizing vaginal tissue compared to similar bacteria that are provided in a planktonic form.
• the degree of improvement of colonization may be measured as an increase in the quantity of bacteria in samples from a tissue treated with biofilm particle -based suppositories compared to a control tissue which is treated with planktonic probiotic bacteria-based suppositories, after a predetermined period of time from administration.
• the bacteria provided herein is generated using the methods as disclosed in PCT/IB2016/000933 and PCT/IL2017/050587 incorporated herein by reference, in its entirety.
• one or more bacterium for generating biofilm provided herein is obtained from a healthy mammal.
• the bacterium is obtained from an animal donor.
• the donor may be screened for their health status and nutrition habits.
• the bacterium is derived from a bacterial strain.
• the bacterium is derived from stored bacterial strain.
• the plurality of bacteria is derived from frozen bacterial strain.
• the bacterium is derived from frozen biofilm.
• the bacterium is derived from lyophilized bacterial strain.
• the biofilm comprises at least one bacterial strain derived from a stored microbiota sample. In one embodiment, the biofilm comprises at least one bacterial strain derived from a bacterial colony.
• the CFU of the composition is 10 4 to 10 15 .
• the CFU of the composition is 10 4 to 10 14 .
• the CFU of the composition is 10 4 to 10 13 .
• the CFU of the composition is 10 4 to 10 12 .
• the CFU of the composition is 10 5 to 10 15 .
• the CFU of the composition is 10 5 to 10 14 .
• the CFU of the composition is 10 5 to 10 13 .
• the CFU of the composition is 10 5 to 10 12 .
• the CFU of the composition is 10 6 to 10 15 .
• the CFU of the composition is 10 6 to 10 14 . According to some embodiments, the CFU of the composition is 10 6 to 10 13 . According to some embodiments, the CFU of the composition is 10 6 to 10 12 . According to some embodiments, the CFU of the composition is 10 7 to 10 15 . According to some embodiments, the CFU of the composition is 10 7 to 10 14 . According to some embodiments, the CFU of the composition is 10 7 to 10 13 . According to some embodiments, the CFU of the composition is 10 7 to 10 12 . According to some embodiments, the CFU of the composition is 10 8 to 10 15 . According to some embodiments, the CFU of the composition is 10 8 to 10 14 . According to some embodiments, the CFU of the composition is 10 8 to 10 13 . According to some embodiments, the CFU of the composition is 10 8 to 10 12 .
• the at least one additional bacterial species is present in the composition in an amount of 10 4 to 10 15 CFU. According to some embodiments, the at least one additional bacterial species is present in the composition in an amount of 10 8 to 10 12 CFU.
• the ratio of Lactobacillus crispatus and the at least one additional bacterial species is selected from 1: 100000 - 100000: 1, 1: 10000 - 10000: 1, 1: 1000 - 100: 1, 1: 100 - 1000: 1, and 1: 100 - 100: 1.
• the biofilm has at least one feature selected from: acid tolerant, antibiotics-resistant, temperature resistance, and any combination thereof.
• the composition further comprises: (i) a first agent comprising an antibiotic agent; (ii) a first lipophilic carrier, (iii) a pH adjusting agent, or any combination thereof.
• the at least one probiotic bacteria in the form of biofilm is homogeneously dispersed within the first lipophilic carrier, the first agent, or any combination thereof, thereby forming a first layer.
• the present invention provides a composition comprising at least one probiotic bacteria in the form of biofilm and a first lipophilic carrier, wherein the at least one probiotic bacterium is 10% to 50% (w/w) of the total composition.
• the at least one probiotic bacterium is 12% to 50% (w/w), 15% to 50% (w/w), 20% to 50% (w/w), 12% to 48% (w/w), 12% to 15% (w/w), 12% to 42% (w/w), 12% to 40% (w/w), 15% to 48% (w/w), 15% to 40% (w/w), 20% to 50% (w/w), 20% to 48% (w/w), 20% to 45% (w/w), or 20% to 40% (w/w), of the total composition.
• the first lipophilic carrier and the second lipophilic carrier are solid at room temperature and are each independently characterized by melting point of at least 25°C. In some embodiments, the first lipophilic carrier and the second lipophilic carrier are solid at room temperature and characterized by melting point of at least 26°C, at least 27°C, at least 28°C, at least 29°C, at least 30°C, at least 31°C, or at least 32 °C, including any value therebetween.
• the first lipophilic carrier and the second lipophilic carrier have each independently a melting point in the range of 25°C to 60°C. In some embodiments, the first lipophilic carrier and the second lipophilic carrier have each independently a melting point in the range of 27°C to 60°C, 30°C to 60°C, 25°C to 58°C, 25°C to 55C, 27°C to 58°C, or 27°C to 55°C, including any range therebetween.
• the first lipophilic carrier and the second lipophilic carrier comprise one or more fatty acids with a saturated content of more than 40%. In some embodiments, the first lipophilic carrier and the second lipophilic carrier comprise one or more fatty acids with a saturated content of more than 41%, more than 45%, more than 48%, or more than 50%, including any value therebetween.
• the first lipophilic carrier and the second lipophilic carrier comprise one or more hydrogenated fats.
• hydrogenated fats refers to fatty acids that have been chemically altered.
• hydrogenated fats are oils whose chemical structures were changed to become solid fats.
• the second lipophilic carrier has a melting point at least 5°C, at least 6°C, at least 7°C, at least 10°C, at least 12°C, or at least 15°C, higher than the first lipophilic carrier.
• the first lipophilic carrier has a melting point at least 5°C, at least 6°C, at least 7°C, at least 10°C, at least 12°C, or at least 15°C, higher than the second lipophilic carrier.
• the melting point of the composition is controlled by controlling the ratio of hydrogenated fats.
• the release time of the probiotic bacteria is controlled by the melting point of the composition.
• the release time of the first agent is controlled by the melting point of the composition.
• the release time of the second is controlled by the melting point of the composition.
• the release of the at least one probiotic bacteria in the form of biofilm is slower than the release of the second agent.
• the first lipophilic carrier and the second lipophilic carrier comprise cacao butter, palm oil, plant wax, vegetable wax, or any combination thereof.
• the first lipophilic carrier and the second lipophilic carrier comprise fatty acids derived from raw materials of vegetable origin.
• excipients are obtained by the esterification of fatty acids with alcohols such as glycerol, polyglycerol, propylene glycol and polyethylene glycol, and by the alcoholysis of vegetable oils and fats with glycerol, polyethylene glycol and propylene glycol.
• alcohols such as glycerol, polyglycerol, propylene glycol and polyethylene glycol
• the composition comprises probiotic bacteria in the form of biofilm a lipophilic carrier, and a first agent, in the form of a first layer.
• the composition further comprises a second layer comprising a second agent.
• any one of the first agent and the second agent is an agent that improves the receptiveness of the vaginal tissue for colonizing probiotic bacteria.
• an agent that may improve the receptiveness of the vaginal tissue for colonizing probiotic bacteria may be a pH modifier.
• the lipophilic carrier is used to release an amount of a pH modifier that is sufficient to decrease the local pH in the vaginal tissue.
• vaginal pH should be modified to about 4 which is optimal for colonization of the probiotic bacteria of the invention.
• the pH modifier can be synthetic.
• the pH modifier can be natural-biological
• any one of the first agent and the second agent is a pH adjusting agent. In some embodiments, any one of the first agent and the second agent is a pH adjusting agent capable of adjusting the pH to 4.
• Non-limiting examples of pH adjusting agents according to the present invention are sodium bicarbonate, ascorbic acid, citric acid, acetic acid, fumaric acid, propionic acid, malic acid, succinic acid, gluconic acid, tartaric acid, lactic acid, boric acid and cranberry extract.
• any one of the first agent and the second agent is an antibiotic.
• the antibiotic is any antibiotic used for treatment of bacterial vaginosis.
• antibiotics include metronidazole (Flagyl), clindamycin (Cleocin), and metronidazole.
• the antibiotic is released first.
• the probiotic bacteria is released after release of the antibiotic.
• the composition further comprises a stabilizer, a preservative, a lubricant, a viscosity modifying agent, a buffering agent, fatty acids, and combinations thereof.
• first lipophilic carrier “first agent” and“second lipophilic carrier”,“second agent” is used herein for ease of reference.
• second agent can be selected to be mixed with the one or more lipophilic carriers and the probiotic bacteria in the form of biofilm in the first layer.
• various systems may comprise more than two lipophilic carriers or agents.
• the probiotic bacteria in the form of biofilm is attached to a particle.
• the average diameter of the particles is in the range of 50 micrometers to 1,500 micrometers (pm). In some embodiments, average diameter of the particles is in the range of 50 pm to 1,200 pm, 50 pm to 1,100 pm, 50 pm to 1,000 pm, 55 pm to 1,200 pm, 55 pm to 1,000 pm, 57 pm to 1,200 pm, or 60 pm to 1000 pm, including any range therebetween. Each possibility represents a separate embodiment of the invention.
• the particle is selected from: MCC, dicalcium phosphate (DCP), seeds, a polysaccharide, or any combination thereof.
• DCP dicalcium phosphate
• a seed is selected from: cranberries, passionfruit, herbals, oat, or any combination thereof.
• polysaccharide encompasses any polymeric of carbohydrates, made from monomeric monosaccharide that are linked to one another via a glycosidic bond.
• the polysaccharide comprises or consists of alginate.
• the particle comprises or consists of a food grade particle.
• food grade particle comprises or consists of a polysaccharide, a fat crystal, a protein, or any combination thereof.
• a food grade particle comprising a fat crystal is selected from: glycerol monooleate, glyceryl stearyl citrate, or a combination thereof.
• a food grade particle comprising polysaccharide is selected from: corn starch, starch nanocrystals, cellulose nanocrystals, microcrystalline cellulose, nano- or methyl cellulose, chitin, chitosan, or any combination thereof.
• a food grade particle comprising a protein is selected from: b-lactoglobulin, lactoferrin, lactoferrin-polysaccharide, bovine serum albumin, gelatin, soy protein isolate, pea protein, Zein, or any combination thereof.
• the food grade particle is selected from: flavonoid (tiliroside), wax, shellac-xanthan gum, or any combination thereof.
• the particles in the composition described herein are adapted, configured or suitable for biofilm formation.
• the composition is formulated for vaginal administration. In some embodiments, the composition is formulated for rectal administration. In some embodiments, the composition is formulated for vaginal administration and rectal administration.
• the composition is adapted to colonize a vagina of a subject in need thereof. In some embodiments, the composition is adapted to colonize a rectum in a subject in need thereof.
• the composition is for use in the treatment of vaginosis, e.g., bacterial vaginosis.
• the composition is for use in treating or preventing a urogenital infection, dysbiosis, or both.
• the composition is for use in treating or preventing a dysbiosis related condition or disease.
• the term“dysbiosis related condition or disease” refers to any disease or a condition or a symptom associated therewith, which is characterized by imbalance of the microbial flora of an organism’s tissue or body.
• the dysbiosis related condition or disease is selected from: bacterial vaginosis, a urogenital infection, ulcerative colitis, inflammatory bowel disease (IBD), and Crohn's disease.
• the composition is for use in treating or preventing yeast vaginitis, viral infection, fungal infection, bacterial vaginosis, urinary tract infection, or any combination thereof, in subject in need thereof.
• the composition is for use in modifying bacterial composition, or restoring the native vaginal flora, gut flora, or both, in a target site of a subject in need thereof.
• modifying bacterial composition in a subject refers to reduction or elimination of an unwanted bacteria, in the subject.
• the at least one probiotic bacteria in the form of biofilm is personalized for the subject.
• the composition is determined or prepared according to the profile of the subject to be treated (e.g., personalized treatment).
• the composition comprises one or more strains selected from Lactobacillus plantamm, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Bifidobacterium breve, Bifidobacterium longum, and Faecalibacterium prausnitzii.
• the composition is an anti-inflammatory composition comprising one or more strains selected from Lactobacillus plantamm, Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. Bulgaricus, Bifidobacterium breve, Bifidobacterium longum, and Faecalibacterium prausnitzii.
• the composition is formulated for vaginal administration.
• the composition is formulated for rectal administration.
• the composition is provided in a form of suppository.
• the composition is provided in a form of vaginal suppository, cream, tablet, capsule, ointment, gel or microcapsule.
• the composition can be administered for treating a medical condition associated with any disease, medical condition, or disorder as described herein throughout in a subject in need thereof.
• the treatment is combined with antibiotics treatment.
• the treatment is prophylactic, i.e., after antibiotic treatment.
• the vaginal tissue is pre-treated with a colonization agent prior to administration of the suppositories, wherein the pre-treatment improves the receptiveness of the vaginal tissue for colonizing probiotic bacteria.
• a method for preventing or treating a dysbiosis related condition or disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the herein discloses composition, thereby preventing or treating a dysbiosis related condition or disease in a subject.
• a method for restoring the native flora in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the herein disclosed composition, thereby restoring the native flora in the subject.
• the subject is afflicted with or at risk of developing bacterial vaginosis, a urogenital infection, dysbiosis, ulcerative colitis, an inflammatory bowel disease (IBD), Crohn's disease, or any combination thereof.
• IBD inflammatory bowel disease
• the present invention provides a method for treating or reducing the risk of urogenital infections, dysbiosis, or both, in a subject, comprising administering an effective amount of a composition as described herein to the subject.
• the present invention provides a method for treating or reducing the risk of ulcerative colitis, inflammatory bowel disease (IBD), Crohn's disease, or any combination thereof, in a subject, comprising administering an effective amount of a composition as described herein to the subject.
• IBD inflammatory bowel disease
• Crohn's disease or any combination thereof
• the release of the at least one probiotic bacteria in the form of biofilm is controlled by the lipophilic carrier and the agent.
• the release of the at least one probiotic bacteria in the form of biofilm is controlled by the melting temperature of the lipophilic carrier.
• different mixtures of lipophilic carriers can be used in order to tune the melting temperature of the composition.
• the present invention provides a process for producing a composition as described herein.
• a method for preparing the composition of the invention comprising the steps of: (a) inoculating a growth medium comprising a particle with a L. crispatus, (b) incubating the particle with L. crispatus from step (a) under conditions suitable for allowing L. crispatus to attach to the particle; (c) inoculating the particle of step (b) with at least one additional bacterial species; and (d) culturing the inoculated particle of step (c) under conditions suitable for forming a biofilm comprising: L. crispatus and at least one additional bacterial species.
• the at least one additional bacterial species is selected from the group consisting of: L. jensenii, L. gasseri, and L. rhamnosus.
• a method for preparing the composition of the invention comprises the steps of:
• the at least one additional bacterial species is selected from: L. jensenii, L. gasseri, and L. rhamnosus.
• the at least one additional bacterial species is selected from the group consisting of: Bif. Longum sub.longum, and Bif. breve.
• the at least one additional bacterial species is selected from the group consisting of: Bl. obeum, Bl. coccoides. Bac. vulgatus, and Dorea (Eubacterium) formicigenerans.
• the inoculating steps are performed simultaneously.
• simultaneously refers to that the particle is inoculated with F. prausnitzii and the at least one additional bacteria at the same time.
• simultaneously refers to that the particle is inoculated with F. prausnitzii and the at least one additional bacteria with no additional step therebetween.
• simultaneously refers to that the particle is inoculated with F. prausnitzii and the at least one additional bacteria with no incubation time therebetween.
• the present invention provides a method or a process for producing a composition as described herein, comprising the steps of (i) mixing at least one probiotic bacteria in the form of biofilm with a first lipophilic carrier, and optionally a first agent, thereby forming a mixture and (ii) heating the mixture to a first heating temperature. [0185] In some embodiments, the process further comprises the step of (iii) adding a second lipophilic carrier and a second agent.
• the ratio of the at least one probiotic bacteria in the form of biofilm and the first lipophilic carrier is in the range of 1: 1 to 1: 10, 1:2 to 1:10, 1:5 to 1: 10, 1: 1 to 1:9, 1: 1 to 1:5, including any range therebetween.
• the ratio of the at least one probiotic bacteria in the form of biofilm and the first agent is in the range of 1:0.1 to 10: 1, 1:0.5 to 10: 1, 1: 1 to 10: 1, 1:2 to 10: 1, 1:0.1 to 9: 1, 1:0.1 to 8:1, 1:0.1 to 1: 1, 1:0.1 to 2: 1, including any range therebetween.
• the first lipophilic carrier and the second lipophilic carrier comprise one or more fatty acids with a saturated content of more than 40%, more than 41%, more than 45%, more than 48%, or more than 50%, including any value therebetween.
• the first lipophilic carrier and the second lipophilic carrier comprise one or more hydrogenated fats.
• the heating temperature is determined according to the melting temperature of the one or more hydrogenated fats.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• the term“treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• strains used in this study were purchase from either ATCC or DSMZ. Strains used in this study were: Lactobacillus crispatus (DSM 20584), Lactobacillus jensenii (DSM 20557), Lactobacillus gasseri (DSM), Lactobacillus rhamnosus (DSM/ATCC), Bifidobacterium longum sub.longum, Bifidobacterium breve, Bifidobacterium adolescentis, Faecalibacterium prausnitzii, Blautia obeum, Blautia coccoides, Bacteroides vulgatus, Dorea (Eubacterium) formicigenerans, Bacteroides uniformis, Ruminococcus gnavus, Blautia producta, Clostridium leptum, Clostridium (Blautia) coccoides, and Blautia (Ruminococcus) obeum.
• L. gasseri and L. rhamnosus were aerobically grown in Animal-based medium (Himedia) or a Nonanimal-based growth medium suited for industrial production (NuCel by Procelys, France).
• L. jensengfii and L. crispatus were grown anaerobically (90% N2, 5%C0 2 , 5% 3 ⁇ 4 atmosphere). Anaerobic experiments were performed in the Bactron anaerobic workstation. Planktonic cultures
• planktonic bacteria resistant to acidity and antibiotics were determined.
• an overnight culture of vaginal bacteria was diluted to achieve 10 5 -10 9 colony forming units (CFU) per mL, based on the number of bacteria in biofilm of the specific strain.
• Planktonic bacteria were exposed to different acidity for lh at 37 °C, similar to the procedure done for bacteria in form of biofilm.
• samples were centrifuges (max. speed, 2 min) and supernatant was removed. Bacteria pellet was then resuspended in phosphate buffered saline (PBS)xl before plating and CFU counting.
• PBS phosphate buffered saline
• a culture of Lactobacillus sp. was started from glycerol stock (12.5%) and was incubated overnight at 37 °C, 150-180 rpm at aerobically or anaerobically conditions, based on the specific strain. The culture was diluted to an OD600 of 0.05, for the final biofilm culture.
• the bacteria in form of biofilm cultures were obtained as described in WO2016181228A2, which is incorporated herein by reference in its entirety.
• Bacteria in form of biofilm were grown at 37 °C with continuous stirring. All bacteria in form of biofilm experiments were carried out in laboratory- scale production either small-scale (volume of 30 mL) or medium-scale (volume of 250 mL or 500 mL in a fermenter of 2 L). Analysis of bacteria in form of biofilm growth was carried out every 24 h incubation during a total of 72 h. Each day, medium was replaced with a fresh medium and the number of bacteria in biofilm was quantified. Measurements of viable cells were done by counting CFU on agar plates.
• bacteria in form of biofilm developmental stage was tested by exposing the bacteria in form of biofilm to extreme conditions such as low pH and antibiotics (for further details see section‘pH and antibiotics resistance assays’). Results were latter compared to planktonic bacteria to demonstrate the advantage of bacteria in form of biofilm over free-living bacteria.
• MCC Microcrystalline cellulose
• DCP Di calcium phosphate
• Cranberries 500-600 pM
• Alginate 1,000 pM
• Bacteria in form of biofilm was grown during 24 h and assessed for their growth development.
• Matrix:Medium ratio - bacteria in form of biofilm growth during 24 h was evaluated as a function of ratio between matrix (MCC) to medium.
• MCC matrix
• a sample from the media-matrix solution were transfer to tubes and then centrifuge at 500 rpm for 3 min at RT. Subsequently, the supernatant was discarded, and the pellet was washed with PBS to remove of planktonic bacteria that precipitated during centrifugation. Samples were centrifuge again at 500 rpm for 3 min at RT. Following centrifugation, supernatant was removed and for each treatment (pH or antibiotics) 1 g of bacteria in form of biofilm was used.
• bacteria in form of biofilm were exposed for 1 h to 5 mL PBS with different acidity: pH 2 and 3.5 (the pH of PBS xl was adjusted to 2 and 3.5, respectively, using 2M HC1) for lh at 37 °C, 100 rpm.
• For antibiotics assay 1 gr of bacteria in form of biofilm were exposed to 3 sequential concentration of antibiotics, which were well above the MIC value of the specific strain. Bacteria in form of biofilm were incubated in 5 mL growth medium with or without antibiotic (the latter was used as a control) for 24 h at 37 °C.
• CFU were determined at the end of each resistance assay. First, serial dilutions were conducted, and bacteria were plated in triplicate onto MRS agar plates. The plates were incubated at 37 °C in aerobic or anaerobic conditions, based on the strain growth condition (see above), for 48-72h prior to CFU counting.
• biofilm formation and development of biofilm on the particles was tested by exposing the bacterial population to extreme conditions such as acidic pH (pH 3.5 and 2), a pH value close to the pH that prevails in a woman vagina (pH 4 5), and antibiotics (CB, carbenicillin; CIP, ciprofloxacin; VAN, vancomycin; NVB, novobiocin). pH resistance assay was conducted daily, and antibiotic assay was performed following 48 hours or 72 hours of incubation. Results from these assays were latter compared to planktonic bacteria of the same species to show the advantage of the produced bacterial population technologies over free-living bacteria.
• extreme conditions pH (pH 3.5 and 2), a pH value close to the pH that prevails in a woman vagina (pH 4 5), and antibiotics (CB, carbenicillin; CIP, ciprofloxacin; VAN, vancomycin; NVB, novobiocin). pH resistance assay was conducted daily, and antibiotic assay was performed following 48 hours or 72 hours of
• a culture of each bacteria strain that were used in the experiment was started from -80 °C stock (5% DMSO) and was incubated in anaerobic conditions overnight at 37 °C. Unless otherwise stated, the culture was diluted to an ODeoo of 0.05 (FaP, 0.03), for the final biofilm culture.
• the mono-culture in each experiment consist of growth medium (BfA, Bifidobacterium medium or Food-grade+casein; FaP, YCFAC or RCM), 20% mono culture technologies particles (MCC:DCP, 1: 1) and planktonic bacteria from the overnight culture.
• Mono-culture were grown at 37 °C with either continuous stirring conditions (small-scale, 80 rpm; fermentor, 300 rpm) or static conditions (only in small-scale). All the experiment was held in anaerobic glove box, however, the growth media for Bifidobacterium spp. was generally aerobic and for the experiments with Faecalibacterium was anaerobic. When continuous stirring was employed, the vessel was first left 5 h in static conditions to allow the bacteria to attach to the matrix. All mono-culture experiments were carried out in laboratory-scale production either small-scale (volume of 50 or 60 mL) or fermentation (volume of 3 L).
• a sample from the media-matrix solution were transfer to tubes and then centrifuge at 500 rpm for 3 min at RT. Subsequently, the supernatant was discarded, and the pellet was washed with PBS to remove of planktonic bacteria that precipitated during centrifugation. Samples were centrifuge again at 500 rpm for 3 min at RT. Following centrifugation, supernatant was removed and for each treatment (pH or antibiotics) 1 g of sample was used. A third wash was done with 10 mL PBS xl. Following centrifugation (500 rpm, 3 min), 9 mL was removed and the biofilm in the 1 mL remaining PBS solution were vortex for 1.5 min at high speed to release bacteria from biofilm that were attached to particles.
• the formulation of the suppositories consists of bacteria in form of biofilm grown as described above, mixed in pharmaceutically acceptable excipients (oil-based carriers) and/or a prebiotic agent (such as, cranberries, ascorbic acid and antibiotics). Bacteria in form of biofilm used was either lyophilized (dry) or wet bacteria in form of biofilm.
• biofilm formation and development of biofilm on the particles was tested by exposing the Bacteria in form of biofilm to extreme conditions such as acidic pH (pH 3.5 and 2) and antibiotics (CB, carbenicillin; CIP, ciprofloxacin; VAN, vancomycin; NVB, novobiocin). pH resistance assay was conducted daily, and antibiotic assay was performed following 48h or 72 h of incubation. Results from these assays were latter compared to planktonic bacteria of the same species to show the advantage of Bacteria in form of biofilm over free-living bacteria.
• extreme conditions pH (pH 3.5 and 2) and antibiotics (CB, carbenicillin; CIP, ciprofloxacin; VAN, vancomycin; NVB, novobiocin). pH resistance assay was conducted daily, and antibiotic assay was performed following 48h or 72 h of incubation. Results from these assays were latter compared to planktonic bacteria of the same species to show the advantage of Bacteria in form of biofilm over free
• Planktonic bacteria produced moderate bacteria yield of ⁇ 10 6 cells/mF at pH 7 (control, Fig. 2). Bacteria yield at pH 3.5 was comparable to control. However, at higher acidic conditions (pH 2) planktonic bacteria did not survived. Finally, exposure to antibiotics showed a MIC of 64 pg/mF of CB and 16 pg/mF of NVB and 4 pg/mF to VAN
• Lactobacillus iners Bacteria inform of biofilm - small-scale experiment
• Bacteria in form of biofilm was tested for resistance to antibiotics (Fig. 5). While Bacteria in form of biofilm demonstrated resistance to all three types of antibiotics compare to planktonic bacteria, the highest resistance was observed for the CB antibiotic. When exposed to CB, bacteria yield was either not affected or slightly affected by antibiotic concentration, even after 24 h incubation. Bacteria in form of biofilm exposure to VAN and NVB antibiotics showed a similar trend of bacteria growth between incubation days: after an initial decrease of ⁇ 4 log, numbers of bacteria did not change significantly with increasing concentrations. When antibiotics resistance data are pooled together, it appears that after 48 h the inventors obtained the highest resistance of biofilm to increasing amount of antibiotics.
• Planktonic bacteria produced bacteria yield of ⁇ 10 6 cells/mL at control conditions (pH 7, Fig. 6). No significant difference was observed in bacteria yield at pH 3.5 compared to control. However, planktonic bacteria did not survive exposure to pH 2.
• planktonic bacteria Exposure of planktonic bacteria to CB and NVB and VAN antibiotics resulted in low bacterial resistance to antibiotic with a MIC of 8 pg/mL, 2 pg/mL and 1.5 pg/mL, respectively. However, planktonic bacteria were not susceptible to CIP and displayed full growth of bacteria cells regardless the employed antibiotic concertation (Table 4).
• Planktonic LCr exhibited similar results to planktonic LJ when exposed to low pH treatments and increased antibiotic concentrations (Fig. 10). Exposure to pH 3.5 did not significantly affected bacteria cells compare to control (pH 7) whereas at pH 2 bacteria did not survive.
• planktonic LCr When planktonic LCr were treated with antibiotics, low bacteria resistance was observed for CB, NVB and VAN with MIC values of 4 pg/mL, 2 pg/mL and 1.5 pg/mL, respectively (Table 5). However, planktonic bacteria were not susceptible to CIP and displayed full growth of bacteria cells regardless the employed antibiotic concertation. Table 5. MIC of different antibiotics for planktonic LCr Values are expressed in pg/mL.
• Bacteria yield in biofilm was slightly higher ( ⁇ l-2 log) at the medium-scale experiment compare with the small-scale experiment (Fig. 12). However, the increase in Bacteria in form of biofilm when exposure to pH 3.5 was comparable to the ones observed in the small-scale experimental set up. High survival of LCr Bacteria in form of biofilm was recorded after the 1 st and 3 rd of incubation, when exposed to the lowest pH treatment. In both experimental set-up, Bacteria in form of biofilm at this pH treatment, perished after
• LCr Bacteria in form of biofilm Resistance of LCr Bacteria in form of biofilm to antibiotics was then investigated (Fig. 13). Similar to LJ Bacteria in form of biofilm, LCr Bacteria in form of biofilm showed high resistance to CBand NVB with a slightly better growth (1 log) of LCr Bacteria in form of biofilm after exposure to NOVO.
• planktonic LG were later tested for their susceptibility to antibiotics (Table 6), MIC values were established; similar to LJ and LCr, planktonic LG were highly sensitive to for CB, NVB and VAN (4 pg/mL, 2 pg/mL and 1.5 pg/mL, respectively) while for CIP bacteria showed full resistance.
• LG planktonic and Bacteria in form of biofilm cultures showed large difference between the two modes of growth in relation to their resistance to extreme conditions.
• the advantage of LG Bacteria in form of biofilm over suspended bacteria is therefore evident.
• Planktonic LRh cells showed similar response to all bacteria strains that are described in this project, when exposed to increase acidity; no difference was detected between the control sample and Bacteria in form of biofilm exposed to pH 3.5 whereas cells disappeared at pH 2 (Fig. 18).
• the formulation of the suppositories consists of Bacteria in form of biofilm, mixed in pharmaceutically acceptable excipients (oil-based carriers) and/or a supplement.
• Bacteria in form of biofilm was used either as lyophilized (dry) powder or as wet Bacteria in form of biofilm (at the end of 72 h incubation).
• a stability assay of the Bacteria in form of biofilm survival in suppositories was preformed once a month, for the duration of 6 months. Each month, one suppository from each formulation was melted in PBS(xl) and bacteria were plated for CFU counting (see Analytical methods).
• the basic formulation of the oil-based carriers included vegetable (palm) butter and cocoa butter, in a ratio of 1:5, respectively as well as few drops of Lecithin to aid with the homogeneity of the mixture.
• the inventors aimed to reduce the volume of oil-based carriers compare to bacteria in form of biofilm, thus increasing the quantity of bacteria in form of biofilm in the suppositories.
• Two ratios of bacteria in form of biofilm to carriers were tested, 1:5 and 1: 10, respectively (Fig. 24). Results showed no difference in Bacteria in form of biofilm growth between the two ratios, thus allowing us to raise Bacteria in form of biofilm quantity in the suppositories composition.
• LG Bacteria in form of biofilm growth and development was as follows Microcrystalline cellulose: Di calcium phosphate (MCC:DCP)> MCC> Alginate> Cranberries.
• LJ and LG were identified as anaerobic/slow-growing bacteria whereas LRh was identified as an aerobic/fast-growing bacteria.
• LJ or LG planktonic cells were co inoculated with LRh, they were added prior to LRh with a gap of about 1 hour between the additions of the strains. No significant difference was recorded in biofilm growth (max. of 1 log) and development (resistance to pH), in comparison to their growth as monoculture (Fig. 27). Number of biofilm-embedded bacteria were between 10 6 -10 7 CFU/mL.
• Bacterial culture of LRh, LCr, LJ and LG, in this experiment is prepared similarly to the described in the Material and Method section, with the following adjustment: when planktonic bacteria from each strain is added to the mixture of growth medium and matrix, at the beginning of the experiment, each strain is added one after the other with a gap of 0.5-1 hour between them. There are two treatments:
• Bacteria strains that is grown in co-culture is compared to monoculture growth and differences in metabolites are analyzed using metabolomics techniques.
• Bacterial population culture of LRh, LCr, LJ and LG, grown either together as coculture or each strain separately, are prepared as described in the Material and Method section. At the end of 72 hours of incubation, the growth medium where the bacterial population are cultured, are centrifuge at maximum speed, for 10 min to remove debris and planktonic bacteria. The supernatant is transferred to a clean tube and are centrifuge again (same conditions). The supernatant is kept at -80 °C till analysis of metabolism. Additionally, the effect of addition of different supplements (for example carbohydrates such as fructooligosaccharide and trace elements such as iron) to the growth media on coculture growth compare to monoculture is investigated.
• supplements for example carbohydrates such as fructooligosaccharide and trace elements such as iron
• LG bacterial population growth and development was as follows Microcrystalline cellulose: Di calcium phosphate (MCC:DCP)> MCC> Alginate> Cranberries. Based on these results the inventors suggest that the combination of both reduced particle size and the type of matrix positively influenced LG Bacteria in form of biofilm growth and development: 1) particle sizes - smaller-particles size (higher surface to volume ratio) may allow more bacteria to attach per particle volume, thus improving bacteria population growth: bacterial population was enhanced on MCC or MCC:DCP combination (80- 150 pm) compare to alginate beads (1000 pm); 2) type of matrix - the inventors suggest here two possible explanations, without wishing to be bound to any particular theory, for the contribution of the specific matrix to biofilm growth and formation. (Fig. 31).
• BfA B. adolescentis
• BfA was added either in parallel with BLL and BfBr (‘control’) or 24 h before the addition of BLL and BfBr (‘Advantage to BfA’). Higher relative abundance of BfA was observed when it was added 24 h prior to the addition of BLL, BB-12 and BfBr (Fig. 36). Improved growth of BfA ( ⁇ 1 log difference) was observed when it was added 24 h prior to the addition of BLL, BB-12 and BfBr (Advantage to BfA) compare to its addition in parallel to the other Bifidobacterium species (Control; Figs. 37- 38).
• FaP F. prausnitzii
• BIO Blautia obeum
• BIC Blautia coccoides
• FaP showed an improved growth when cocultured with either BIO, BIC or both for 48 h, compare to its growth alone (Fig. 39).
• FaP also showed an improved growth when cocultured with either Bacteroides vulgatus ( BaV ), or BaV and Dorea (Eubacterium) formicigenerans (DoF), compare to its growth alone (Fig. 40).
• the inventors further examined FaP growth compared to its growth with either BIP, BfA or Bacteroides thetaiotaomicron (BaT). Also, the inventors examined the effect of higher initial OD of FaP on its growth. FaP was found to have an improved growth when coculture with either BfA or BaT for 48 h (Fig. 41).
• the inventors further examined FaP relative abundance during 72 h of incubation when coculture with few species from the Clostridiales and Bacteroides families. When FaP was cocultured with Bacteroides spp. a clear advantage was observed compared to its growth in coculture with only Clostridiales spp. (Fig. 42).
• FaP relative abundance in planktonic and biofilm phase during 72 h of incubation when coculture with few species from the Clostridiales and Bacteroides families. FaP was found to have a higher relative abundance in biofilm (Fig. 43B) compared to the planktonic form (Fig. 43A) during 72 h of growth.

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