US20220088094A1 - Probiotic compositions comprising lactobacillus reuteri strains and methods of use - Google Patents

Probiotic compositions comprising lactobacillus reuteri strains and methods of use Download PDF

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US20220088094A1
US20220088094A1 US17/502,716 US202117502716A US2022088094A1 US 20220088094 A1 US20220088094 A1 US 20220088094A1 US 202117502716 A US202117502716 A US 202117502716A US 2022088094 A1 US2022088094 A1 US 2022088094A1
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composition
isolated
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lactobacillus reuteri
animal
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Arvind Kumar
Dharanesh Mahimapura GANGAIAH
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Biomedit LLC
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Elanco US Inc
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • A61K35/741Probiotics
    • A61K35/744Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747Lactobacilli, e.g. L. acidophilus or L. brevis
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
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    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
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    • A23K20/163Sugars; Polysaccharides
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/70Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75Feeding-stuffs specially adapted for particular animals for birds for poultry
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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    • A61K35/66Microorganisms or materials therefrom
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    • A61K35/741Probiotics
    • A61K35/742Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04Antibacterial agents
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
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    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor
    • C12N1/205Bacterial isolates
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00Lactic or propionic acid bacteria
    • A23V2400/11Lactobacillus
    • A23V2400/173Reuteri
    • A23Y2220/71
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/01Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225Lactobacillus
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to probiotic compositions and methods for improving animal health.
  • the probiotic compositions include one or more isolated strains of Lactobacillus sp which colonizes the gastrointestinal tract to increase the health of an animal.
  • Direct fed microbials are microorganisms which colonize the gastrointestinal tract of an animal and provide some beneficial effect to that animal.
  • the microorganisms can be bacterial species, for example those from the genera Bacillus, Lactobacillus, Lactococcus , and Entercoccus.
  • the microorganisms can also be yeast or even molds.
  • the microorganisms can be provided to an animal orally or mucosally or, in the case of birds, provided to a fertilized egg, i.e. in ovo.
  • the beneficial activity provided by a DFM can be the synthesis of vitamins or other nutritional molecules needed for a healthy metabolism of the host animal.
  • a DFM can also protect the host animal from disease, disorders, or clinical symptoms caused by other, pathogenic microorganisms.
  • the DFM may naturally produce factors having inhibitory or cytotoxic activity against certain species of pathogens, such as deleterious or disease-causing bacteria.
  • the present invention provides compositions and methods for improving animal health.
  • the invention provides a composition having at least one of an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain, wherein said composition increases animal health when an effective amount is administered to an animal, as compared to an animal not administered the composition.
  • the first isolated first Lactobacillus reuteri strain includes at least one of: a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, and a nucleic acid that encodes for an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.
  • the second isolated second Lactobacillus reuteri strain includes at least one of: a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28, and a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29.
  • the invention provides a method of increasing animal health.
  • the method includes administering an effective amount of the above composition to an animal.
  • FIG. 2 depicts antimicrobial effect of L. reuteri strain 3632.
  • Pathogenic bacteria but not nonpathogenic Lactobacillus acidophilus are killed by L. reuteri strain 3632, as evidenced by a clear “halo” surrounding strain 3632.
  • Pathogenic bacteria shown are Clostridium perfringes, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Salmonella Typhimurium, F. necrophorum , and Campylobacter jejuni.
  • FIG. 3 depicts colonization of L. reuteri strain 3632 in chickens, via oral administration route. Left shows the experimental timeline. Right shows colony formation and PCR & sequence confirmation.
  • FIG. 4 depicts experimental timeline to determine colonization of L. reuteri strain 3632 in chickens, via in ovo administration route. Chicks were harvested at day 3 and day 7, and the strain is characterized.
  • FIG. 5 depicts colonies from chicks sacrificed at day 3.
  • Strain 3632 is found in the tissues of chicks at 3 days post hatching, while colonization of a control strain declines after day 3.
  • FIG. 7 depicts characterization of L. reuteri strain 3630 and 3632 colonization in chicks via on ovo(spray) and in ovo administration route. Left shows the experimental timeline. Right shows CFU of L. reuteri strains 3630 and 3632 of chicks sacrificed at 3 days and 7 days.
  • FIG. 8 depicts data from birds vaccinated with coccidiosis vaccine on day 0 and fed with L. reuteri in feed from day 0 to day 42 @ 10 ⁇ circumflex over ( ) ⁇ 7 CFUs/Kg of feed or with BMD_55.
  • FCR Top Feed conversion Ratio
  • FIG. 9 depicts quantification of in vitro kill effect of L. reuteri strain 3632 on Clostridium perfringens .
  • L. reuteri appears to inhabit C. perfringens growth almost by 4 logs.
  • CFU counts were determined 24 hours after coculture in BHI broth.
  • L. reuteri were enumerated on MRS agar plates and C. perfringens was enumerated on OPSP plates. This data is from one representative experiment of 3 replicates.
  • FIG. 10 depicts the effect of prebiotics on the growth of Lactobacillus reuteri strains 3630 & 3632.
  • the prebiotics tested include fructooligosaccharides (FOS), arabinoxylan oligosaccharides (AXOS), mannan-oligosaccharides (MOS), or galacto-oligosaccharides (GOS). Increased growth is observed with GOS.
  • FOS fructooligosaccharides
  • AXOS arabinoxylan oligosaccharides
  • MOS mannan-oligosaccharides
  • GOS galacto-oligosaccharides
  • FIGS. 11A-11B depict biochemical metabolic profile of two Lactobacillus reuteri strains.
  • the figure represents metabolic data obtained by principal component analysis (PCA) of two strains of Lactobacillus reuteri that were cultured individually and together.
  • FIG. 11A represents the cell pellet of the culture
  • 1_P-30 is the pellet from strain 3630
  • 2_P-32 is the pellet from strain 3632
  • 3_P-30-32 is the pellet from a co-culture of strain 3630 and strain 3632.
  • 11B represents the supernatant of the culture
  • 1_S-30 is the supernatant from strain 3630
  • 2_S-32 is the supernatant from strain 3632
  • 3_S-30-32 is the supernatant from a co-culture of strain 3630 and strain 3632.
  • 4_Medial is AOF-MRS media control with no glucose but 0.5% GOS.
  • data represents subject molecule that is secreted into the supernatant.
  • Lactobacillus reuteri strain 3630 2) Lactobacillus reuteri strain 3632, 3) co-culture of Lactobacillus reuteri strain 3630 and Lactobacillus reuteri strain 3632, and 4) media control.
  • Data are scaled such that the median value measured across all samples was set to 1.0.
  • FIG. 12 depicts levels of dimethylglycine, allo-threonine, and 1-methyl-4-imidazoleacetate secreted by Lactobacillus reuteri strains.
  • FIG. 13 depicts levels of 4-imidazoleacetate, lysine, and N6-methyllysine secreted by Lactobacillus reuteri strains.
  • FIG. 14 depicts levels of N6, N6-dimethyllysine, 5-aminovalerate, and tyrosine secreted by Lactobacillus reuteri strains.
  • FIG. 15 depicts levels of 4-hydroxyphenylpyruvate, indolacetate, and gamma-glutamylglutamine secreted by Lactobacillus reuteri strains.
  • FIG. 16 depicts levels of glucose 6-phosphate, 4-hydroxyl-2-oxoglutaric acid, and myo-inositol secreted by Lactobacillus reuteri strains.
  • FIG. 17 depicts levels of Uridine 5′-monophosphate (UMP), Cytidine 5′-monophosphate (5′-CMP), and 3′-5′-uridylyluridine secreted by Lactobacillus reuteri strains.
  • UMP Uridine 5′-monophosphate
  • CMP Cytidine 5′-monophosphate
  • 3′-5′-uridylyluridine secreted by Lactobacillus reuteri strains 3′-5′-uridylyluridine secreted by Lactobacillus reuteri strains.
  • FIG. 18 depicts levels of O-sulfo-L-tyrosine, indole 3 acetamide, and indolin-2-one secreted by Lactobacillus reuteri strains.
  • FIG. 19 depicts levels of daidzein secreted by Lactobacillus reuteri strains.
  • the present invention provides compositions and methods for increasing animal health.
  • the invention provides a composition having at least one of an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain.
  • the isolated first Lactobacillus reuteri strain is strain 3632.
  • strain 3632 has a nucleic acid or amino acid sequence including at least one of SEQ ID NOs: 1-24 and 26, sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs: 1-24 and 26.
  • Lactobacillus reuteri strain 3632 was deposited on 19 Jun. 2020 according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Va., 20110, USA. The deposit has been assigned ATCC Patent Deposit Number PTA-126788.
  • the isolated second Lactobacillus reuteri strain is strain 3630.
  • strain 3630 has a nucleic acid sequence including at least one of SEQ ID NOs: 25 and 27-43, sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs: 25 and 27-43.
  • Lactobacillus reuteri strain 3630 was deposited on 19 Jun. 2020 according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Va., 20110, USA. The deposit has been assigned ATCC Patent Deposit Number PTA-126787.
  • the isolated strains of the present disclosure are not genetically modified by recombinant or genetically engineered means.
  • the composition disclosed herein includes an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain at a ratio of approximately 0.75-1.5:1. In a preferred embodiment, the composition includes about equal amounts of the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain.
  • compositions disclosed herein can be formulated as animal feed, feed additive, food ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof.
  • the composition includes water.
  • compositions disclosed herein includes the isolated first Lactobacillus reuteri strain in an amount of about 10 ⁇ circumflex over ( ) ⁇ 2-10 ⁇ circumflex over ( ) ⁇ 8 CFU/kg of the composition, about 10 ⁇ circumflex over ( ) ⁇ 4-10 ⁇ circumflex over ( ) ⁇ 7 CFU/kg of the composition, about 10 ⁇ circumflex over ( ) ⁇ 3-10 ⁇ circumflex over ( ) ⁇ 5 CFU/kg of the composition, about 10 ⁇ circumflex over ( ) ⁇ 2 CFU/kg of the composition, about 10 ⁇ circumflex over ( ) ⁇ 3 CFU/kg of the composition, or about 10 ⁇ circumflex over ( ) ⁇ 7 CFU/kg of the composition.
  • compositions of the disclosure exhibit markedly different functional and/or structural characteristics/properties, as compared to their closest naturally occurring counterpart.
  • the Lactobacillus reuteri strain of the disclosure are structurally different from a Lactobacillus reuteri strain as it naturally exists in a gastrointestinal tract.
  • the Lactobacillus reuteri strain can be isolated and purified, such that it is not found in the milieu of the gastrointestinal tract, the Lactobacillus reuteri strain can be present at concentrations that do not occur in the gastrointestinal tract, the Lactobacillus reuteri strain can be associated with acceptable carriers that do not occur in the gastrointestinal tract, the Lactobacillus reuteri strain can be formulated to be shelf-stable and exist outside the gastrointestinal tract, and said microbe can be combined with other microbes at concentrations that do not exist in the gastrointestinal tract. Further, the Lactobacillus reuteri strain of the disclosure are functionally different from a Lactobacillus reuteri strain as it naturally exists in a gastrointestinal tract.
  • Lactobacillus reuteri strain when applied in an isolated and purified form can lead to modulation of the gastrointestinal microbiome, increased weight gain, increased feed utilization, decreased amounts of microbial pathogens, and decreased pathogen-associated gastro-intestinal lesions.
  • the present disclosure also provides methods of increasing animal health, wherein the method includes administering an effective amount of the composition to an animal.
  • composition disclosed herein and above increases animal health by providing positive health benefits when administered to an animal, as compared to an animal that has not been administered the composition.
  • animal includes bird, poultry, a human, or a non-human mammal. Specific examples include chickens, turkey, dogs, cats, cattle and swine. The chicken may be a broiler chicken or egg-laying or egg-producing chicken.
  • Positive health benefits include decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate.
  • the compositions disclosed herein decreases feed conversion ratio by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein increases poultry weight by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease pathogen-associated lesion formation in the gastrointestinal tract by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease pathogen colonization by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein reduce inflammation by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease mortality rate by at least 1%, at least 5%, at least 25%, or at least 50%.
  • following values may be combined in any manner to create a minima, a maxima, or a range for decreasing feed conversion ratio, increasing poultry weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, and decreasing mortality rate, 1%, 5%, 25%, 50%, and 75%.
  • the decrease in pathogen-associated lesion formation may be decreased by approximately 1% to 5%, and more preferably between approximately 5% to 50%.
  • pathogen includes Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus , and E. coli bacterium. Further examples of pathogens include Salmonella typhimurium, Salmonella infantis, Salmonella Hadar, Salmonella enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptoccus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni , and Fusobacterium necrophorum.
  • compositions may be administered orally, parentally, nasally, or mucosally.
  • administration includes feeding the poultry, or spraying onto the poultry. In other aspects, administration includes on ovo administration or in ovo administration,
  • the animal is vaccinated in conjunction with administration.
  • the animal may be vaccinated prior to administration of the compositions disclosed herein.
  • the animal may be vaccinated with an coccidiosis vaccine.
  • Coccidiosis vaccines are known in the art, for example, COCCIVAC.
  • administration is by way of injection or infusion.
  • the composition is administered to a cow by way of intra-mammary infusion.
  • strains disclosed herein demonstrate certain phenotypic properties. Without wishing to be bound by theory, it is believed that these phenotypic properties at least contribute to increasing animal health.
  • the isolated strains secrete at least one of cyclic dipeptides (cyclo(his-phe) and cyclo (phe-pro), short chain fatty acids (2-hydroxy-3-methylvalerate and alpha-hydroxyisocaproate), betaine, dimethylglycine, essential amino acids (e.g., allo-threonine, phosphothreonine, histidine, lysine, phenylalanine, tryptophan, leucine, isoleucine, and cysteine s-sulfate), nucleotides (e.g., adenosine 5′-monophosphate (AMP), uridine 5′-monophosphate (UMP), cytidine 5′-monophosphate (5′-CMP), and cytidine 2′3′-cyclicmonophosphate), myo-inositol, and indolin-2-one.
  • cyclic dipeptides cyclo(his-phe) and cyclo (p
  • the composition including the isolated first Lactobacillus reuteri strain (strain 3632) and the isolated second Lactobacillus reuteri strain (strain 3630) in combination will secrete certain beneficial molecules in larger quantities than when individually cultured. See for example, FIGS. 12-19 .
  • the animal administered the composition exhibits a shift in the microbiome content of the gastrointestinal tract. For example, there may be an increase in the amount of bacteroidaceae bacteria in the gut of an animal that has been administered the composition described herein, as compared to an animal that was not administered the composition.
  • the invention provides a method of treating, ameliorating the effects of, or preventing necrotic enteritis in an animal by administering a composition disclosed herein to an animal in need thereof.
  • the invention provides a method of treating, ameliorating the effects of or preventing bovine mastitis.
  • the method includes administering a composition disclosed herein to an animal in need thereof.
  • isolated means that the subject isolate has been separated from at least one of the materials with which it is associated in a particular environment. Thus, an “isolate” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture in association with an acceptable carrier.
  • individual isolates should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.
  • the isolated Lactobacillus reuteri strain exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular Lactobacillus reuteri strain, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual Lactobacillus reuteri strain in question. The culture can contain varying concentrations of said isolated Lactobacillus reuteri strain. The present disclosure notes that isolated and biologically pure microbes often necessarily differ from less pure or impure materials.
  • the composition includes a combination of two isolated Lactobacillus reuteri strains.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, in some embodiments as injectable solutions.
  • the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant.
  • a binder for compressed pills
  • a glidant for compressed pills
  • an encapsulating agent for a glidant
  • a flavorant for a flavorant
  • a colorant for a colorant.
  • the choice of carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. See Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2nd Ed. CRC Press. 504 pg.); and E. W. Martin (1970. Remington's Pharmaceutical Sciences. 17th Ed. Mack Pub. Co.).
  • delivery means the act of providing a beneficial activity to a host.
  • the delivery may be direct or indirect.
  • An administration could be by an oral, nasal, or mucosal route.
  • an oral route may be an administration through drinking water
  • a nasal route of administration may be through a spray or vapor
  • a mucosal route of administration may be through direct contact with mucosal tissue.
  • Mucosal tissue is a membrane rich in mucous glands such as those that line the inside surface of the nose, mouth, esophagus, trachea, lungs, stomach, gut, intestines, and anus.
  • administration may be in ovo, i.e. administration to a fertilized egg. In ovo administration can be via a liquid which is sprayed onto the egg shell surface, or an injected through the shell.
  • treating include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • a treatment may also be applied prophylactically to prevent or reduce the incidence, occurrence, risk, or severity of a clinical symptom, disorder, condition, or disease.
  • a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • each member may be combined with any one or more of the other members to make additional sub-groups.
  • additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
  • SEQ ID NO: 6 1 MILTTFIILI LMGCFINGHR RGLLTMTLML GTYIVAWIVA RQGAQLIGGW LKSLLPSIGT 61 PATFSESLLA NVNSNLFFYN GIAFMIIFTI VSILCHWGIR QLNWIKRIPV VGTVDKIAGG 121 LISFLIGYLI IYVVLLIMQL FPAGWWQMQI ANSELARFMI NQTPGIAHLV IDTLVQGG Agglutinin receptor precursor-1 (ARP-1) (natural) SEQ ID NO: 7 1 MNEYNAEMAK LNQGANAPVI TTNSVNQALS LKPENNATVD IEALNPRITF KRVEEGTKYA 61 GYQIFDKNNA YVNNIDGEFL RVTYTNLKNS TYKGSKISKI VVTYSDSTPT GNRITQSGLN 121 AVTEGANDNF LVVFEDPVRG DMHSTTVTAT YQYYDANGNL IDFS
  • Lactobacillus reuteri ( L. reuteri ) strains were isolated from older birds at the Research Center, Hannover from the cecal contents received from the Poultry Clinic, University of Hannover. All the seven strains are identified to be L. reuteri by 16S rRNA sequencing.
  • Antimicrobial susceptibility of bacterial isolates were tested using the AVIPRO® PLATE. All strains are resistant against colistin, doxycycline, enrofloxacin, erythromycin, neomycin, oxacillin, penicillin G, trimethroprim-sulfamethoxazole, tetracycline, tilmicosin and tylosin. All strains are resistant to streptomycin except strain 3632, and to tiamulin except strain 2098.
  • strains 2091, 2095, 2097 and 3630 resistance to cefpodaxime-proxetil is observed with strains 2091, 2095, 2097 and 3630; resistance to cefotaxime is observed with strains 2091, 2095 and 2097; and resistance to lincomycin is observed with strains 3630 and 3632. No strain was found to be resistant against amoxicillin, ceftiofur, erythromycin D, lincomycin-spectinomycin, and rifampicin under tested concentrations.
  • the L. reuteri isolates were tested for various desirable probiotic anti-infective properties, such as growth kinetics, ability to produce hydrogen peroxide, autoaggregation, enzyme profile, survival in the presence of ox bile and pancreatic enzymes, and sensitivity to heat shock and pH changes.
  • the L. reuteri strains are also tested for safety using a haemolytic assay.
  • strain 3632 which shows some unique properties, including the ability to autoaggregate in liquid media (comparable to that of the well-characterized human probiotic strain L. reuteri ATCC 23272).
  • L. reuteri 3632 also appears to produce an orange pigment, which resembles beta carotene in color. None of the other strains, including the human L. reuteri strain ATCC 23272 and L. acidophilus , produced orange colored pigment. None of the strains is found to be hemolytic on blood agar plates, suggesting that these isolates are less likely to be pathogenic to humans.
  • L. reuteri 3632 colonizes the intestine of 16-day old embryos.
  • 16 day-old embryos were obtained from Charles River and inoculated with 10 ⁇ circumflex over ( ) ⁇ 8 CFU/embryo of L. reuteri 3632 by air cell, yolk sac and allantoic routes. Embryos were sacrificed on 18 days and the intestines were harvested and plated on MRS agar plates. An uninoculated group was included as a control. L. reuteri was recovered from the intestines of the majority of the embryos, suggesting that L. reuteri 3632 colonizes the intestine of 16-day old embryos. L. reuteri 3632 was not recovered from any of the control (uninoculated) embryos, even after 2-days of enrichment in liquid culture.
  • L. reuteri and C. perfringens were cultured in BHI broth. CFU counts were determined 24 hours after coculture in BHI broth. L. reuteri were enumerated on MRS agar plates and C. perfringens was enumerated on OPSP plates. This data is from one representative experiment of 3 replicates. Co-culture- L. reuteri, L. reuteri recovered from co-cultured sample using L. reuteri -specific media. Co-culture- C. perfringens, C. perfringens recovered from co-culture sample using C. perfringens -specific media. See FIG. 9 .
  • Feed conversion ratio and European Broiler index were calculated. Birds that were fed L. reuteri demonstrated a reduction in feed conversion ratio and European Broiler index as compared to those fed BMD_55. See FIG. 8 .
  • L. reuteri strains 3630 & 3632 were grown individually and in combination, in the presence of several different prebiotics, including fructooligosaccharides (FOS), arabinoxylan oligosaccharides (AXOS), mannan-oligosaccharides (MOS), or galacto-oligosaccharides (GOS).
  • FOS fructooligosaccharides
  • AXOS arabinoxylan oligosaccharides
  • MOS mannan-oligosaccharides
  • GOS galacto-oligosaccharides
  • strains grown in the presence of GOS demonstrated increased growth as compared to strains grown in the presence of FOS, AXOS, or MOS.
  • An identified strain can effectively function as a direct feed microbial only if the strain is able colonize the host gastrointestinal tract.
  • the ability of L. reuteri strains to colonize chickens is assessed following different routes of administration.
  • L. reuteri is administered orally to 10-day old chicks. At various times post inoculation, chicks are sacrificed and the presence of L. reuteri is measured in the crop, small intestine, and cecum. Strain 3632 is able to colonize all three tissues and remain detectable for at least 18 days.
  • L. reuteri is administered by in ovo inoculation (i.e. injection into the egg) 3 days before hatching.
  • Strain 3632 is found in the tissues of chicks at 3 and 7 days post hatching, while colonization of a control strain declines after day 3.
  • L. reuteri is administered in ovo by spraying a liquid containing bacteria onto the egg surface.
  • Strains 3630 and 3632 are able to colonize and remain present in chicks 3 and 7 days post hatching. Inoculation by spray is just as efficient as in ovo injection but requires less manipulation of the incubating egg.
  • Table 2 shows quantifies the effect of L. reuteri 3632 treatment on survival.
  • the difference score between strain 3632 and the control is 3.3 and the difference between BMD and control is 2.5.
  • Strain 3632 provides increased survival over BMD treated chics.
  • a global metabolomics analysis of L. reuteri strains 3632 and 3630 were conducted. The strains were grown individually and in combination, and various molecules were analyzed in the cell pellet and the supernatant of the cultures. The strains were grown in AOF-MRS media control with no glucose but 0.5% GOS. The molecules in the supernatant represent molecules that are secreted by the cell.
  • PCA Principal component analysis
  • the data is shown in FIGS. 12-19 .

Abstract

The present invention relates to probiotic compositions and methods for increasing animal health. The probiotic compositions include one or more isolated strains of novel Lactobacillus reuteri strains which colonizes the gastrointestinal tract to increase the health of an animal.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of U.S. Ser. No. 17/428,510 filed on 4 Aug. 2021, which is a National Stage Application claiming priority from co-pending PCT Application No. PCT/US2020/016668 filed on 4 Feb. 2020, which in turn claims priority to U.S. Provisional Application No. 62/801,307, filed on 5 Feb. 2019, the contents of which are hereby incorporated by reference.
  • SEQUENCE LISTING
  • This application contains a Sequence Listing, which was submitted in ASCII format via EFS-Web, and is hereby incorporated by reference in its entirety. The ASCII copy, created on 4 Feb. 2020, is named “X30027-SeqListing_ST25.txt” and is 121 KB in size.
  • FIELD OF THE INVENTION
  • The present invention relates to probiotic compositions and methods for improving animal health. The probiotic compositions include one or more isolated strains of Lactobacillus sp which colonizes the gastrointestinal tract to increase the health of an animal.
  • BACKGROUND OF THE INVENTION
  • Direct fed microbials (DFMs), often also called probiotics, are microorganisms which colonize the gastrointestinal tract of an animal and provide some beneficial effect to that animal. The microorganisms can be bacterial species, for example those from the genera Bacillus, Lactobacillus, Lactococcus, and Entercoccus. The microorganisms can also be yeast or even molds. The microorganisms can be provided to an animal orally or mucosally or, in the case of birds, provided to a fertilized egg, i.e. in ovo.
  • The beneficial activity provided by a DFM can be the synthesis of vitamins or other nutritional molecules needed for a healthy metabolism of the host animal. A DFM can also protect the host animal from disease, disorders, or clinical symptoms caused by other, pathogenic microorganisms. For example, the DFM may naturally produce factors having inhibitory or cytotoxic activity against certain species of pathogens, such as deleterious or disease-causing bacteria.
  • There is a need in the art for probiotic compositions and methods that provide improved delivery of beneficial molecules to the gastrointestinal tract of an animal and improve animal health.
  • SUMMARY OF THE INVENTION
  • The present invention provides compositions and methods for improving animal health.
  • In one embodiment, the invention provides a composition having at least one of an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain, wherein said composition increases animal health when an effective amount is administered to an animal, as compared to an animal not administered the composition.
  • In one embodiment, the first isolated first Lactobacillus reuteri strain includes at least one of: a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, and a nucleic acid that encodes for an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8.
  • In one embodiment, the second isolated second Lactobacillus reuteri strain includes at least one of: a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28, and a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29.
  • In one embodiment, the invention provides a method of increasing animal health. The method includes administering an effective amount of the above composition to an animal.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 depicts antimicrobial effect of L. reuteri strain 3630. Pathogenic bacteria, but not nonpathogenic Lactobacillus acidophilus are killed by L. reuteri strain 3630, as evidenced by a clear “halo” surrounding strain 3630. Pathogenic bacteria shown are gram-positive pathogens Clostridium perfringes, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis; gram negative pathogens are Escherichia coli, Salmonella Typhimurium, and Campylobacter jejuni.
  • FIG. 2 depicts antimicrobial effect of L. reuteri strain 3632. Pathogenic bacteria, but not nonpathogenic Lactobacillus acidophilus are killed by L. reuteri strain 3632, as evidenced by a clear “halo” surrounding strain 3632. Pathogenic bacteria shown are Clostridium perfringes, Staphylococcus aureus, Streptococcus uberis, Streptococcus suis, Escherichia coli, Salmonella Typhimurium, F. necrophorum, and Campylobacter jejuni.
  • FIG. 3 depicts colonization of L. reuteri strain 3632 in chickens, via oral administration route. Left shows the experimental timeline. Right shows colony formation and PCR & sequence confirmation.
  • FIG. 4 depicts experimental timeline to determine colonization of L. reuteri strain 3632 in chickens, via in ovo administration route. Chicks were harvested at day 3 and day 7, and the strain is characterized.
  • FIG. 5 depicts colonies from chicks sacrificed at day 3. Strain 3632 is found in the tissues of chicks at 3 days post hatching, while colonization of a control strain declines after day 3.
  • FIG. 6 depicts colonies from chicks sacrificed at day 7. Strain 3632 is found in the tissues of chicks at 7 days post hatching, while colonization of a control strain declines after day 7.
  • FIG. 7 depicts characterization of L. reuteri strain 3630 and 3632 colonization in chicks via on ovo(spray) and in ovo administration route. Left shows the experimental timeline. Right shows CFU of L. reuteri strains 3630 and 3632 of chicks sacrificed at 3 days and 7 days.
  • FIG. 8 depicts data from birds vaccinated with coccidiosis vaccine on day 0 and fed with L. reuteri in feed from day 0 to day 42 @ 10{circumflex over ( )}7 CFUs/Kg of feed or with BMD_55. Top Feed conversion Ratio (FCR) of chickens administered L. reuteri 3632, as compared to chickens administered BMD_55 (bacitracin methylene disalicylate).
  • FIG. 9 depicts quantification of in vitro kill effect of L. reuteri strain 3632 on Clostridium perfringens. In a co-culture experiment, L. reuteri appears to inhabit C. perfringens growth almost by 4 logs. CFU counts were determined 24 hours after coculture in BHI broth. L. reuteri were enumerated on MRS agar plates and C. perfringens was enumerated on OPSP plates. This data is from one representative experiment of 3 replicates. Co-culture-L. reuteri, L. reuteri recovered from co-cultured sample using L. reuteri-specific media. Co-culture-C. perfringens, C. perfringens recovered from co-culture sample using C. perfringens-specific media.
  • FIG. 10 depicts the effect of prebiotics on the growth of Lactobacillus reuteri strains 3630 & 3632. The prebiotics tested include fructooligosaccharides (FOS), arabinoxylan oligosaccharides (AXOS), mannan-oligosaccharides (MOS), or galacto-oligosaccharides (GOS). Increased growth is observed with GOS.
  • FIGS. 11A-11B depict biochemical metabolic profile of two Lactobacillus reuteri strains. The figure represents metabolic data obtained by principal component analysis (PCA) of two strains of Lactobacillus reuteri that were cultured individually and together. FIG. 11A represents the cell pellet of the culture, 1_P-30 is the pellet from strain 3630, 2_P-32 is the pellet from strain 3632, and 3_P-30-32 is the pellet from a co-culture of strain 3630 and strain 3632. FIG. 11B represents the supernatant of the culture, 1_S-30 is the supernatant from strain 3630, 2_S-32 is the supernatant from strain 3632, and 3_S-30-32 is the supernatant from a co-culture of strain 3630 and strain 3632. 4_Medial is AOF-MRS media control with no glucose but 0.5% GOS.
  • For FIGS. 12-19, data represents subject molecule that is secreted into the supernatant. 1) Lactobacillus reuteri strain 3630, 2) Lactobacillus reuteri strain 3632, 3) co-culture of Lactobacillus reuteri strain 3630 and Lactobacillus reuteri strain 3632, and 4) media control. Data are scaled such that the median value measured across all samples was set to 1.0.
  • FIG. 12 depicts levels of dimethylglycine, allo-threonine, and 1-methyl-4-imidazoleacetate secreted by Lactobacillus reuteri strains.
  • FIG. 13 depicts levels of 4-imidazoleacetate, lysine, and N6-methyllysine secreted by Lactobacillus reuteri strains.
  • FIG. 14 depicts levels of N6, N6-dimethyllysine, 5-aminovalerate, and tyrosine secreted by Lactobacillus reuteri strains.
  • FIG. 15 depicts levels of 4-hydroxyphenylpyruvate, indolacetate, and gamma-glutamylglutamine secreted by Lactobacillus reuteri strains.
  • FIG. 16 depicts levels of glucose 6-phosphate, 4-hydroxyl-2-oxoglutaric acid, and myo-inositol secreted by Lactobacillus reuteri strains.
  • FIG. 17 depicts levels of Uridine 5′-monophosphate (UMP), Cytidine 5′-monophosphate (5′-CMP), and 3′-5′-uridylyluridine secreted by Lactobacillus reuteri strains.
  • FIG. 18 depicts levels of O-sulfo-L-tyrosine, indole 3 acetamide, and indolin-2-one secreted by Lactobacillus reuteri strains.
  • FIG. 19 depicts levels of daidzein secreted by Lactobacillus reuteri strains.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides compositions and methods for increasing animal health.
  • In one embodiment, the invention provides a composition having at least one of an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain.
  • In one embodiment, the isolated first Lactobacillus reuteri strain is strain 3632. As disclosed herein, strain 3632 has a nucleic acid or amino acid sequence including at least one of SEQ ID NOs: 1-24 and 26, sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs: 1-24 and 26. Lactobacillus reuteri strain 3632 was deposited on 19 Jun. 2020 according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Va., 20110, USA. The deposit has been assigned ATCC Patent Deposit Number PTA-126788.
  • In one embodiment, the isolated second Lactobacillus reuteri strain is strain 3630. As disclosed herein, strain 3630 has a nucleic acid sequence including at least one of SEQ ID NOs: 25 and 27-43, sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with at least one of SEQ ID NOs: 25 and 27-43. Lactobacillus reuteri strain 3630 was deposited on 19 Jun. 2020 according to the Budapest Treaty in the American Type Culture Collection (ATCC), ATCC Patent Depository, 10801 University Boulevard, Manassas, Va., 20110, USA. The deposit has been assigned ATCC Patent Deposit Number PTA-126787.
  • The isolated strains of the present disclosure are not genetically modified by recombinant or genetically engineered means.
  • In some embodiments, the composition disclosed herein includes an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain at a ratio of approximately 0.75-1.5:1. In a preferred embodiment, the composition includes about equal amounts of the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain.
  • The compositions disclosed herein can be formulated as animal feed, feed additive, food ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof. In one embodiment, the composition includes water.
  • The compositions disclosed herein includes the isolated first Lactobacillus reuteri strain in an amount of about 10{circumflex over ( )}2-10{circumflex over ( )}8 CFU/kg of the composition, about 10{circumflex over ( )}4-10{circumflex over ( )}7 CFU/kg of the composition, about 10{circumflex over ( )}3-10{circumflex over ( )}5 CFU/kg of the composition, about 10{circumflex over ( )}2 CFU/kg of the composition, about 10{circumflex over ( )}3 CFU/kg of the composition, or about 10{circumflex over ( )}7 CFU/kg of the composition.
  • The compositions of the disclosure exhibit markedly different functional and/or structural characteristics/properties, as compared to their closest naturally occurring counterpart. For instance, the Lactobacillus reuteri strain of the disclosure are structurally different from a Lactobacillus reuteri strain as it naturally exists in a gastrointestinal tract. To elaborate, the Lactobacillus reuteri strain can be isolated and purified, such that it is not found in the milieu of the gastrointestinal tract, the Lactobacillus reuteri strain can be present at concentrations that do not occur in the gastrointestinal tract, the Lactobacillus reuteri strain can be associated with acceptable carriers that do not occur in the gastrointestinal tract, the Lactobacillus reuteri strain can be formulated to be shelf-stable and exist outside the gastrointestinal tract, and said microbe can be combined with other microbes at concentrations that do not exist in the gastrointestinal tract. Further, the Lactobacillus reuteri strain of the disclosure are functionally different from a Lactobacillus reuteri strain as it naturally exists in a gastrointestinal tract. To elaborate, the Lactobacillus reuteri strain when applied in an isolated and purified form can lead to modulation of the gastrointestinal microbiome, increased weight gain, increased feed utilization, decreased amounts of microbial pathogens, and decreased pathogen-associated gastro-intestinal lesions.
  • Method
  • The present disclosure also provides methods of increasing animal health, wherein the method includes administering an effective amount of the composition to an animal.
  • The composition disclosed herein and above increases animal health by providing positive health benefits when administered to an animal, as compared to an animal that has not been administered the composition. As used herein, “animal” includes bird, poultry, a human, or a non-human mammal. Specific examples include chickens, turkey, dogs, cats, cattle and swine. The chicken may be a broiler chicken or egg-laying or egg-producing chicken.
  • Positive health benefits include decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, reducing inflammation, and decreasing mortality rate.
  • In some embodiments, the compositions disclosed herein decreases feed conversion ratio by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein increases poultry weight by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease pathogen-associated lesion formation in the gastrointestinal tract by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease pathogen colonization by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein reduce inflammation by at least 1%, at least 5%, at least 25%, or at least 50%. In some embodiments, the compositions disclosed herein decrease mortality rate by at least 1%, at least 5%, at least 25%, or at least 50%.
  • In some embodiments, following values may be combined in any manner to create a minima, a maxima, or a range for decreasing feed conversion ratio, increasing poultry weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, and decreasing mortality rate, 1%, 5%, 25%, 50%, and 75%.
  • For example, the decrease in pathogen-associated lesion formation may be decreased by approximately 1% to 5%, and more preferably between approximately 5% to 50%.
  • As used herein, pathogen includes Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus, and E. coli bacterium. Further examples of pathogens include Salmonella typhimurium, Salmonella infantis, Salmonella Hadar, Salmonella enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptoccus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, and Fusobacterium necrophorum.
  • The compositions may be administered orally, parentally, nasally, or mucosally.
  • In some aspects, administration includes feeding the poultry, or spraying onto the poultry. In other aspects, administration includes on ovo administration or in ovo administration,
  • In some aspects the animal is vaccinated in conjunction with administration. The animal may be vaccinated prior to administration of the compositions disclosed herein. The animal may be vaccinated with an coccidiosis vaccine. Coccidiosis vaccines are known in the art, for example, COCCIVAC.
  • In some embodiments, administration is by way of injection or infusion. In one embodiment, the composition is administered to a cow by way of intra-mammary infusion.
  • The strains disclosed herein demonstrate certain phenotypic properties. Without wishing to be bound by theory, it is believed that these phenotypic properties at least contribute to increasing animal health.
  • In some embodiments, the isolated strains secrete at least one of cyclic dipeptides (cyclo(his-phe) and cyclo (phe-pro), short chain fatty acids (2-hydroxy-3-methylvalerate and alpha-hydroxyisocaproate), betaine, dimethylglycine, essential amino acids (e.g., allo-threonine, phosphothreonine, histidine, lysine, phenylalanine, tryptophan, leucine, isoleucine, and cysteine s-sulfate), nucleotides (e.g., adenosine 5′-monophosphate (AMP), uridine 5′-monophosphate (UMP), cytidine 5′-monophosphate (5′-CMP), and cytidine 2′3′-cyclicmonophosphate), myo-inositol, and indolin-2-one. Some of the aforementioned molecules provide beneficial characteristics to the host, including increased weight, pro-inflammatory effects, and antibiotic effects.
  • In some embodiments, the composition including the isolated first Lactobacillus reuteri strain (strain 3632) and the isolated second Lactobacillus reuteri strain (strain 3630) in combination, will secrete certain beneficial molecules in larger quantities than when individually cultured. See for example, FIGS. 12-19.
  • In some embodiments, the animal administered the composition exhibits a shift in the microbiome content of the gastrointestinal tract. For example, there may be an increase in the amount of bacteroidaceae bacteria in the gut of an animal that has been administered the composition described herein, as compared to an animal that was not administered the composition.
  • In some embodiments, the invention provides a method of treating, ameliorating the effects of, or preventing necrotic enteritis in an animal by administering a composition disclosed herein to an animal in need thereof.
  • In some embodiments, the invention provides a method of treating, ameliorating the effects of or preventing bovine mastitis. The method includes administering a composition disclosed herein to an animal in need thereof.
  • As used herein, “isolated” means that the subject isolate has been separated from at least one of the materials with which it is associated in a particular environment. Thus, an “isolate” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture in association with an acceptable carrier.
  • As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.
  • In certain aspects of the disclosure, the isolated Lactobacillus reuteri strain exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular Lactobacillus reuteri strain, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual Lactobacillus reuteri strain in question. The culture can contain varying concentrations of said isolated Lactobacillus reuteri strain. The present disclosure notes that isolated and biologically pure microbes often necessarily differ from less pure or impure materials.
  • In embodiments of the present invention, the composition includes a combination of two isolated Lactobacillus reuteri strains.
  • As used herein, “carrier”, “acceptable carrier”, or “pharmaceutical carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, in some embodiments as injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. The choice of carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. See Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2nd Ed. CRC Press. 504 pg.); and E. W. Martin (1970. Remington's Pharmaceutical Sciences. 17th Ed. Mack Pub. Co.).
  • As used herein, “delivery” or “administration” means the act of providing a beneficial activity to a host. The delivery may be direct or indirect. An administration could be by an oral, nasal, or mucosal route. For example without limitation, an oral route may be an administration through drinking water, a nasal route of administration may be through a spray or vapor, and a mucosal route of administration may be through direct contact with mucosal tissue. Mucosal tissue is a membrane rich in mucous glands such as those that line the inside surface of the nose, mouth, esophagus, trachea, lungs, stomach, gut, intestines, and anus. In the case of birds, administration may be in ovo, i.e. administration to a fertilized egg. In ovo administration can be via a liquid which is sprayed onto the egg shell surface, or an injected through the shell.
  • As used herein, the terms “treating”, “to treat”, or “treatment”, include restraining, slowing, stopping, reducing, ameliorating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. A treatment may also be applied prophylactically to prevent or reduce the incidence, occurrence, risk, or severity of a clinical symptom, disorder, condition, or disease.
  • Unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • Any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.” In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
  • Throughout this specification, quantities are defined by ranges, and by lower and upper boundaries of ranges. Each lower boundary can be combined with each upper boundary to define a range. The lower and upper boundaries should each be taken as a separate element. Two lower boundaries or two upper boundaries may be combined to define a range.
  • SEQUENCE LISTING 
    mersacidin-E1 cDNA 
    SEQ ID NO: 1
    1 atggacaaag aagaattaga aaaaattgta ggtaataact ttgaggaaat gagtttacaa 
    61 aaaatgacag aaattcaagg tatgggtgaa taccaagtgg attcaacacc agcagcttct 
    121 gcgatttcac gggcaacaat tcaagtatca cgtgcatctt ctggaaaatg tctaagttgg 
    181 ggtagtggtg cagcatttag tgcttatttt actcataaaa gatggtgcta g 
    mersacidin-E1 amino acid (natural) 
    SEQ ID NO: 2
    1 MDKEELEKIV GNNFEEMSLQ KMTEIQGMGE YQVDSTPAAS AISRATIQVS RASSGKCLSW 
    61 GSGAAFSAYF THKRWC 
    mersacidin-E2 cDNA 
    SEQ ID NO: 3
    1 atggaagaaa aagaattaga aggtgtaata gggaattcgt ttgaaagtat gactgtagag 
    61 gaaatgacaa aaattcaagg tatgggtgaa tatcaagtag attcgacgcc tggatatttt 
    121 atggaaagtg ctgccttttc agctcttaca gccaatataa caagacatgc tatgcatcat 
    181 cattaa 
    mersacidin-E2 amino acid (natural) 
    SEQ ID NO: 4
    1  MEEKELEGVI GNSFESMTVE EMTKIQGMGE YQVDSTPGYF MESAAFSALT ANITRHAMHH 
    61 H 
    Capreomycidine synthase (natural). 
    SEQ ID NO: 5
    1 MVEIAHFGVE AWLNKWEKSA TYDISQSTIA SLSMHDLLNL DGNNGEEFYE MLDKQQMNYG 
    61 WIEGSPEFKE EVAKLYHHVD PENILQTNGA TGANILALYA LINPGDHVIA EYPSYQQLYD 
    121 IPKSLGADVD YWHIHEEDNW YPRIDDLKAM VKPNTKMICL NNANNPTGTV LDKEFLEQVV 
    181 EIAKSVDAYV LVDEVYLPLD HPEKFAQIID LYDKGISTNS LSKTYSVPGV RIGWTATNAE 
    241 VADIFRKFRD YTMICGGVFN DQLATYVLRH RDQVLARNRK LVLGNLAIYK DWIDHEDRAS 
    301 VIMPQAVSTS FPKLDVPVDI HTFCENLLHD EGVLLVPGDA FDTPGHVRLG YCAPEATLKE 
    361 GLKRLSKYMH QYD 
    Colicin V production protein (natural). 
    SEQ ID NO: 6
    1 MILTTFIILI LMGCFINGHR RGLLTMTLML GTYIVAWIVA RQGAQLIGGW LKSLLPSIGT 
    61 PATFSESLLA NVNSNLFFYN GIAFMIIFTI VSILCHWGIR QLNWIKRIPV VGTVDKIAGG 
    121 LISFLIGYLI IYVVLLIMQL FPAGWWQMQI ANSELARFMI NQTPGIAHLV IDTLVQGG 
    Agglutinin receptor precursor-1 (ARP-1) (natural) 
    SEQ ID NO: 7
    1 MNEYNAEMAK LNQGANAPVI TTNSVNQALS LKPENNATVD IEALNPRITF KRVEEGTKYA 
    61  GYQIFDKNNA YVNNIDGEFL RVTYTNLKNS TYKGSKISKI VVTYSDSTPT GNRITQSGLN 
    121 AVTEGANDNF LVVFEDPVRG DMHSTTVTAT YQYYDANGNL IDFSGTNNAW LSVGSLNFDQ 
    181 GNDYQGGKNE GNPTSGISEG VKLISGAQIK QLAGSSISVH DDGWAYAGFN NYSGTGMNNG 
    241 INTDNGGSGW DMDGSPNAYY GAIVFQLTGS SVSLRQGLVS WGGADIASQY NNQFLNNAWF 
    301 TAGTTLPETQ IKQPIRKTSE THYHYNPSVI RL 
    Agglutinin receptor precursor-2 (ARP-2) (natural) 
    SEQ ID NO: 8
    1 MAQKLMSANS TDKNFKMYKS KKSWVFAYST TLALAAVAGI TLSTTNVHAD TTNGGDNQVN 
    61 ATAVTQNTTS NTVDQIAANT AQTDNTSTSI NIRSLMDDLA SGDDTSSSQN GQEQSQNYAS 
    121   SNQNSQTQQE NGTTGQSTAS QNGTTSDQTN SDQSDKNYYV ISTRDLDKNG NVNYLTQKNY
    181 TSIKGQEVAD GTVVTWPLSV SALPANRAQD LKSHVISETL DPHLEYLHYR AYLTNTDGTV 
    241 TDVTNHVNLN RSGQTLIFTD DNYLLSIYNN NRYRVQNLPV IKLVTKANGN GYIIPNAFKS 
    301 SYVFNDGSHD VSFTTTSNNV QIKTFNPGNS KDVEIGGNVQ GDPSGTINGQ VVADGSVVTW 
    361 PMSVGDLPAN RAQDVLSHIE TDTLYNGLNY EGYHAYLPQA DGSFQDVSSH INVQQNGQDL 
    421 TFIADDYLIG LYNQDKSTAF KMPIIDLITS VHGTSIIAPN KFNSQLAFKD GNGQTVINNT 
    481 SNQVQISTYH PTNTKDVELG GNVQGDTPNS INDKVVANGA IVTWPMASSE LPANRVQDLQ 
    541 SRVISETLDS HLQYQGYKAW LQNADGKYTD VTSHVKLTQD GQNLTFADDE YLLNLYNSNK 
    601 GTAYKLPIID LVTKVNGAGI TAPNSYTTKY VYSDGDGNTT INVTSNTVKI STFNPTTNKD 
    661 VELGDNIHGD TESSIAGKLV SEGTIVTWPL STSDLPANRA QDVVSHTAVD ALEPTLQYIS 
    721 YTAWLPDSNG QLQDVTSHVK MTRDGQKLTF TDDDYLIGLY NQNKDIALKM PIIDLVTKAT 
    781 GNTKLLPNSF DSQFVYNDVD GNTIINVSSN KPTVETFDPT VHKDVELGGN NVQGDTPNSI 
    841 DGKIVAQGTV VTWPMSTSDL PANRTQDVVS HSTSETLNQN LQYVGYHAYM PDANGKLQDV 
    901 TSHVQLQQNG QNLVFTDDSY LINLYNQDKS IAFKMPIIDL MTKAISDSAT IPNTFESQYV 
    961 FNDGNGNTTF KSTSNTVQII TYKPKTTKDV ELGDNIHGDT NASIAGQMIT DGTVVTWPMS 
    1021 TSDLPANRTQ DLQQHVVTDN LNDNLIFQGY TAWLPTANGL VDVTNHIELT RDGQNLTFTD 
    1081 DAYLLNLYNQ NKDTAYKLPI IDLVTKANGN TKLIPNNFDS MFVYNDGDQQ TTVNVTSNTV 
    1141 NISTYDPTAT KDVELGDDIE GDTADTINNL MVQIGTKMTY PLTVSDLPAN RADEITAHQS 
    1201 VDTLSDYLEY QGYKAYLPDA DGKLQDITEH VNLKREGQKL SFNDDDYLIN LYNNSKATKQ 
    1261 ALPVIDLVAK VTGSNDGKKV HIIPNHFDST ITTKDGKINT TSNTVVINSN DPEAVKDVEL 
    1321 GDNVVGDTPN SVTGTTVADG TIVTWPMSVG SLGANRAQNV IKHTETENLD SGLTYLSFKA 
    1381 YLPDADGKMQ DITEHINIQQ DGQKLVFTDD DYLISLYNKD KSQRFALPVI DLVTRVNGDN 
    1441 KIIPNTFVSQ FTFNDGKGNT ITSVTSNQVN VSTFKSNPEK HVTLGTDIEG DDAENADGTV 
    1501 VAQGSEVTWP LSDKSPLPAN RSQDVKSHTL VDKLDDNLQY NSYKAYLKGT DGKLQDVTDH 
    1561 IKLTRDGQNL TFIDDDYLLD LYNKDKSTAF NLPIIDLVTT VVGNDKLIPN KFDSNFVFSD 
    1621 GNKDTSMKTT SNEVSISTYT PVTNKDAELG DNVVGDTSDS IANETVPDGT IVTWPLSVSS 
    1681 LPANRSQDVF KHVIEDILDG NLTYNSFKAY LKDAAGNLQE VTDHVKLAQE GQHLTFTDDD 
    1741 YLINLYNSSK NKEQSLPIID LVTTVHGDSK LIPNEFDNVF VFKDGKGQTT VKTTSNKVTI 
    1801 KTASLPTPTK EETDDQGNNI NGNEVKAGEH VNYTLNWDLS NDKDVKATPE MIKKGFFFID 
    1861 PIDSRALSVD DLSKAKVVDQ NGNKVDGISF HLYNSLSEVP EFIQEQVKAN NLQDKITGPF 
    1921 VVAQADDLQA FFDKYVKTGA KLKVTIPTIV KSGFTGEFSN TAYQFGFGKA TPTNTVTNYV 
    1981 KPMHKPASPE TPAAIAPQVI SATAQPMTSD APVTPSEKTA KLPQTGNADE GALLGLAAVS 
    2041 LVGSLGLAAL GLKQNRNDD 
    Xylulose-5-phosphate phosphoketolase promoter (natural) 
    SEQ ID NO: 9
    1 TTAAAGTATT AAAATAGATG TAAAATTTAT TTTTTTCAAA AGAAATTTTA ATTGTACACT 
    61 GTTGGTATTG AACGGGGTTA AACAAAGGTA AATTAGCATT TCTGCGGATT AAGATAAATA 
    121 GAAAAATGTT AAAGAACACC TTAAAAAGAT TAATTTTTTA TAATTGGACC GTATCAATTT 
    181 GTAAAAAGGT TGACTTTTTG AAAAAAAAGT TTATCATTAA CATTGTAAAT TTAATGATTT 
    241 ACGTTATGTT GTTATAGAGC ACAGGACGTA TTGATTTATA TAGAAGGAGT GTTTATTAGA 
    Elongation factor TU promoter (natural) 
    SEQ ID NO: 10
    1 ATGAATGGAC AGATGTTTTA ATCGCTAGAA TAGAAGGAAA GAAAGTCGCA ACAAATACGG
    61 TTTCTAGTAC GTGGCAGGAA CGACTAGGTA AGCAGATTGA CGAATTAATA GAAAAACATT 
    121 AGTCAAATAC ATTTACAAAT GAACAGATAG TTGATATTAT ATTTAAGAAT TCTTCTTCAG 
    181 AGCCTAAGAT TAAAGCTTTC AATTGGCGAA AAGAAGTTGT ACAATATGTA TAAAGGTATG 
    241 TCAGTCACCG AATCAGATGA TCTGGCATTA TACTTGTAAA TTATCAGGAG GTTTTCATTA
    Glyceraldehyde-3-phosphate dehydrogenase promoter (natural) 
    SEQ ID NO: 11
    1 ATCTCACGTG CGATCCATTA CACTAAGGGC GCGTCAACAA ATATTATACT ATCTTAAATA 
    61 AGAATGAATT GCAAGCATTA TTTGAAAATT TTAATTAAAA TAACGCTTAC ATCAGAAAAA 
    121 TGTTGTGATT GAATAGACAA TTTTTTTGAA GATGGTATCA TAAGTATCGT AGGAGTTGTA 
    181 TTATTGCTTA GACCTTACCA CTGCGTCACT TACAATGGTT GAGAGTTGCG ATGCTGATGT 
    241 AATGTGATAA ACTAAGCAAG TACACTAATT ATGTTTTTTC CTAAAGGAGG AATTTGCAGT 
    Glucose-6-phosphate dehydrogenase promoter (natural) 
    SEQ ID NO: 12
    1 TTGTTTAAGA TATCTTTCAA AGCTGCGGAA TTTTTCCCAG CTTTTTTAGT TAGTTTTGTT 
    61 TTCATAAGCT ATAATTTTAA CCGATTCCAA ATTTCTTTTA AAAGTTTTTT TGATCTAGAC 
    121 CATTAATTGA TAAACGCTTA CCAAAGACTA ATCAACAAGC CATTTAGCGG TAGTGGTCCA 
    181 TTTTAACTTT CTAAGACATC TTCTCAGAAA ACGTTTCCTT TGATAGTGCA GATTGTGCTT 
    241 TAAGAGTATA TAATTGTCAC GGTATAAGAA TTTTCTGAAA TTTCAGAAGG AGTGAACATT 
    L-lactate dehydrogenase promoter (natural) 
    SEQ ID NO: 13
    1 CTCCTCTATT ATTATTCCTG ATCAATTTTA AATTAATCTC CCTAGATAGG TATATTTTAG 
    61 CACAGGTCAC CAACGTTCCA AAGTTTAATC TATGTTTAAA CTTTAATTTT CAAAAAAATG 
    121 CTATACTATG TTCACGATAC TTTAAGGAAA GGTGATTACA ATAGTGAGTC TCTTAATTGC 
    181 TATTCTTATC TGCTGGTTGC TATGGAAGAT TGGGGGTTTA ACGGTTAAGT TCATTGGTCT 
    241 AATCCTTCTT ATTCTATTAA TCGGGACATT AATTCATGTT TTACTTTGGC CAGCGATCCT 
    301 TTTAGCAGTT ATTATCTTAG GAGCAGGTTT ATTCACTAAC TAATTTATCT ATAAAATCTT 
    361 ATAGTAATTT TTCTGCGGAA TGTTATAATC ATTACTGTGA GAGAAATCTC AAATAATGTA 
    421 TACATAAGAT GAAAGGGAGA CTGTTTATT 
    tuf promoter (natural) 
    SEQ ID NO: 14
    1 ACAAATACGG TTTCTAGTAC GTGGCAGGAA CGACTAGGTA AGCAGATTGA CGAATTAATA 
    61 GAAAAACATT AGTCAAATAC ATTTACAAAT GAACAGATAG TTGATATTAT ATTTAAGAAT 
    121 TCTTCTTCAG AGCCTAAGAT TAAAGCTTTC AATTGGCGAA AAGAAGTTGT ACAATATGTA 
    181 TAAAGGTATG TCAGTCACCG AATCAGATGA TCTGGCATTA TACTTGTAAA TTATCAGGAG 
    241 GTTTTCATTA 
    C protein alpha-antigen precursor (natural) 
    SEQ ID NO: 15
    1 MVSKNNHQFY QQKHAERKQR WGIRKLSVGV ASVLLGTTFM LYGNHAVLAD TVTSPSDDVT 
    61 RSTTTQGGNK DKVTEGTTEG TTSTPQTSGD STDKQANGQN VNQQVPTTDT EEATNHQDTP 
    121 QGQDTTQNTT NVDKKDTEVT PANDATTPTT QKITAKFTTA KFTTAKFTAA KFKVLAARPV 
    181 MKVAGTASLP ISNQDIKLDS QPMLTEIINK PTDNWVYNNL KWYQDTSTEK IKEILQNHTA 
    241 NDESGRYYFA GVANYNEHYH AIYLLARSNN LNDNSLYVTI LHTGLGKNIQ EAVVAPGESK 
    301 KVEYSGTTHT PIFTNYDGTS ASIDLDGIEK GDNIYGMVVG FAYGHNTGIK GDPASMGNGF 
    361 VMTPIPTKMT TTIHYIDQAT GDEIAVPKSF EGVAYQKYTI TGEAPTIDGY TLKKSPETTG 
    421 YISPYKVGES YDFRLDKHVV IKQTVIDAQG LVRVTAYYDG EVLNNTTRYL GNKLNVNDRM 
    481 SFISHGKWYT YINQITSTND GIVYYYAKDG SEDKSEVRVH YIDVTGSKNS IFVPGDGEEV 
    541 ATDKISGKLG ENYNYDVNLP TDYNLATNQA NTVNGTYTID HHDEYVYVVK KTSAELDPTV 
    601 PAKTKVDNPT SLTADEKKTI EDKIVEANKD KFPEGTGVTV ANDGKATITY PDKSVDTIEG 
    661 NQLVEEKTSA EKLDPTVPAK TKVDNPTSLT ADEKKTIEDK IVEANKDKFP EGTGVTVAND 
    721 GKATITYPDK SVDTIEGNQL VEEKTSAEKL DPTVPAKTKV DNPTSLTADE KKTIEDKIVE 
    781 ANKDKFPEGT GVTVANDGKA TITYPDKSVD TIEGNQLVEE KTSAEKLDPT VPAKTKVDNP 
    841 TSLTADEKKT IEDKIVEANK DKFPEGTGVT VANDGKATIT YPDKSVDTIE GNQLVEEKTS 
    901 AEKLDPTVPA KTKVDNPTSL TADEKKTIED KIVEANKDKF PEGTGVTVAN DGKATITYPD 
    961 KSVDTIEGNQ LVEEKTSAEK LDPTVPAKTK VDNPTSLTAD EKKTIEDKIV EANKDKFPEG 
    1021 TGVTVANDGK ATITYPDKSV DTIEGNQLVE EKTSAEKLDP TVPAKTKVDN PTSLTADEKK 
    1081 TIEDKIVEAN KDKFPEGTGV TVANDGKATI TYPDKSVDTI EGNQLVEEKT SAEKLDPTVP 
    1141 AKTKVDNPTS LTADEKKTIE DKIVEANKDK FPEGTGVTVA NDGKATITYP DKSVDTIEGN 
    1201 QLVEEKTSAE KLDPTVPAKT KVDNPTSLTA DEKKTIEDKI VEANKDKFPE GTGVTVANDG 
    1261 KATITYPDKS VDTIEGNQLV EEKTSAEKLD PTVPAKTKVD NPTSLTADEK KTIEDKIVEA 
    1321 NKDKFPEGTG VTVANDGKAT ITYPDKSVDT IEGNQLVEEK TSAEKLDPTV PAKTKVDNPT 
    1381 SLTADEKKTI EDKIVEANKD KFPEGTGVTV ANDGKATITY PDKSVDTIEG NQLVEEKTSA 
    1441 EKLDPTVPAK TKVDNPTSLT ADEKKTIEDK IVEANKDKFP EGTGVTVAND GKATITYPDK 
    1501 SVDTIEGNQL VEEKTSAEKL DPTVPAKTKV DNPTSLTADE KKTIEDKIVE ANKDKFPEGT 
    1561 GVTVANDGKA TITYPDKSVD TIEGNQLVEE KTSAEKLDPT VPAKTKVDNP TSLTADEKKT 
    1621 IEDKIVEANK DKFPEGTGVT VANDGKATIT YPDKSVDTIE GNQLVEEKTS AEKLDPTVPA 
    1681 KTKVDNPTSL TADEKKTIED KIVEANKDKF PEGTGVTVAN DGKATITYPD KSVDTIEGNQ 
    1741 LVEEKTSAEK LDPTVPAKTK VDNPTSLTAD EKKTIEDKIV EANKDKFPEG TGVTVANDGK 
    1801 ATITYPDKSV DTIEGNQLVE EKTSAEKLDP TVPAKTKVDN PTSLTADEKK TIEDKIVEAN 
    1861 KDKFPEGTGV TVANDGKATI TYPDKSVDTI EGNQLVEEKT SAEKLDPTVP AKTKVDNPTS 
    1921 LTADEKKTIE DKIVEANKDK FPEGTGVTVA NDGKATITYP DKSVDTIEGN QLVEEKTSAE 
    1981 KLDPTVPAKT KVDNPTSLTA DEKKTIEDKI VEANKDKFPE GTGVTVANDG KATITYPDKS 
    2041 VDTIEGNQLV EEKTSAEKLD PTVPAKTKVD NPTSLTADEK KTIEDKIVEA NKDKFPEGTG 
    2101 VTVANDGKAT ITYPDKSVDT IEGNQLVEEK TSAEKLDPTV PAKTKVDNPT SLTADEKKTI 
    2161 EDKIVEANKD KFPEGTGVTV ANDGKATITY PDKSVDTIEG NQLVEEKTSA EKLDPTVPAK 
    2221 TKVDDPTKLT NDEKKEVEDN IRDHNTGLPE GTKIAVGDNG DTTITYPDKS VDTIEGNQLV 
    2281 EEKTSAEKLD PTVPAKTKVD DPTKLTNDEK KEVEDNIRDH NTGLPEGTKI AVGDNGDTTI 
    2341 TYPDNSVDTI PGDKVVEGKS DAAKNEPKVP GDKVKVDDPN KLTEDEKSEV VKAVEDANKD 
    2401 ENGKSTLPEG SKVTVGDNGD VTVTYPDGSK DTIPGDKVVE GKGTEGQTDA DKNEPKVPGD 
    2461 KVKVDDPNKL TEDEKSEVVK AVEDANKDEN GKSTLPEGSK VTVGDNGDVT VTYPDGSKDT 
    2521 IPGDKVVEGK GTEGQTDADK NEPKVPGDKV KVDDPNKLTE DEKSEVVKAV EDANKDENGK 
    2581 STLPEGSKVT VGDNGDVTVT YPDGSKDTIP GDKVVEGRGT EGQTDADKNE PKVPGDKVKV 
    2641 DDPTKLTEDE KSDVEQAIKD ANKDENGKST LPEGSKVTVG DNDDVTVTYP DGSKDTIPGD 
    2701 KVVEGKGTEG QTDADKNEPK VPGDKVKVDD PNKLMEDEKS DVEQAIKDAN KDENGKSTLP 
    2761 EGSKVTVSDN GDVTITYPDG SKDTIPGDQV IEGKSDADKN TPNVPGGDKV KVDDPTKLTD 
    2821 NEKNAVKDKV DEANSNLPDG TKVTVGDDGT TTITYPDGST NTISGHDLVT GKTDADKYPL 
    2881 NPGQAVNVVD PNHLTQAEQD QVKEAIQTTN PTAPIATITV DTAGNVQVTF ADGSTTTLQA 
    2941 NLHKHVTEAT TGSAIKPGVG TNGGQTKGAT STNQTATKQQ AQQHLPQTGD QPATWAMLSG 
    3001 LGVAFLGLLG LKKKRED 
    Arabinogalactan endo-1, 4-beta-galactosidase (natural) 
    SEQ ID NO: 16
    1 MEIKKHFKLY KDGKKWCCAA IATTVLGIGL AIGSPSVLAD ADTITSTSDA NNSLVKNDNT 
    61 SDTDSNSEST FTDTNKNSTN EKEINENKNI DSSQQINQEQ TKSNNSEEQT TPVNVKAENT 
    121 DIKDSIPEKS TPNSFKEING STYYYGENGD LYRNQFYNNW GRTYYFQANG ARLDNGFYNN 
    181 WGRTYYFGSD GARWDNRFYN NWGRTYYFQN DGSRLDNSFY NNWGRTYYFG VDGARWDNRY 
    241 MVKWGRAYYF GNDGALLQNQ LKSINGINYW INNEGIIPLK NQFLTANENQ LFYFDGNGSL 
    301 VVNKFYHNWG HTYYFGSDGA RYTDQFLNRD GKVYYFDNQG IMYQDQYYKN WGHTYYFGSD 
    361 GARYTDQFLN RDGKVYYFDN QGIMYQDQYY KNWGHTYYFG SDGARYTDQF LNRDGKVYYF 
    421 DNQGIMYQDQ YYKNWGHTYY FGSDGARYTD QFLNRDGKVY YFDNQGIMVT NQVRVIDGKG 
    481 YEFNDNGEAT ETSDMGQTRD TVAKEVAQAL TNQGIKGVKY DWRNTNNDYQ ELALHDIAQE 
    541 VAQGDTNPDK NVIEKKLQAN NLLSGKVLVV YSTDFTNDDP QKITNTFMNS YDFTNADNSV 
    601 LGVGADLNKN KLVIILFKPG EKAEQPQATS TISASISDIF KKAGVNVDVD NGLTKGSVVN 
    661 SADLGNALTN GTAELLKGDK GTIISQEVLK AIFAAFAGNT SAVEGTKNYY NGNDAYHYEF 
    721 WLEGQSADDK LNNFLALNKG AKYGDQLKVN YTATLVFGQE TGTNSNESKV PASERTDEQL 
    781 DLAYKTGTDT GLRYDSVKVE KIPGMTDDMV RGVDVSSYQA LINAGVKFYD FNGQESNLFK 
    841 ILKDSGVNWV RLRVWNDPYN AQGQPYAGGD NNEENLIKMA KEASDNGLKL LIDFQYSDFW 
    901 TDPAQQILPK AWRNLSHGEM SQEVYLYTSK ILNDLQKAGA SVKMVQIGNE ITNGAFGLYT 
    961 GRNGGGNWAS LWETSDGDQV AKYIQAGSSA VRRIDPTIKV AIQLETPEIN KYRGIMNVLK 
    1021 KNNVDYDYLG TSYYPFWSTT QGNGWYDNVD LGYGANTPVN LEAIEKMAWN EFGKRTVILE 
    1081 SGWLNNTNDA DGTHNSVGEN NETTNIDRYS ADPQGQVDEI EDMYNAIIAQ KGLGAFYWEP 
    1141 AWIPVKAGWN NWQYNKLMSN IYGSGWASQY AKGYAPDSVL YYDGKEAWGG SSWDNISLFD 
    1201 DHGHPLQSLN VYNGMLNGYE SPKNVKSSLS TQLVKIWNET DVIPNDGLTE GTKLSTDLFG 
    1261 TTQLSGNDGQ SIGNAELTKL AGRLKDGISS KVYTAANGAR YHYIYWLEGG NNKVNTFVSA 
    1321   NKDAKYGQPL IANYSATVVV DSEPGTQVAT SPLQIKISQV WNTVNNEEIK IDNPLKQGDL
    1381 ITDKSDNAFS GILNSKDIKE ALTGEKGKDV SESTVNDVKS LLPKEVKGSK TYTTADGNQY 
    1441 YYDFWLASVE TSNVNYGEPI IVNYTASLKW LG 
    Chromosome segregation protein (natural) 
    SEQ ID NO: 17
    1 MEKTMKKKAL VATTAVAGIT LVGEVTTVHA ADNVQQPVNE QNVNQSSQEE KQAAQNLQNA 
    61 QSDVNTATEA NSNAQDNLAS ANNNLSNAKK AVSDQAAKVA DATKAQSDAS TKVDNDNKVV 
    121 ADAQQKADQA TPANIENAKQ AIEGQNKVID QDNENIKYSN TDQDKAQNTL NNAQSNEDKA 
    181 NATLSNKKSS QASAQNNVKQ AEDALNGTHL VEAQNAFNQA QSNVENAQSK YDQANNQLSD 
    241 AQKKVTTNQN DLTAKNKALD NINNQVDTDQ NNVNSNQATA DSASSATQVA QNAVDQTKQS 
    301 LDKVIEELNG FSENTIKVPA GAQEAYEAFI DAVDNNADQS QLDSLAKKMY DTLHQGQGTN 
    361 GINHFNSSKY DQNQLVDVDH LTTDQLNELT QFAADMINSA RKAWGSDKNA GTLIPTQGVS 
    421 EMAQQIAKGY VSDNWHISQG HDVKRVTAAA GLIGLNDAGQ FYEDASEGYV HAWPWEKDSY 
    481 TMDNLKEAVY DSILGMLFAD DNSGNGHMTD LLGLHVNRKE DHQYFGLSTN MCPGSYMGQL 
    541 HFIIVENDPA YIKDPQTFNA KGGTTKIEYI DPKVQLNQQK DILTTTLSTQ QADLATKQDA 
    601 LNKANQNLAN AKKQLSEDQD LQTVAQQNRD SAQKALNDAT AKVSNLQATV NSLSQDLNSA 
    661 KATLDQAKKT LESYTADHKA KLDNYNNAKA ALDDANKAVA EAQSAVDTAV NETKIAQNNL 
    721 DQKKQAVTDA QNKLANDQEY LATLKQNLAD LQNAPQNLQK AKDQLAKDQI ALDNANKDLQ 
    781 NQKDSLDELN KKLEDAQVKV NEAQSAANVT KATLDQAQAK LSDAEATWKE LHNDAHRYGN 
    841 VVKVTPITME AGTSLPDPVI ENGFTVNTGT NQLFVSLAAI DSSNNNIPQG TKASWANRSK 
    901 ALTDSQNAGS YSEDILITFP DNSTVTVPVD LTVTAKKITE DQKATEGGYH IVNGSVVDKQ 
    961 NNLVSGWTVK NGQMVDPEGN VIKTTMSTAQ GVTIEKNNSK SGNTKTNMIQ TSLTIANNKA 
    1021 TTNKDNQLPQ TGNYNNNTKV LGLAGIALAS ALTMFGYKKR QHN 
    D-gamma-glutamyl-meso-diaminopimelic acid endopeptidase Cwls (natural) 
    SEQ ID NO: 18
    1 MKSTTKKILA SSLGVAGAMA MGTVTAKADT TVTVNAGDSL NGIAQKYNVS ADDIATANHL 
    61 QNKELIFVGQ KLTIPTKDKN ETPANNAEKK DQASKNSQSL QDSVNKAMSY LGTPYVWGGN 
    121 KPGGFDCSGL VQYCYGIPQR TTYEQQALGP HIHDNVLNAP YGALVFYGSD DAPYHVAISL 
    181 GDGRIIQAPN ENETVKITDQ QYFPGNYYVV MH 
    N-acetylmuramoyl-L-alanine amidase sle1 precursor (natural) 
    SEQ ID NO: 19
    1 ATGCGTAATC AATTCATCGA TGTTTCAAGT TATCAACCAG ATACTGTTGC CTTTTTCCAA 
    61 GCTGCTAAAG CTCAGGGTGC ATTAGGGGTC GTTGTTAAGT TAACGGAAGG GTCCGAAGAT 
    121 GGTTCGGCTT ATGTTAATCC ACGTGCGGCC GCTCAAATTC GTAATGCCTT AGCGGTTGGC 
    181 TTGCGCGTTT CCTGTTACCA CTTTGCTCGT TATACATCAG TGACTGATGC ACAAAATGAA 
    241 GCTCGATTCT TCGTTAAAAT CGCTAAGCAA TTTGGTATGT ATGACGATAC TTTGATGATT 
    301 GATGATGCGG AAGTTCATTC AACTGCAGAT TATCAATCAG TATCCTTAGC CTTTCTTCAA 
    361 GAAGTAGAAG CTCTTGGTTA CAAGAATACT GGGATTTACT CCATGAAGTC CTTCTTCACT 
    421 GGCGGTATTC TTAATTCACA TGGCTTTGAT TCCCGGAAGA TTTGGATTGC TGGCTATGGT 
    481 GTGACTGAAC TGGGGATTGA TAATGCAAGT GCTTGGCAAT ATTCTGATCA TAGCATCATG 
    541 GGAATTGATA CTAGTTATGA CTTTGACGGT GCCTTTACGA CTGGTTTAGT ATCAGGCAAT 
    601 GTTCCGCAAG CTGTTATTCC AGCACCACAG CCGGTTCAAC ATATTGGTCA CCCAGCTACT 
    661 GGAACCTACA TTGTTCAGCC GGGCGATACA TTGAGTGGAA TTGCAGAAAA ATACGGGACT 
    721 ACTTATCAGA ACCTAGCAGC AATCAATGGT ATTGGTAATC CAAACCAGAT CAATGTCGGC 
    781 CAAGTCCTCA AAGTCACCGG AAAAGTATCA AACGAAAATA CTTACTTTGT TCAATCAGGC 
    841 GATACGTTAT CCGGAATTGC CACCAAATTC GGCACCACTG TCTCAGACCT CGTAAGCCGT 
    901 AATCACATTA CTAACCCGAA TGTGATCTAC GTTGGGCAAA AACTCTACTT AGCCGGCAAC 
    961 GGACAATCCA ATGCTTATAC TGTCCAAGCA GGGGACACAC TAAGCGGAAT TGCGGCTAAG 
    1021 TTTGGCAAGA CCTGGCAAGC ATTAGCTCAA AAGAATGGCA TCGCAAATCC TAATATGATT 
    1081 TTCATTGGTC AAACAATTCA GATTTAA 
    Peptidase family M23 (natural) 
    SEQ ID NO: 20
    1 GTGTACCGAA TTATTGGTTA TAATGAACCA ACAGATAAAG CAGGATTTAT TGTACTGGAT 
    61 CCCCGAGTTA ATCGTCATAT TAGTTCGGGA AAACTCACGC TTAAAGAATC TAATATTGAT 
    121 GATTTGACTA TTACGGTTAA TCAAGCAAGT CCATTATGGG ACAACGTAAG GCCTTATCAT 
    181 ACTCATGTTA ACGTTTATGA TGATAATGAA CTTATTTTTC GTGGACGAGC TATCAAACCT 
    241 AAAAAGTCGA TGGAAGAAAG CGGACAATTC ATTCGTGAAT ATGTTTTTGA AGATATTGAA 
    301 GCATATCTCA TGGATAGCAC CCAAAGATTT TATGAAGGTG TTGGTCAAAC GCCCAAAGAA 
    361 TTTTTACAAA CTTTAATCGA TGTTCATAAT TCACAGGTTC CTGACTATAA AAAGTTTCAA 
    421 GTCCGGAATG TAAATGTCAC TAATAATAAG GATGACCAAT ATCGACAAAT TGATTATCCC 
    481 AAAACTAGCG ATGCTATTAA TGATAAATTA GTTAAATCTC TTGGTGGTTA TATTGTGACT 
    541 ACTTACAACG CTAACGGAAT AAACTACATT GACTACTTAA CGGATATTGG GGTTGATCAT 
    601 AAAGATGATA CTCCTATTCA GTTAGCTAAA AATATGAAGT CTGCAAGTAT GCAAATTGAT 
    661 CCTACTAAGG TGATTACAAG ACTGATTCCA CTGGGAAAGA CACTAGAACC ATCAAAAGTT 
    721 GATGTAAGTG ATGATGATGG AGAGGGCGGT TCTGGATCAT TAGATAGCCC TGAAGAATTT 
    781 TGTAAATCAG AAATTAATGC TACTTGGGGT AGTGATATTA ATAATATGAA ACAAGATTTT 
    841 GCCGCTCGTT CTTCGAGAGT TCGGGCTTGG GGAGTGGACG TTAATCGTTT ATATGATGTG 
    901 GTGAAAAATG CTGGAGTAAG TCCTGAATGG TTCTTTGCTT ATGAACTTCA AGAACAAGGA 
    961 ACTTACTATG GATGGCTTAA CCATACTTAT CGACACGGTG ATGCGTATAG TGATGCGCAA 
    1021 TCTGTTTGTG AGTGGATTAA AAATTGTTCA AATAGTAATT CCATTAATCC AGCATGGAGC 
    1081 GCACCGGAAG GATCAATGGC GCCGAATCAA GCATTAGCGG ATAAATGGAA TCAAGAGTTT 
    1141 GGAAAAGGTA CTATTGGCCG CGTTTATTTA CAAGGGACTG CCGCTGCTGT TTGGGATTTA 
    1201 GCTGGTCAAA CGCCTAATCC AGCTATTGGA AAGCCAATTA GTGGATGCAT TTCTTGTATT 
    1261 AAACGTTGGG GTGGTCATTC TAATGCAGCT GGTGGTACAT GGGGATGGCC TTTTCCTGAT 
    1321 GTTGGGGAAG GTCATTTTTC TCAAGTTCAG AGTTTCGGAA ATGATGGCGG ATATCGTCAA 
    1381 AATAGTTATC ACGATGGTGT GGATTTTGGA TCAATAGATC ATCCTGGTAG AGAAGTGCAT 
    1441 TGTATTCATG GTGGAACGGT AACTATCAAA TCAGCTATGG GTGGCTTAGG TAATTTTGTG 
    1501 GTTATTCATA CGCCGGAAGG ATTCAATATC GTTTATCAAG AAGCTTTTAG TTCTCCCTCT 
    1561 AATATTATTG TTAGTGTTGG GCAAAAAGTA AAAACTGGTG ATGTAATTGG ATATCGTGAT 
    1621 ACAGACCATG TTCATATTGG CGTAACTAAG CAAGATTTTT ATCAAGCAGT TCGAAATTCT 
    1681 TTTTCTCCTG CAGGTGGTTG GCTAGATCCA GTAAAACTAA TTAAAGAAGG TGGCGATGGG 
    1741 TCTAAACCAC AAGAAGGAAA GAAAGATCAA ACTGTTGATA ATAGTAATGC TGCACGTCCT 
    1801 AAATTAACCA TTACTACTGT CAATAACGGT AGAGACTATA TTGATATTCC TGATTTACAA 
    1861 AAAGAATTCG GTATTATTGA GGGAACTGTT GAATTTGATA ATGTAGATGA TCCGAATGTT 
    1921 TTAATGCAAC AAGCTCAAAC ATGGATAAAG GCTCAAAGAA TACCTCAAAG TTGGGAAGTT 
    1981 ACAGCTTTAG AATTACATAT GACAAACTTC AAATCTTTTA AGGTTGCTGA TAGGTACATG 
    2041 TTTATTAATC CAAATGTTGC AAAACCCCAA TTATTACGAA TTACTCAAAA AGAAATTGAT 
    2101 TTACTAAAGC CCCATGCGTC TTCATTAACG ATTGGTGATA AGACGATGGG GCTTACTGAT 
    2161 TATCAGTTAG AAAATCAAGT CAATTTTCAA CAATTTAAGG AAATTCGAGT GATGGTTAAT 
    2221 CAGGTTGTCC AAACCCAAGA GCAATCTGCT AATAACAATA ATAAGGTTAT GCAAAATTTT 
    2281 GCTAGTAGTG CTGATCTTGC ACAAATGAGA CAGGATCTAA GAAATCTTCA AGATGATAAC 
    2341 GATCGTGCTC GCAAAGGAAT GGTTTCCTTA GAAGAATTCA ATAAACTAAA GGAACAAGTA 
    2401 GAAAAACTAA CAACAGGAGG CGATGATAAT GGCAAGTGA 
    Chromosome Partition Protein Smc (natural) 
    SEQ ID NO: 21
    1 MNKANQKVAD DTTAVNNKQT DVNNAAEAKK NADEALKNAN DAQTSAQKNK DAKQAIADEA 
    61 SVALADANTA VKDAQAKVDA INDKLANFNT ITLPAGYKDD LIAYYNYFGN SNYNQDEANN 
    121 LAQDLLKYRD QAMSQNKFKD NLSDDRVVDI DNLNSTDRAE LSQFVASLIN QVRTQMGTNL 
    181 VISSPAADDY AEQVSQNYNK DNWNSADNGK HDQSALNNAT DQLNISWNGE NMGLDQSIFT 
    241 TDYTVLTDGT KLPTGNKQTI NDLKHLIYDD FISMMFDDAD SAWGHATNFA GIDNFAAEKQ 
    301 AVGFSLDKFY NTHYDLVEAN QKVEENSYTL PSINALTQKL ADAKDDLSIK QTDQASKQKA 
    361 NDDAQNALSS ANQVLVAAQN DVKDKTATAQ EANDNLTTAQ NDLATLQNQL SADQANQKQA 
    421 QTTFDSFDAD LATKQANLQK ATDSLKAEQG RLAIAQADLD NANKALSDAN NNLAQKKQVV 
    481 ENDNETLKVD NDKLVQLQNN LSDLQNAPKL LAAAKEQVAT AQKALADAQE AYNVANDKLT 
    541 SLKQTAAGTT TNVSKAQQAL AEAKNNEDAA KEVLDQAQQA LTELRQKEAL AKQVAEEQAK 
    601 LAAEKEAKDN GYHIENNQVV DAKGNSVNGW TVKGNQIVSP TNATVDPAVS VTTNVNVDSK 
    661 GQVQPQTSVT ANSVKTVAAT ESANPVATTT VQTREQYKQQ LKSNNQLPQT GNNDSAVLSL 
    721 AGVALAAMLS LFGIKKREY 
    Cwls endopeptidase promoter (natural) 
    SEQ ID NO: 22
    1 AAATTAAAAG GCTGGATTTT TTCGGCCTTT TTTTAGTGCA AATAATTATT TTTTACGTAT 
    61 TTATATTATA GGGCTAATCA CTAAACTAAT AATTAGTGGT TGAAGCGCTG AAAATTTTCT 
    121 GCTATTTTAT TAATAGTTTG ATAATAAAAT AATGATATTT AATATAAAGA GGGATAAACG 
    181 AAATA 
    chromosome segregation protein promoter (natural) 
    SEQ ID NO: 23
    1 GAAGTACAAA GTTACTTTAA CTATAATGAA AAACAAGACA ATATAAAGAA AACAACATAT 
    61 AAGGTTCAGT TCATAACTGA TTAGATTTAT AATAAATATT GTAAATCGGA CAAAAATAAA 
    121 TTAATTTTCA ATTAATTCAA AAAAACCATA TTTTTTTCGT TTTGGCATAT TTGGATTTGC 
    181 TACACTAAAG ATGATCAAGA AAGGGGAAAA GATAATCTTC AATCTTGTGT ACTTAGTTTG 
    241 TTAATTAATT TATAAATTTA GGGAGGAAAC CTATC 
    Chromosome Partition Protein Smc promoter (natural) 
    SEQ ID NO: 24
    1 GCAATGCACA AGATGCTGAA ACAAAGGCAC AACAAAATGC AGATCAAGCT TCACCAGCTA 
    61   ATATTCAAAA GGCACAAGAT GCTATTGCTA ATCAAGAAAC TCAAATTAGT AAAGACACCG
    121 ATGCTATTAA TGACGCTAAC AAAGCCGTTA GCGATGCACA AAGCACAGTT GATGCAGCGC 
    181 AAAAAAAGTT AATGATGCAA CTACTGCTCG TGACAATCAA CAAAAGAATG TTGATACTGC 
    241 TAGTGATGCA GTTAAGAATG CTCAAGCTAT TCTTGACAAC AGTGATCAGG CTAAAAAGGA 
    301 AGCCCAAGAT GCT 
    Lactobacillus reuteri 3630 F1 16S 
    SEQ ID NO: 25
    1 GCCGCGGGTG CCTATACATG CAGTCGTACG CACTGGCCCA ACTGATTGAT GGTGCTTGCA 
    61 CCTGATTGAC GATGGATCAC CAGTGAGTGG CGGACGGGTG AGTAACACGT AGGTAACCTG 
    121 CCCCGGAGCG GGGGATAACA TTTGGAAACA GATGCTAATA CCGCATAACA ACAAAAGCCA 
    181 CATGGCTTTT GTTTGAAAGA TGGCTTTGGC TATCACTCTG GGATGGACCT GCGGTGCATT 
    241 AGCTAGTTGG TAAGGTAACG GCTTACCAAG GCGATGATGC ATAGCCGAGT TGAGAGACTG 
    301 ATCGGCCACA ATGGAACTGA GACACGGTCC ATACTCCTAC GGGAGGCAGC AGTAGGGAAT 
    361 CTTCCACAAT GGGCGCAAGC CTGATGGAGC AACACCGCGT GAGTGAAGAA GGGTTTCGGC 
    421 TCGTAAAGCT CTGTTGTTGG AGAAGAACGT GCGTGAGAGT AACTGTTCAC GCAGTGACGG 
    481 TATCCAACCA GAAAGTCACG GCTAACTACG CCCCCCACCA GCCCCAA 
    Lactobacillus reuteri 3632 F1 16S 
    SEQ ID NO: 26
    1 TGCTGGGGTT TGCCTATACA TGCAGTCGTA CGCACTGGCC CAACTGATTG ATGGTGCTTG 
    61 CACCTGATTG ACGATGGATC ACCAGTGAGT GGCGGACGGG TGAGTAACAC GTAGGTAACC 
    121 TGCCCCGGAG CGGGGGATAA CATTTGGAAA CAGATGCTAA TACCGCATAA CAACAAAAGC 
    181 CACATGGCTT TTGTTTGAAA GATGGCTTTG GCTATCACTC TGGGATGGAC CTGCGGTGCA 
    241 TTAGCTAGTT GGTAAGGTAA CGGCTTACCA AGGCGATGAT GCATAGCCGA GTTGAGAGAC 
    301 TGATCGGCCA CAATGGAACT GAGACACGGT CCATACTCCT ACGGGAGGCA GCAGTAGGGA 
    361 ATCTTCCACA ATGGGCGCAA GCCTGATGGA GCAACACCGC GTGAGTGAAG AAGGGTTTCG 
    421 GCTCGTAAAG CTCTGTTGTT GGAGAAGAAC GTGCGTGAGA GTAACTGTTC ACGCAGTGAC 
    481 GGTATCCAAC CAGAAAGTCA CGACTAACTA CGCCCCACAC CCCAGCCGCA A 
    Lactobacillus reuteri 3630_00184 
    SEQ ID NO: 27
    1 ATGGATATGA AAATAAAAAA AGAACCACCA CAACAAGTAA AATTAGTTGA AGTAATTCAG 
    61 GTTATAACCT CTCGTGGAGC TGGAACAAAG GAGGATCCGA TAAGAAAGAT TATTCAGTAT 
    121 TGGAGCAAAG AAGGCACATT ATTAGCAGAA AGTTTTGGAA ACTAA 
    Lactobacillus reuteri 3630_00491 
    SEQ ID NO: 28
    1 ATGGCTGGTA TCAAAAGTAT CGCAAAAGCG GTAATGACCC AGAATCACTT CGTGATCGCC 
    61 GAGGCAAAGC TAAGCCAGAA GAGAAGTGGA CGGAAGTTGA CCGACTCAAG GCAGAAAATC 
    121 GCTTATTAA 
    Lactobacillus reuteri 3630_00497 
    SEQ ID NO: 29
    1 ATGGCTAAAT ACACTGTTGA ATTAAGTGAA GAAGATATCC AAATGATCAA GGATTGTCAT 
    61 TCAAAGAATC CTTCTATCAT GAAGGCAATG AACGACGCTA AAAAAGTTGA AGATTAA 
    Lactobacillusreuteri 3630_00533 
    SEQ ID NO: 30
    1 GTGGATGCGG ATTCGCTGGC ACTCGTTGAT GCACTTTCAC TTGCGCTCGT TGATGCCGAT 
    61 TCACTGGCAC TCGTTGATGC ACTTTCGCTA GCACTAGTAG AAGCTGACTC ACTTGCACTT 
    121 GTCGATGCCG ATTCGCTTGC ACTTGTCGAT GCTGATTCGC TTGCGCTCGT TGAGGCTGAT 
    181 TCGCTAGCAC TAGTAGAAGC TGATTCACTG GCACTCGTTG AGGCACTTTC ACTTGCGCTT 
    241 GTTGAAGCCG ACTCACTTGC GCTCGTTGAT GCACTTTCGC TGGCACTTGT CGATGCCGAT 
    301 TCACTTACGC TCGTTGAGGC CGATTCGCTG ACACTCGTTG ATGCACTTTC ACTTACGCTC 
    361 GTTGAGGCCG ATTCGCTGAC ACTCGTTGAT GCACTTTCAC TTACGCTCGT TGAGGCCGAT 
    421 TCGCTGACAC TCGTTGATGC ACTTTCGCTG GCACTTGTCG AGGCACTTTC ACTTACGCTC 
    481 GTTGAGGCCG ATTCACTTAC GCTCGTTGAG GCCGATTCAC TTGCGCTTGT GGATGCGGAC 
    541 TCACTTGCAC TCGTTGAGGC ACTTTCACTT GCACTTGTCG ATGCAGATTC GCTGGCACTC 
    601 GTTGATGCAC TTTCACTTGC ACTCGTTGAT GCACTTTCAC TTGCACTTGT CGATGCGGAT 
    661 TCACTTGCAC TTGTTGAAGC GGATTCGCTA GCACTTGTCG ATGCGGATTC ACTTGCGCTT 
    721 GTTGAAGCGG ATTCGCTGGC GCTTGTTGAA GCCGACTCAC TTGCACTCGT TGAGGCTGAT 
    781 TCACTTGCAC TCGTTGAGGC ACTTTCGCTG GCGCTCGTTG ATGCAGATTC GCTGGCACTC 
    841 GTTGAGGCCG ATTCGCTGGC GCTTGTTGAG GCCGATTCGC TTGCACTTGT CGATGCTGAT 
    901 TCACTTGCAC TGGTTGAAGC AGATTCGCTG GCACTCGTTG AGGCACTTTC GCTGGCACTC 
    961 GTTGAGGCCG ATTCACTTGC GCTTGTTGAA GCTGATTCAC TTGCACTTGT CGATGCTGAT 
    1021 TCACTTGCGC TAGTAGATGC CGATTCGCTG GCACTCGTTG AAGCAGATTC ACTTGCGCTT 
    1081 GTTGAAGCCG ATTCACTTGC GCTTGTTGAA GCCGACTCAC TTGCACTCGT TGAGGCACTT 
    1141 TCGCTGGCGC TCGTTGAAGC AGATTCGCTG GCACTCGTTG AAGCAGATTC GCTGGCGCTT 
    1201 GTTGAGGCCG ATTCGCTTGC ACTTGTCGAT GCTGATTCAC TTGCACTGGT TGAAGCAGAT 
    1261 TCGCTGGCAC TCGTTGAGGC ACTTTCGCTG GCACTCGTTG AGGCCGATTC ACTTGCGCTT 
    1321 GTTGAAGCTG ATTCACTTGC ACTTGTCGAT GCTGATTCAC TTGCGCTAGT AGATGCCGAT 
    1381 TCGCTGGCAC TAGTAGAAGC AGATTCACTT GCGCTTGTTG AAGCCGATTC ACTTGCGCTT 
    1441 GTTGAAGCCG ACTCACTTGC ACTCGTTGAT GCCGATTCAC TTGCACTTGT GGATGCGGAC 
    1501 TCACTTGCAC TCGTTGATGC ACTTTCACTT GCACTTGTGG ATGCGGATTC GCTGGCACTC 
    1561 GTTGATGCAC TTTCACTTGC GCTCGTTGAT GCCGATTCAC TGGCACTCGT TGATGCACTT 
    1621 TCGCTAGCAC TAGTAGAAGC TGACTCACTT GCACTTGTCG ATGCCGATTC GCTTGCACTT 
    1681 GTCGATGCTG ATTCGCTTGC GCTCGTTGAG GCTGATTCGC TAGCACTAGT AGAAGCTGAT 
    1741 TCACTGGCAC TCGTTGAGGC ACTTTCACTT GCGCTTGTTG AAGCCGACTC ACTTGCGCTC 
    1801 GTTGATGCCG ATTCACTGGC ACTCGTTGAG GCACTTTCAC TTGCGCTTGT TGAAGCCGAC 
    1861 TCACTTGCGC TCGTTGATGC ACTTTCGCTG GCACTTGTCG ATGCCGATTC ACTTGCGCTT 
    1921 GTGGATGCGG ACTCACTTGC ACTCGTTGAA GCCGATTGGC TTGCACTAGT AGAAGCTGAT 
    1981 TCACTGGCGC TCGTTGAGGC TGATTCGCTG GCGCTCGTTG AGGCTGATTC ACTGGCACTC 
    2041 GTTGAGGCCG ATTCGCTGGC GCTCGTTGAT GCAGATTCGC TGGCACTAGT AGAAGCTGAT 
    2101 TCACTGGCGC TCGTTGATGC CGATTCACTT GCACTTGTCG ATGCGGACTC ACTTGCACTC 
    2161 GTTGATGCAC TTTCGCTTGC ACTTGTGGAT GCGGATTCGC TGGCACTCGT TGATGCACTT 
    2221 TCGCTAGCAC TTGTCGATGC CGATTCACTT GCACTCGTTG ATGCACTTTC GCTAGCACTC 
    2281 GTTGATGCAC TTTCACTTGC ACTTGTGGAT GCGGATTCGC TGGCACTTGT TGATGCGGAC 
    2341 TCACTTGCAC TTGTTGAAGC CGATTCACTG GCGCTCGTTG ATGCCGATTC ACTTGCACTT 
    2401 GTCGATGCCG ATTCACTGGC GCTTGTTGAT GCGGACTCAC TTGCACTCGT TGATGCACTT 
    2461 TCACTTGCAC TTGTGGATGC GGATTCGCTG GCACTAGTAG AAGCTGACTC ACTTGCACTT 
    2521 GTCGATGCCG ATTCACTGGC ACTCGTTGAA GCACTTTCAC TTGCGCTTGT TGATGCGGAC 
    2581 TCACTTGCAC TCGTTGAAGC CGATTCGCTA GCACTTGTCG AAGCTGATTC ACTGGCGCTC 
    2641 GTTGATGCTG ATTCACTGGC GCTTGTTGAA GCCGACTCAC TTGCGCTCGT TGATGCACTT 
    2701 TCACTTGCGC TCGTTGATGC CGATTCACTA GCACTAGTAG AAGCTGATTC ACTGGCGCTT 
    2761 GTTGATGCAG ATTCGCTGGC ACTTGTCGAT GCCGATTCGC TAGCACTAGT AGAAGCTGAT 
    2821 TCACTGGCGC TTGTTGATGC AGATTCGCTG GCACTTGTCG ATGCCGATTC GCTAGCACTA 
    2881 GTAGAAGCTG ATTCACTTGC ACTTGTCGAT GCCGATTCAC TGGCGCTCGT TGATGCCGAT 
    2941 TCGCTGGCAC TAGTAGAAGC TGACTCACTT GCACTTGTCG ATGCCGATTC ACTGGCACTC 
    3001 GTTGATGCAC TTTCGCTAGC ACTTGTAGAT GCGGATTCAC TTGCACTCGT TGATGCACTT 
    3061 TCACTGGCAC TCGTTGATGC ACTTTCGCTA GCACTCGTTG AAGCACTTTC ACTTGCGCTT 
    3121 GTTGATGCGG ATTCGCTGGC ACTCGTTGAT GCACTTTCAC TTGCGCTCGT TGATGCCGAT 
    3181 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTCGTTG ATGCACTTTC GCTTGCACTT 
    3241 GTGGATGCGG ATTCGCTGGC ACTAGTAGAA GCAGATTCAC TTGCGCTCGT TGATGCCGAT 
    3301 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTCGTTG ATGCACTTTC ACTTGCACTT 
    3361 GTGGATGCGG ATTCGCTGGC ACTAGTAGAA GCTGACTCAC TTGCACTCGT TGAAGCACTT 
    3421 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTTGTCG ATGCTGATTC GCTAGCACTA 
    3481 GTAGAAGCTG ATTCACTTGC ACTCGTTGAT GCACTTTCAC TTGCGCTTGT TGAAGCCGAC 
    3541 TCACTTGCGC TCGTTGATGC CGATTCACTT GCACTCGTTG AGGCACTTTC ACTTGCGCTT 
    3601 GTTGAAGCCG ACTCACTTGC GCTCGTTGAT GCACTTTCGC TGGCACTTGT CGATGCCGAT 
    3661 TCGCTGGCAC TAGTAGATGC GGACTCACTT GCGCTCGTTG AAGCCGATTG GCTTGCACTA 
    3721 GTAGAAGCTG ATTCACTGGC GCTCGTTGAG GCTGATTCAC TGGTACTCGT TGATGCACTT 
    3781 TCGCTAGCAC TCGTTGAAGC ACTTTCACTT GCGCTTGTGG ATGCGGACTC ACTTGCACTA 
    3841 GTAGAAGCAG ATTCACTTGC GCTCGTTGAT GCCGATTCAC TTGCACTTGT CGATGCCGAT 
    3901 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTCGTTG ATGCACTTTC GCTTGCACTT 
    3961 GTGGATGCGG ATTCGCTGGC ACTAGTAGAA GCAGATTCAC TGGCGCTCGT TGATGCCGAT 
    4021 TCACTTGCAC TTGTCGATGC CGATTCACTT GCACTCGTTG ATGCACTTTC ACTGGCACTC 
    4081 GTTGATGCAC TTTCGCTAGC ACTCGTTGAA GCACTTTCAC TTGCGCTTGT TGATGCGGAC 
    4141 TCACTTGCAC TTGTTGAAGC CGATTCACTG GCGCTCGTTG ATGCACTTTC ACTTGTGCTT 
    4201 GTTGAAGCCG ACTCACTTGC GCTCGTTGAT GCACTTTCAC TTGCGCTCGT TGATGCCGAT 
    4261 TCACTAGCAC TAGTAGAAGC TGATTCACTG GCGCTTGTTG ATGCCGATTC GCTGGCACTT 
    4321 GTCGATGCCG ATTCGCTAGC ACTAGTAGAA GCTGATTCAC TGGCACTTGT CGATGCCGAT 
    4381 TCACTGGCGC TCGTTGATGC CGATTCGCTG GCACTAGTAG AAGCTGACTC ACTTGCACTT 
    4441 GTCGATGCCG ATTCACTGGC ACTCGTTGAT GCACTTTCGC TAGCACTCGT TGATGCACTT 
    4501 TCGCTAGCAC TCGTTGATGC ACTTTCACTT GCGCTCGTTG ATGCAGATTC GCTAGCACTA 
    4561 GTAGAAGCTG ACTCACTTGC ACTTGTCGAT GCCGATTCAC TGGCACTCGT TGATGCACTT 
    4621 TCGCTAGCAC TCGTTGATGC ACTTTCACTT GCGCTTGTTG AAGCCGACTC ACTTGCGCTC 
    4681 GTTGATGCAG ATTCGCTAGC ACTAGTAGAA GCAGATTCAC TTGCGCTCGT TGATGCCGAT 
    4741 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTCGTTG ATGCACTTTC ACTTGCACTT 
    4801 GTGGATGCGG ATTCGCTGGC ACTAGTAGAA GCTGACTCAC TTGCACTCGT TGAAGCACTT 
    4861 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTTGTCG ATGCTGATTC GCTAGCACTA 
    4921 GTAGAAGCTG ATTCACTTGC ACTCGTTGAT GCACTTTCAC TTGCGCTTGT TGAAGCCGAC 
    4981 TCACTTGCGC TCGTTGATGC CGATTCACTT GCACTCGTTG AGGCACTTTC ACTTGCGCTT 
    5041 GTTGAAGCCG ACTCACTTGC GCTCGTTGAT GCACTTTCGC TGGCACTTGT CGATGCCGAT 
    5101 TCGCTGGCAC TAGTAGATGC GGACTCACTT GCGCTCGTTG AAGCCGATTG GCTTGCACTA 
    5161 GTAGAAGCTG ATTCACTGGC GCTCGTTGAG GCTGATTCAC TGGTACTCGT TGATGCACTT 
    5221 TCGCTAGCAC TCGTTGAAGC ACTTTCACTT GCGCTTGTGG ATGCGGACTC ACTTGCACTA 
    5281 GTAGAAGCAG ATTCACTTGC GCTCGTTGAT GCCGATTCAC TTGCACTTGT CGATGCCGAT 
    5341 TCACTTGCGC TTGTGGATGC GGACTCACTT GCACTCGTTG ATGCACTTTC GCTTGCACTT 
    5401 GTGGATGCGG ATTCGCTGGC ACTAGTAGAA GCAGATTCAC TTGCGCTCGT TGATGCCGAT 
    5461 TCACTTGCAC TTGTCGATGC CGATTCACTT GCACTTGTTG AAGCAGATTC ACTTGCGCTC 
    5521 GTTGAGGCCG ATTCGCTGGC ACTCGTTGAT GCCGATTCGC TAGCACTAGT AGAAGCTGAT 
    5581 TCACTTGCGC TCGTTGATGC CGATTCACTT GCACTTGTCG ATGCCGATTC ACTGGCGCTT 
    5641 GTGGATGCGG ACTCACTTGC ACTCGTTGAT GCACTTTCAC TTGCACTTGT GGATGCGGAT 
    5701 TCGCTGGCAC TTGTCGATGC CGATTCACTG GCGCTTGTTG ATGCGGACTC ACTTGCACTC 
    5761 GTTGATGCAC TTTCACTTGC ACTTGTGGAT GCGGATTCGC TGGCACTAGT AGAAGCTGAC 
    5821 TCACTTGCAC TTGTTGAAGC CGATTCACTG GCGCTCGTTG ATGCACTTTC ACTTGTGCTT 
    5881 GTTGAAGCCG ACTCACTTGC GCTCGTTGAT GCACTTTCAC TTGCGCTCGT TGATGCCGAT 
    5941 TCACTAGCAC TAGTAGAAGC TGATTCACTG GCGCTTGTTG ATGCAGATTC GCTGGCACTT 
    6001 GTCGATGCCG ATTCGCTAGC ACTAGTAGAA GCTGATTCAC TTGCGCTCGT TGATGCCGAT 
    6061 TCGCTAGCAC TTGTCGAAGC TGATTCACTG GCACTCGTTG ATGCACTTTC ACTTGCACTC 
    6121 GTTGATGCCG ATTCGCTAGC ACTTGTCGAA GCTGATTCAC TTGCGCTCGT TGATGCTGAT 
    6181 TCACTGGCGC TTGTTGAAGC CGACTCACTT GCGCTCGTTG ATGCACTTTC ACTTGCGCTC 
    6241 GTTGATGCCG ATTCACTAGC ACTAGTAGAA GCTGATTCAC TGGCGCTTGT CGATGCCGAT 
    6301 TCGCTAGCAC TTGTCGAAGC TGATTCACTT GCACTCGTTG ATGCTGATTC ACTTGCACTT 
    6361  GTCGATGCTG ATTCACTTGC GCTCGTTGAT GCTGATTCGC TGGCACTTGT CGATGCCGAT 
    6421 TCACTTGCGC TTGTTGAAGC TGATTCACTT GCGCTTGTTG ATGCGGATTC GCTTGCACTC 
    6481 GTTGATGCAC TTTCACTTGC GCTCGTTGAT GCCGATTCGC TGGCGCTCGT TGATGCACTT 
    6541 TCACTTACGC TCGTTGATGC ACTTTCACTT ACGCTCGTTG ATGCACTTTC ACTTACGCTC 
    6601 GTTGATGCAC TTTCACTTAC GCTCGTTGAT GCACTTTCAC TTACGCTTTT TGATGCCGAT 
    6661  TCGCTGGCAC TCCTCGTTGA AGCGGATTCG CTGGCACTCC TCGTTGAAGC GGATTCACTT 
    6721 ACGCTCGTTG ATGCGCTAGT CGAAGTACTC GTCGAGGTGG ATTCCTGTTC ACTCTTACTC 
    6781 GTCAATTGA 
    Lactobacillus reuteri 3630_00804 
    SEQ ID NO: 31
    1 ATGGAGATAT TGGTAACGTT ATTTGATTTG GTGTTTTTTA TTACGTTTAT AGTCGCAATT 
    61  GTCTATGGTA TTAGGTGGTT TAAAGGAAGA AAAGATAAAG AAAATGAATC TCTTAAGAAA 
    121 CGCCGTTTGT AA 
    Lactobacillus reuteri 3630_00827 
    SEQ ID NO: 32
    1 ATGATTAACT TAAACTTAGC AGGCTTAGAT TTTGTAATGA CGGCACTTTT TATTGTGTTG 
    61 TTTACAGAAC AGTTGAAAAA TGCCCGAACT CAGCGTGATG CTCTGATTGG TTTAGCATTT 
    121 GCAATTATTT GTTTACTATT TTGCAACAAG AATGTTTTTC TATTAGTGAC ATTAGTAACA 
    181 CTTGTCGCAC TGTTTTCATT AAATTACTTA ATCACGAGGA GAAAAAATGA CATTAACTGA 
    Lactobacillus reuteri 3630_00947 
    SEQ ID NO: 33
    1 ATGTCAGAGA TGAAAAATCG CGTAATTAAT TTTCGGAATG ATAACTTAGC CAAACTTATA 
    61 GTCAATTATT ATGGAAATAG TCAATTAAGT GTGCATATTA CTAATAATAT GTTTTTTGAA 
    121 TGA 
    Lactobacillus reuteri 3630_01261 
    SEQ ID NO: 34
    1 ATGAAACGCA AAATTGCATT AGCTCAACTT GATATTCAAT TAGGAAATCC TGCCGAAAAT 
    61 TATCAAAAAG CTAAACAAGC GATTGAAGAA GCTGCTAGTC ACCATGCAGA TATCGTTGTC 
    121 TTGCCGGAGA TGTGGAATGC TGGCTATGCC TTAGATCAAT TAGCAGAATT GGCAGATGAA 
    181 AACGGTCAAA AGACACAAAA ATTTCTTAGT GAGTTAGCGT TAGAAAATCA AATTAACATT 
    241 GTCGGTGGTT CAGTAGCGGT GAGATGTGGA CAATCTTTTT TCAATACAAC CTATGTTTAT 
    301 GATCAAAAGG GAAATCTAAT TAGCAGTTAC GAGAAGGTGC ATTTATTTGG ACTAATGAAT 
    361 GAAGACCGAT ATCTAAAAGC CGGGCAAAAA GAAAATCACT TTGAATTAGC TGGGGTTCCG 
    421 AGTGCAAGTT TTATTTGTTA TGATTTGCGA TTCCCTGAAT GGATTAGAAC AGTCACTCGT 
    481 TATGGAACTG ATATCTTATA TTTTTCGGCA GAATGGCCAA GCAAACGGAT TAAACAATGG 
    541 AAAATAATGC TTCAGTCACG GGCAATTGAA AATCAAGCCT TTGTAGTCGC GGTCAATCGT 
    601 GTTGGGACGG ATTTAGAGAA TAGCTTTAAT GGTCATTCGT TAGTAATAGA TCCGCTTGGG 
    661 CAGATTATCC ATGATGCAGG AGAAGTTGAA CAAGTAAGTT ATGCAGAAAT TGACTTAGCG 
    721 CAGTTAGCAC AGGTTCGGGG GCCGATTCCG GTGTTTAAGG ATCGCCGACC AAGTCTTTAT 
    781 CATTAA 
    Lactobacillus reuteri 3630_01695 
    SEQ ID NO: 35
    1 ATGCAAAATA AAGATGCTTG TACATCAATT ATGGTCGGTA AAAAGGCTTC TCTCGACGGT 
    61 GCTAATTATA TTGCTCGTAA TGAAGATCGC GTAAAAGCAA TTGAACCCAA GCGATTTTTA 
    121 GTAAAACCGG CAGTAAAAGG ACGCCACGAA ACCTACGTAT CACCTTACAA TAAAGTAACT 
    181 GTAGCTTTGC CGGAAGAGAG AATGCGTTAT ACTTCTACGC CTACCCTTGA TCAAACAGCC 
    241 GGACCTAATG AAGAAGATGG AATTAATGAA GCAAATGTGG CAGCTTCCTT TACTGAGAGT 
    301 GTTTATGCAA ATGATCGGGT GTTAGCATAT GATCCATACG TAAAAAATGG CCTGGCAGAA 
    361 GACTCACTTT GTACTTTAGT ATTACCGTAT ATTCATTCTG CCCGTGAAGG AGTTGAATAT 
    421 ACTGGAAAAT TAATTGCTGA ATTGGGCTCT GCTGAGGGAA ATGGAATGCA ATTTGCAGAT 
    481 GCAGATGATA TTTGGTATAT GTAA 
    Lactobacillus reuteri 3630_01889 
    SEQ ID NO: 36
    1 ATGAATTATT TTATCGGTGT TGATGTTGGA ACTACTTCTA CAAAGGCAGT TCTATATGAC 
    61 CAAAATGCAA CTGTGTTAGA TCAATTTAGC CAAGGTTATT CCCTTTACCG CGATGCTAGT 
    121 GGAATGGCTG AACAAAACCC AACTGCAATT GTCGAAGCAG TCGAAAAAGT TATTCATGAT 
    181 GCAGCACAAA AAGCAGATTT AACAAATGGA AAATTGTTAG CGGTATCATT TTCTAGTGCT 
    241 AACCAAAGTG TGATTATGCT CGACAAGAAT TTCAATCCCC TTTCACGGGT CATCACTTGG 
    301 GCTGATACCC GTGCACGTGA TGTCGCCAAC GAATTAAAGA ATAGTCCTGC TGGTCAGCAA 
    361 ATCTATGCTA AAACAGGTAC ACCTATTCAT CCAATGTCCC CATTGACCAA GATTATGTGG 
    421 CTCAATAAGA CACAAGCAGA TAAGGTTGCT CAAACTGCAT ATTTTGGCGA TATCAAATCC 
    481 TACCTCTTCC ACCAGTTTTT CAATACATTT AAGGTTGATG TTTCCATCGC TTCATGTACC 
    541 GGAATGATGA ATGTCAATAC GTGTGACTGG GACGATCAAG CATTGGAACT CGCTAACGTC 
    601 GACTGTTCCC AATTACCAGA AATCGTGAAC GGAACAACCC AAGCGATTGG CCTAACAGCA 
    661 GCGGCGCAAG CAAAAATGGG TATCCCCGCT GACACGCCAT TTGTCTATGG TGCCTTTGAC 
    721 GGTGCTTTAT CTAATTTAGG TGTGGGGGCA ATTAAGCAAA ATACTGTTGC CATTACGATT 
    781 GGAACTTCGG CTGGTGTTCG GGTAGTAACT GACCATCCAG TGATCGATCC TCAGCAACGA 
    841 CTCTTCTGTT ACGCCGTGGA TAAAGGTTTA TGGGTCATCG GCGGTCCGCT TAATAATGGT 
    901 GGCGATGTCT ATCAGTGGGC CGTTGAACAC TTAGTTGACG CTAGTGCAGT TAAAAATGAA 
    961 AATATTGATC CCTACACTCT TGCTAACCGA GTTATTGAAG GTGTTCCCGC CGGAGCTCAC 
    1021 GGTTTGCTCT TCCACCCATT CCTTGGCGGT GAACGGGCAC CATTATGGGA CGCTAATGCG 
    1081 CGCGGTAGTT TCTTTGGACT TTCCCACATT CATACTCGTG CCGATATGCT GCGCTCAGTA 
    1141 ATGGAAGGAA TTTGTATGAA TATTGCAACT GTTTTCCAAG CGGTTCGTGA TCTTGTTGGT 
    1201 AATCCTGCAA GCGTAACTGC AACTGGCGGT TTTGCGCGAG CTGAAGTTTG GCGGCAAATG 
    1261 TTAGCAGATG TCTTGAACTG TCCGGTCAAT ATCCCGAACT CATTTGAATC TGGTTGTCTC 
    1321 GGTGCAATCA CCATGGCAAT GAAGAGTTTA GGAATGATTG AAAACTATGA AATCATTAAA 
    1381 ACATTAGTTG GTGATATCAG TTCTTATCAG CCAAATCAAG ATGCGGTTAA TGTTTATCAA 
    1441 AATTACTTAC CACTTTTTAA GCAGGTCGAA GGATTATTAA CACCAGCCTA TTCGACCATC 
    1501 GCTAAATTAC AACAACAATC TACTACTCAT TAG 
    Lactobacillus reuteri 3630_01932 
    SEQ ID NO: 37
    1 ATGACAACAT CAATGATCCA CAGTAGAAGT ATGTTGGCGA AAGTGATTGC AGAATCACCT 
    61 TCACCTTTTA TTATACCAAT TTTTTGTCCT AATGTAATAA AGATTATCTT TTTATCTATA 
    121 AATTATTTTA TGAAAAGAGT GGAAAAGGCA AGAAGAGCAA TCAAAAAGCC AATTTTAACA 
    181 ATTTTATTAA GTTGA 
    Lactobacillus reuteri 3630_02243 
    SEQ ID NO: 38
    1 ATGAGCAAAC TATTACTTGA TGAAAGACCG TTACAAGTTC AGGCATCGTT AGCTGGAGCG 
    61 TTGAAAAGCT TAGACGAAGC TGTTATTCTC CAGCAGCTTC ACTATTGGCT TCAACGTTCT 
    121 AATACAGTAA GAGACAATCA CAAATGGGTC TATAACAGCA TGGCTGATTG GAATAAACAG 
    181 TTCCCTTGGC TTTCTAGAAA GGCTCTATCG AACCACTTTA AGAAATTAGA AAAACGAGGA 
    241 CTAATTATTA CAGGCAACTA TAATAAATTA TCTTTTGACA AAACAAAGTG GTATCGAATT 
    301 GACTATGACG CATTTTCCCA TTTGGAACAA CGATTGGGTA GAAACTACCC AACGAATGGG 
    361 AAGAATCTAC CCAATGGAGA CGGTAAAAAC TGCCCAATCG GAGAGGAAGA ATCTACCCAA 
    421 CCAATACCAA TAGACTACCA AGAGACTACA CAAAAGACTA CTACAAGAGA TAAAGGGCAG 
    481 GCACAGCCAG CCCAACCTTC CATTGCTGCA CAGCGGCGAG AAGTTGTTGA ATATCTCAAT 
    541 CAAAAAAACT GGCAAGCACT TCAAGCCTGA 
    Lactobacillus reuteri 3630_02365 
    SEQ ID NO: 39
    1 TTGAATAATT TTCAAAAGGC AATTTTCTTG TTGCAGAATA TCGACAAGCT TAAACAGCTT 
    61 AATGGTAAAG CGATGACTCT TACTGAGTTC TCTAAAATAA CTGATGTTTC ACGGCCAACG 
    121 TTGTATAAAT ACATTCAGCA TCCAGAAACA ATGAGTAGTT CGTTTGTAAA TAAAGCGGCC 
    181 ATGCTCTACG ACAAGGTTGT TAAATTTCAA GATATTCTTG ATACAGTTCA GCGTGAAGAT 
    241 AAACAATTTA AGACTACCAG GCAGGAATTG ATTAAGCTTT TAGAGTCTAA TGTAGCTAAT 
    301 ATTGAAGTTA CAGATTATAC AAAAGCAATC GCGACAGTAA TTATTAGTGA CTTAAAAGAA 
    361 GAAAATTCAA GTCTGCTAAA AGCGTTAAGT AAGCAATTAC CATTTAAACC AAATTTAAAT 
    421 GATAATTTGT CAAAATAG 
    Lactobacillus reuteri 3630_02435 
    SEQ ID NO: 40
    1 GTGAAGATGA ATAGTATGAC AAACAACCAA AAAGAAAGTT GGAACGTTGG CAACTATAAA 
    61 ATCAATGTAT TACCAGATGA TGAGTTCCAG CAATTGTTAA AGAACCAACG CCAACTTCAA 
    121 CAGATCATTG AAAGTATGCC ACTACCAACC GACCCCAATG TTGATCTAGT TAAAAAGATT 
    181 CATTCCCAAC TCCCTATTAC AAACTGGGCT TGGGAATTAA CTAAACAACG AGAACATGAG 
    241 GAAAAGTTAA AGAAACAAAA GCAGCGAATT GCACAGCAAT CGCTTAACTA TCCAACAAAC 
    301 CTCAAGAAAC CGGATAATGG CCTTTCCCTA TAA 
    Lactobacillus reuteri 3630_02436 
    SEQ ID NO: 41
    1 ATGAATTCTA ATTTGAAAAA GAATTTGATT ATCGCAAATG GATTTCTACT ACTAATAATA 
    61 ATATTTTATG TTTTATTACA TATGGGTCCA TTAAATATGA AAGTCTTATT AGTAGGATTG 
    121 GTATTAATGA ATCTGACAGT AATATTTAAA TAA 
    Lactobacillus reuteri 3630_02437 
    SEQ ID NO: 42
    1 ATGGATAAAT TAACATTGAA AAAACTAATT ATTATTACAT TAGGCATGCT AATGGTATTT 
    61 CTTTTAGGTA TGCACCTTCA TCAAAAGACA CACTTTAACA AAAACGTGAA GATTAATAAT 
    121 ATTCCAGTCG GAGGTCTTAC TGTTCAACAG GCATATAATA AAGTAAGTAA TACTAAAAGA 
    181 AAATCAAAAA TCTACATTAA CAAAAAGTTA GTTTATTCAG GTAAAAGTAC TGACTCAGGT 
    241 TTTAAGTTAT CTGATAAAGA AAGATTTAGT AAGGCGTTAC ATTACCAATA CACTTTTTTT 
    301 CCCTCACGAA AACATGAAAA TTTGCTAGTT GAGCCAGCTG ATTTAGATAA GTCAGCGTTA 
    361 AATAATATTG ATTCGGCAAT AGTAGCTAGG ATTCATCAGC TCAATATAGG CAGAAAGGCC 
    421 CCACGTGATG CATACGCCGT TTACCAGAAT AATAAAGTTT CAGTTATTCC AGCAATTGAT 
    481 GGGACACGAT ATAGTGAACA AGGACTTTGT AATATTGCTA ATAAGGAATT TGTTAATGGG 
    541 ACAATTCATT TAACTCCTAA GGTTATTACT CCTTTATCGG CAAACAGTAA AGTAGTTCAA 
    601 GATGAAAAGA AACACCTGAG TAAACTACAA AATCGATCGG TTGTTTATCA GGTTCAGAAA 
    661 ACAAAATATA ATTTTAAAGC GTCTAATGTC ATTTCTAAAG CAACTTATCA GCATGGGAAA 
    721 TACCATTTTG AAACTGACAA CGTTAAATCC AAGATTGCCA ATATAAATAA TAAGCAAGCA 
    781 ACATTAGGAA AGAGCTTTAA ATTTAGAACT GATTCTGGAA AAGTTATTTC TACATCTAAT 
    841 CAGGGAACAT ATGGTTGGAA AATAAGTAGC AAGCAGGCAG GACAAACACT CTCTAAAGCG 
    901 TTAGCTAATA ATGTTAAGAG CGTTAATGCC GAAAATGATA TTTACGGTAA AGGCTATAGT 
    961 CATCTTGGTA CTGGATATTC GGCTGTGAAT AATCATGGGC TTGGTAATAC TTATGTGGCT 
    1021 GTATCATTAG CTAAACAGCA TGCTTGGTTT TATAAAAATG GAAAATGTGT ACTGAGTACA 
    1081 GATATTGTTA GTGGATCAGA TGACGCTAAT AATAGGACTC CTAAAGGTGT TTGGTATATC 
    1141 ATGTATCAAC AAACGCCATC AGTTTTACGT GGGACTAATG ATGATGGTTC CAAGTATAGT 
    1201 AGTCCTGTTC AGTATTGGTC TCCGTTTACT TTATCAGGGT GTGGCTTTCA TGATGCTAGT 
    1261 TGGAGGCATA ATTGGTCTAA AACAGCTTAT AAACAGACTC ATGGTGGCTC ACATGGCTGT 
    1321 ATTAACATGC ATCCGGAAAA TGCAGGAGAC GGTTTCCATG CCCTTACTAA AGGAGAACCG 
    1381 GTAATAATTT ATTAG 
    Lactobacillus reuteri 3630_02438 
    SEQ ID NO: 43
    1 ATGCAATTAA ACCATAAATT AGGAGTTTTC TTGGCAGCGC CATTTGCTCT ACTGGTTTTA 
    61 TCAGCTACAA ACGTGCATGC CGATAACATT CAAAGTAATA GTAACCAGAC AATCAGTAAT 
    121 ATGAGTTTGC AAACTAATGA CACAAAGACT CAACAAAATG TTGTAATGTC AAACGATGCA 
    181 AAAGCTCAAA TTACTGTAAA TCCTAGTTCT AATGCTAATT CTAGTTCTGT AGCAAAGATA 
    241 AATGAAAAGA ATAATGTAAA ATCGGATACT GACAATACTA ATGTCGAATC AAATGCTGAT 
    301 AATATTGGGA ACATTGCTTC TAGCGATTCC ACGGCAGTGG CTAATTCTGC TAGTTCCGAT 
    361 AATATTCAGT CATTTAACGT AAATACGCAG GAACAGCCTG CAATAAATGT ATCTGAACTA 
    421 ACAACCGAAG AGTATGTTAC GAATTACACT CAACAACAGA TCAATAATGC GACGACTATT 
    481 CATGATTACT TTATAAATCA AGGATGGACA CCAAATGCTA TTGCTGGAAT GCTTGGTAAC 
    541 TTTGTTTCAG AGTCAGGTTT AATCCCAGAC TTACATCAAT ATGGTGGTGG GCCTGGTTAT 
    601 GGGTTAGCTC AATGGCCATT TAATAGTGTA GTAAATTGGT GTCGTAATAA TGGATATGAT 
    661 TATCGTACTT TGCAAGGACA ATGTGCATAT ATTGAATATC AAATGACTCA TGGACAGCAG 
    721 TATTATCCAT CAGCTTACTC TAGAATGACC GCTAATGAAT ATATGCATAG TTATGCTTCA 
    781 GCATATACTT TAGGTATGAT TTGGCTTAAT AACTTTGAGC GACCTGCAAA TAGGAATCAG 
    841 CCAGCTCGTG GTCAACAGGC TCAATACTGG TATCAGTATT TCCAAAGTCA TGGTTCTACA 
    901 TCAGCACCGG TACAACAAAA TCCTAGTACA CCAGCAACAA CTCCTAGCTC AAGTCGAATG 
    961 AGTCAACACG GGACATTCAA AGTTGCTTAT GGATTAAATG TACGCCAAGC ACCAAGTACA 
    1021 TCGGCAGCTA TTGTAACGTA TTACAATGGT GGTCAAAGCT TTACATATGA TTCAAAGATT 
    1081 GAAGCTAACG GGTATCTTTG GGTATCATAC ATGAGTTATA GTGGCGTACG TCGTTATGTT 
    1141 GCAATTAAGA ATTTGAATAA TGGAACGACT TACGGTTATG ATTCGAATAA CTTCTCATAC 
    1201 AGTGCTCCTG CATCTTCAAC ACCATCTACT AATGTGCCAA GTACGCCAGC ACCAAGTACA 
    1261 TCTACTTCAT CAACTGAGAA GCAATATGGA ACATTCAAAG TTGCTTATGG ATTAAATGTA 
    1321 CGCCAAGCAC CAAGTACATC GGCAGCTATT GTAACGTATT ACAATGGTGG TCAAAGCTTT 
    1381 ACATATGATT CAAAGATTGA AGCTAATGGG TATCTTTGGG TATCATACAT GAGTTATAGT 
    1441 GGCGTACGTC GTTATGTTGC GATTAAGAAT TTGAGTAATG GAACAACTTA CGGTTACGAT 
    1501 TCAAATAACT TTTCATTTAA TGGGACTCCA GTAACATCAA ATAATAATCC TTCTAGTACT 
    1561 CCGGCAGTTC CGCAAGGTAA TAAGGGCCAA CAAGTTGTTG CTCTTGCACG TCAACAAATA 
    1621 GGTAAACCTT ATGTTTGGGG AGCAACCGGT CCTAATTCGT TTGATTGTTC AGGACTCGTG 
    1681 CAGTATGTTT ATCGTCAAGT TGGTGTTAAC TTACCACGGA CTACAACTCA ACAAGAATAT 
    1741 TGTGGACATG CTGTAAGCTT TAATAATCTT CAACCTGGAG ATCTAATGTT CTGGGGAAAG 
    1801 TATGGTAGTG CATATCACGT TGGAATCTAT ACCGGAAACG GTAATGTTTT ATTTGCACCG 
    1861 CAACCTGGTC AAACAGTTAA GGAACAACCA ATGCGCTATT ACATGCCTGC CTTTGCAAGA 
    1921 AGAGTATTGT AA 
  • The present disclosure may be better understood with reference to the examples, set forth below. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure.
  • Example 1. L. reuteri Strain Identification
  • Seven Lactobacillus reuteri (L. reuteri) strains were isolated from older birds at the Research Center, Hannover from the cecal contents received from the Poultry Clinic, University of Hannover. All the seven strains are identified to be L. reuteri by 16S rRNA sequencing.
  • While only limited growth is observed for most strains under aerobic conditions in MRS broth and agar (de Man, J. D.; Rogosa, M.; and Sharpe, M. E. “A Medium for the Cultivation of Lactobacilli”. J Appl Bact. 23: 130-135 (1960)), all isolates show very good growth on MRS agar and MRS broth under anaerobic conditions at 39° C. Culturing the bacterial strains on blood agar under anaerobic conditions results mostly in limited growth. None of the strains is able to grow in Mueller Hinton broth under anaerobic conditions. For all further analysis, bacterial strains are grown in MRS medium under anaerobic conditions at 39° C.
  • Antimicrobial susceptibility of bacterial isolates were tested using the AVIPRO® PLATE. All strains are resistant against colistin, doxycycline, enrofloxacin, erythromycin, neomycin, oxacillin, penicillin G, trimethroprim-sulfamethoxazole, tetracycline, tilmicosin and tylosin. All strains are resistant to streptomycin except strain 3632, and to tiamulin except strain 2098. In addition, resistance to cefpodaxime-proxetil is observed with strains 2091, 2095, 2097 and 3630; resistance to cefotaxime is observed with strains 2091, 2095 and 2097; and resistance to lincomycin is observed with strains 3630 and 3632. No strain was found to be resistant against amoxicillin, ceftiofur, erythromycin D, lincomycin-spectinomycin, and rifampicin under tested concentrations.
  • Example 2. L. reuteri Strain Selection and Anti-Infective Activity
  • To select the best strain for further use as a priobiotic, the L. reuteri isolates were tested for various desirable probiotic anti-infective properties, such as growth kinetics, ability to produce hydrogen peroxide, autoaggregation, enzyme profile, survival in the presence of ox bile and pancreatic enzymes, and sensitivity to heat shock and pH changes. The L. reuteri strains are also tested for safety using a haemolytic assay.
  • In general, all strains behave very similar in terms of probiotic properties, including growth kinetics and ability to produce hydrogen peroxide, except for strain 3632, which shows some unique properties, including the ability to autoaggregate in liquid media (comparable to that of the well-characterized human probiotic strain L. reuteri ATCC 23272). In addition to autoaggregation, L. reuteri 3632 also appears to produce an orange pigment, which resembles beta carotene in color. None of the other strains, including the human L. reuteri strain ATCC 23272 and L. acidophilus, produced orange colored pigment. None of the strains is found to be hemolytic on blood agar plates, suggesting that these isolates are less likely to be pathogenic to humans.
  • Whole-genome sequencing was performed for L. reuteri strains 2091, 3632, 3630, and an independently isolated L. reuteri strain 170331 of European origin, using PACBIO® sequencing (Amplicon Express). Sequencing, assembly and annotation statistics are summarized in TABLE 1. Genomic structures and organization differs among the tested strains.
  • TABLE 1
    Strain 170331 2091 3632 3630
    Genome size 2,090,596 bp 2,231,245 bp 2,482,713 bp 2,399,045 bp
    Contigs 9 11 7 5
    Coding 2,154 2,280 2,595 2,467
    Sequences
    (ORFs)
    Ribosomal 2,231 2.354 2,680 2,467
    Binding Sites
    Transcription 1,123 1,123 1,375 1,339
    Terminators
    Operons 452 472 541 475
    tRNA 75 61 77 73
    rRNA 18 19 13 18
    Other RNAs 39 99 89 N/A
    CRISPR 1 0 0 0
    Prophages 7 1 8 5
    Islands 30 22 24 9
    Bacteriocins 0 1 4 3
  • Example 2.5. Colonization of L. reuteri 3632 in 16-Day Old Embryos
  • The goal of this study was to test if L. reuteri 3632 colonizes the intestine of 16-day old embryos. To this end, 16 day-old embryos were obtained from Charles River and inoculated with 10{circumflex over ( )}8 CFU/embryo of L. reuteri 3632 by air cell, yolk sac and allantoic routes. Embryos were sacrificed on 18 days and the intestines were harvested and plated on MRS agar plates. An uninoculated group was included as a control. L. reuteri was recovered from the intestines of the majority of the embryos, suggesting that L. reuteri 3632 colonizes the intestine of 16-day old embryos. L. reuteri 3632 was not recovered from any of the control (uninoculated) embryos, even after 2-days of enrichment in liquid culture.
  • Example 3. Co-Culture of L. reuteri and C. perfringens in Brain Heart Infusion (BHI) Broth
  • L. reuteri and C. perfringens were cultured in BHI broth. CFU counts were determined 24 hours after coculture in BHI broth. L. reuteri were enumerated on MRS agar plates and C. perfringens was enumerated on OPSP plates. This data is from one representative experiment of 3 replicates. Co-culture-L. reuteri, L. reuteri recovered from co-cultured sample using L. reuteri-specific media. Co-culture-C. perfringens, C. perfringens recovered from co-culture sample using C. perfringens-specific media. See FIG. 9.
  • Example 4. In Vivo Effects of L. reuteri
  • Birds were vaccinated with CocciVac on day 0 and fed with L. reuteri in feed from day 0 to day 42 @ 10{circumflex over ( )}7 CFUs/Kg of feed. A separate group of birds were fed with feed supplemented with bacitracin methylene disalicylate (BMD_55).
  • Feed conversion ratio and European Broiler index were calculated. Birds that were fed L. reuteri demonstrated a reduction in feed conversion ratio and European Broiler index as compared to those fed BMD_55. See FIG. 8.
  • Example 4. The Effect of Prebiotics on the Growth of Lactobacillus reuteri
  • L. reuteri strains 3630 & 3632 were grown individually and in combination, in the presence of several different prebiotics, including fructooligosaccharides (FOS), arabinoxylan oligosaccharides (AXOS), mannan-oligosaccharides (MOS), or galacto-oligosaccharides (GOS).
  • As shown in FIG. 10, strains grown in the presence of GOS demonstrated increased growth as compared to strains grown in the presence of FOS, AXOS, or MOS.
  • Example 5. In Vivo Effects of L. reuteri
  • An identified strain can effectively function as a direct feed microbial only if the strain is able colonize the host gastrointestinal tract. The ability of L. reuteri strains to colonize chickens is assessed following different routes of administration.
  • L. reuteri is administered orally to 10-day old chicks. At various times post inoculation, chicks are sacrificed and the presence of L. reuteri is measured in the crop, small intestine, and cecum. Strain 3632 is able to colonize all three tissues and remain detectable for at least 18 days.
  • L. reuteri is administered by in ovo inoculation (i.e. injection into the egg) 3 days before hatching. Strain 3632 is found in the tissues of chicks at 3 and 7 days post hatching, while colonization of a control strain declines after day 3.
  • L. reuteri is administered in ovo by spraying a liquid containing bacteria onto the egg surface. Again, Strains 3630 and 3632 are able to colonize and remain present in chicks 3 and 7 days post hatching. Inoculation by spray is just as efficient as in ovo injection but requires less manipulation of the incubating egg.
  • Table 2 shows quantifies the effect of L. reuteri 3632 treatment on survival. The difference score between strain 3632 and the control is 3.3 and the difference between BMD and control is 2.5. Strain 3632 provides increased survival over BMD treated chics.
  • TABLE 2
    Treatment conditions Difference Score
    Lr_3632 Ctrl 3.333333
    BMD_55 Ctrl 2.5   
  • Example 6. Necrotic Enteritis Model
  • L. Reuteri administered in ovo and in drinking water daily @ 10{circumflex over ( )}8 CFU/bird. The birds were orally gavaged with E. maxima on day 14. The birds were orally gavaged with C. perfringes on day 18, 19, and 20 with 10{circumflex over ( )}8 CFUs/bird.
  • TABLE 3
    Summary of intestinal lesion score, total mortality percent (d15-d28),
    and necrotic enteritis mortality percent.
    Lesion Total NE
    No. Score Mortality Mortality
    Treatment Cages Mean (SE) Percent (SE) Percent (SE)
    T1. Unchallenged, 4 0.25a (0.25)  0.0a (3.4)  0.0a (2.5)
    untreated
    T2. E. maxima alone 4 0.50a (0.25)  4.2a (3.4)  0.0a (2.5)
    T3. E. maxima + C. 4 0.50a (0.25)  20.8b (3.4) 20.8b (2.5)
    perfringens
    T4. Lactobacillus 5 0.20a (0.22)  0.0a (3.0)  0.0a (2.3)
    reuteri alone (in
    ovo + oral)
    T5. E. maxima + L. 5 0.20a (0.22)  3.3a (3.4)  0.0a (2.3)
    reuteri (in ovo +
    oral)
    T6. E. maxima + C. 5 0.20a (0.22)  6.7a,b (3.0)  6.7a (2.3)
    perfringens + L.
    reuteri
    (in ovo + oral)
    T7. E. maxima + C. 1  0.0a (0.50) 16.7a,b (6.8) 16.7a,b (5.1) 
    perfringens + L.
    reuteri (spray +
    oral)
    P-value 0.881 0.003 <0.001
    Lesion scores and cage-level mortality percentages tested by one-way ANOVA. Within columns, means and percentages with a superscript in common do not differ with a level of significance of 5% over all comparisons.
  • Example 7. Global Metabolomics Analysis
  • A global metabolomics analysis of L. reuteri strains 3632 and 3630 were conducted. The strains were grown individually and in combination, and various molecules were analyzed in the cell pellet and the supernatant of the cultures. The strains were grown in AOF-MRS media control with no glucose but 0.5% GOS. The molecules in the supernatant represent molecules that are secreted by the cell.
  • Samples were subject to global untargeted metabolic profiling. Welch t-test and Principal Component Analysis (PCA) were used to analyze the data. Principal component analysis (PCA) is a mathematical procedure that reduces the dimensionality of the data while retaining most of the variation in a dataset. This approach allows visual assessment of the similarities and differences between samples. Populations that differ are expected to group separately and vice versa.
  • The data is shown in FIGS. 12-19.
  • EMBODIMENTS OF THE INVENTION
    • 1. A composition comprising at least one of: an isolated first Lactobacillus reuteri strain and an isolated second Lactobacillus reuteri strain;
      • wherein said composition increases animal health when an effective amount is administered to an animal, as compared to an animal not administered the composition.
    • 2. The composition according to embodiment 1, wherein the first Lactobacillus reuteri strain comprises at least one of:
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26,
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1,
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, and
      • a nucleic acid that encodes for an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8;
      • wherein the isolated second Lactobacillus reuteri strain comprises at least one of:
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25,
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27,
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28, and
      • a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29.
    • 3. The composition according to any one of embodiments 1-2, wherein the isolated first Lactobacillus reuteri strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26.
    • 4. The composition according to any one of embodiments 1-3, wherein the isolated second Lactobacillus reuteri strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25.
    • 5. The composition according to any one of embodiments 1-4, wherein the composition comprises an isolated first Lactobacillus reuteri strain comprising a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25; and a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1,
    • 6. The composition according to any one of embodiments 1-5, wherein the isolated first Lactobacillus reuteri strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26; and
      • wherein the isolated second Lactobacillus reuteri strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25.
    • 7. The composition according to any one of embodiments 1-6, wherein at least one of the first lactobacillus strain and a second lactobacillus strain secrete at least one of cyclic dipeptides, short chain fatty acids, betaine, dimethylglycine, essential amino acids, nucleotides, myo-inositol, and indolin-2-one.
    • 8. The composition according to any one of embodiments 1-7, wherein the composition comprises a ratio of isolated first Lactobacillus reuteri strain to isolated second Lactobacillus reuteri strain of 0.75-1.5:1.
    • 9. The composition according to any one of embodiments 1-8, wherein the composition comprises about equal amounts of the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain.
    • 10. The composition according to any one of embodiments 1-9, wherein the composition is formulated as animal feed, feed additive, food ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or combinations thereof.
    • 11. The composition according to any one of embodiments 1-10, wherein the composition comprises animal feed.
    • 12. The composition according to embodiment 11, wherein the composition comprises the isolated first Lactobacillus reuteri strain in an amount of about 10{circumflex over ( )}2-10{circumflex over ( )}8 CFU/kg of the composition, about 10{circumflex over ( )}4-10{circumflex over ( )}7 CFU/kg of the composition, or about 10{circumflex over ( )}3-10{circumflex over ( )}5 CFU/kg of the composition.
    • 13. The composition according to any one of embodiments 10-12, wherein the composition comprises isolated second Lactobacillus reuteri strain in an amount of about 10{circumflex over ( )}2-10{circumflex over ( )}8 CFU/kg of the composition, about 10{circumflex over ( )}4-10{circumflex over ( )}7 CFU/kg of the composition, or about 10{circumflex over ( )}3-10{circumflex over ( )}5 CFU/kg of the composition.
    • 14. The composition according to any one of embodiments 10-13, wherein the composition comprises the isolated first Lactobacillus reuteri strain in an amount of about 10{circumflex over ( )}7 CFU/kg of the composition.
    • 15. The composition according to any one of embodiments 10-14, wherein the composition comprises isolated second Lactobacillus reuteri strain in an amount of about 10{circumflex over ( )}7 CFU/kg of the composition.
    • 16. The composition according to any one of embodiments 1-15, wherein the composition comprises water.
    • 17. The composition according to any one of embodiments 1-16, wherein the animal is bird, poultry, a human, or a non-human mammal.
    • 18. The composition according to any one of embodiments 1-17, wherein the animal is poultry and increases poultry health comprises at least one of: decreasing feed conversion ratio, increasing weight, increasing lean body mass, decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, and decreasing mortality rate.
    • 19. The composition according to embodiment 18, wherein increases poultry health comprises decreasing feed conversion ratio by at least 1%, at least 5%, at least 25%, or at least 50%.
    • 20. The composition according to any one of embodiments 17-19, wherein increases poultry health comprises increasing poultry weight by at least 1%, at least 5%, at least 25%, or at least 50%.
    • 21. The composition according to any one of embodiments 17-20, wherein increases poultry health comprises decreasing pathogen-associated lesion formation in the gastrointestinal tract by at least 1%, at least 5%, at least 25%, or at least 50%.
    • 22. The composition according to any one of embodiments 17-21, wherein increases poultry health comprises decreasing mortality rate by at least 1%, at least 5%, at least 25%, or at least 50%.
    • 23. The composition according to any one of embodiments 18-22, wherein the pathogen comprises at least one of Salmonella, Clostridium, Campylobacter, Staphylococcus, Streptococcus, and E. coli bacterium.
    • 24. The composition according to any one of embodiments 18-23, wherein the pathogen comprises at least one of Salmonella typhimurium, Salmonella infantis, Salmonella Hadar, Salmonella enteritidis, Salmonella Newport, Salmonella Kentucky, Clostridium perfringens, Staphylococcus aureus, Streptoccus uberis, Streptococcus suis, Escherichia coli, Campylobacter jejuni, and Fusobacterium necrophorum.
    • 25. The composition according to any one of embodiments 17-24, wherein administered comprises in ovo administration.
    • 26. The composition according to any one of embodiments 17-25, wherein administered comprises spray administration.
    • 27. The composition according to any one of embodiments 17-26, wherein the poultry is a chicken.
    • 28. The composition according to any one of embodiments 17-27, wherein the poultry is a broiler chicken.
    • 29. The composition according to any one of embodiments 17-27, wherein the poultry is an egg-producing chicken.
    • 30. The method according to any one of embodiments 1-29, wherein the animal administered the composition, further exhibits at least one improved gut characteristic, as compared to an animal not administered the composition; wherein improved gut characteristics includes:
      • increased cyclic dipeptides, increased short chain fatty acids, increased betaine, increased dimethylglycine, increased essential amino acids, increased nucleotides, and increased myo-inositol.
    • 31. The composition according to any one of embodiments 1-30, wherein the animal administered the composition exhibits a shift in the microbiome content of gastrointestinal tract.
    • 32. The method according to embodiment 31, wherein the shift comprises an increase in the amount of Bacteroidaceae bacteria.
    • 33. The composition according to any one of embodiments 1-32, wherein administered comprises administration of a vaccine.
    • 34. The composition according to any one of embodiments 1-33, wherein the animal is poultry and the poultry is administered a vaccine prior to the administration of the composition.
    • 35. The composition according to any one of embodiments 1-34, wherein the animal is poultry and the poultry is administered a vaccine concurrently with the administration.
    • 36. The composition according to any one of embodiments 33-35, wherein the animal is poultry and the poultry is administered a vaccine, wherein said vaccine comprises a vaccine that aids in the prevention of coccidiosis.
    • 37. The composition according to any one of embodiments 1-36, wherein administered comprises administration of galacto-oligosaccharides (GOS).
    • 38. A composition according to any one of embodiments 1-37 for use in therapy.
    • 39. A composition according to any one of embodiments 1-38 for use in improving animal health.
    • 40. A composition according to any one of embodiments 1-39 for use in reducing colonization of an animal by a pathogenic bacterium.
    • 41. A composition according to any one of embodiments 1-40 for use in the manufacture of a medicament for reducing colonization of an animal by a pathogenic bacterium.
    • 42. A method for increasing animal health, the method comprising:
      • administering to an animal an effective amount of a composition according to any one of embodiments 1-41.
    • 43. The method according to embodiments 42, wherein the animal is poultry.
    • 44. The method according to any one of embodiments 42-43, wherein increasing animal health includes decreasing pathogen-associated lesion formation in the gastrointestinal tract, decreasing colonization of pathogens, and decreasing mortality rate.
    • 45. A method of treating, ameliorating the effects of, or preventing necrotic enteritis in poultry by administering a composition according to any one of embodiments 1-29 to a poultry in need thereof.

Claims (21)

1.-45. (canceled)
46. A probiotic composition for increasing animal health comprising an isolated first Lactobacillus reuteri strain 3632 (ATCC PTA-126788) and an isolated second Lactobacillus reuteri strain 3630 (ATCC PTA-126787).
47. The composition of claim 46, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are present at a ratio of 0.75-1.5:1.
48. The composition of claim 46, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are included in an amount of 102-108 CFU/kg of the composition.
49. The composition of claim 47, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are included in an amount of 104-107 CFU/kg of the composition.
50. The composition of claim 46, formulated as animal feed, feed additive, food ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel or injection.
51. The composition of claim 50, formulated as animal feed.
52. The animal feed composition of claim 51, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are present in an amount of 102-108 CFU/kg of animal feed.
53. The animal feed composition of claim 52, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are present in an amount of 107 CFU/kg of animal feed.
54. The composition of claim 46, formulated for oral administration, nasal administration, or in ovo administration.
55. The composition of claim 46, wherein each isolated individual strain is free of other living organisms and the composition contains only the individual strains Lactobacillus reuteri strain 3632 (ATCC PTA-126788) and strain 3630 (ATCC PTA-126787).
56. The composition of claim 46, wherein the isolated first Lactobacillus reuteri strain and the isolated second Lactobacillus reuteri strain are formulated to be shelf-stable and exist outside the gastrointestinal tract.
57. The composition of claim 46, further comprising a prebiotic, wherein the prebiotic is galacto-oligosaccharides (GOS).
58. The composition of claim 46, wherein the animal is a bird, a human, or a non-human mammal.
59. The composition of claim 58, wherein the bird is poultry.
60. The composition of claim 58, wherein the animal is selected from chickens, turkey, dogs, cats, cattle and swine.
61. The composition of claim 58, wherein the animal is a human.
62. A probiotic composition for increasing animal health comprising an isolated first Lactobacillus reuteri strain 3632 (ATCC PTA-126788) and an isolated second Lactobacillus reuteri strain 3630 (ATCC PTA-126787), wherein the isolated first strain and the isolated second strain are present at a ratio of 0.75-1.5:1.
63. The composition of claim 62, formulated as animal feed, feed additive, food ingredient, water additive, water-mixed additive, consumable solution, consumable spray additive, consumable solid, consumable gel or injection.
64. The composition of claim 62, wherein each isolated individual strain is free of other living organisms and the composition contains only the individual strains Lactobacillus reuteri strain 3632 (ATCC PTA-126788) and strain 3630 (ATCC PTA-126787).
65. A probiotic composition for increasing animal health comprising an isolated first Lactobacillus reuteri strain 3632 (ATCC PTA-126788) and an isolated second Lactobacillus reuteri strain 3630 (ATCC PTA-126787), wherein the isolated first strain and the isolated second strain are present at a ratio of 0.75-1.5:1 and are included in an amount of 102-108 CFU/kg of the composition.
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