<i>Lactobacillus </i>sp. strain having ability to inhibit growth of vaginal pathogenic microorganisms
12070477 ยท 2024-08-27
Assignee
Inventors
Cpc classification
A23L33/40
HUMAN NECESSITIES
A23C9/1234
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
International classification
A23C9/123
HUMAN NECESSITIES
A23L33/00
HUMAN NECESSITIES
A23L33/135
HUMAN NECESSITIES
Abstract
The present invention relates to a novel Lactobacillus sp. isolation strain having an activity to inhibit the growth of vaginitis pathogens, and a pharmaceutical composition, a health functional food, and a cleansing product, comprising the strain as an active ingredient. Therefore, the present invention exhibits an effect of inhibiting the growth of Sneathia spp. pathogens associated with the infection with human papillomavirus (HPV) and the incidence of bacterial vaginitis, Gardnerella vaginalis as a vaginitis pathogen, and Candida albicans as a causative yeast of Candidal vaginitis, and an effect of recovering and maintaining the vaginal microflora, and thus can be used for the prevention and treatment of female vaginitis.
Claims
1. A method for treating vaginitis comprising administering a Lactobacillus sp. to a subject in need, wherein the Lactobacillus sp. is at least one selected from the group consisting of Lactobacillus crispatus SNUV 220 deposited under accession No. KCTC18374P, Lactobacillus fermentum SNUV 175 deposited under accession No. KCTC18371P, Lactobacillus jensenii SNUV 360 deposited under accession No. KCTC18372P, and Lactobacillus gasseri SNUV 281 deposited under accession No. KCTC18375P; and wherein the vaginitis is caused by at least one pathogenic vaginal microorganism selected from the group consisting of a Candida sp., a Sneathia spp., a Gardnerella sp., and human papillomavirus.
2. The method of claim 1, wherein the Sneathia spp. is Sneathia amnii or Sneathia sanguinegens; the Candida sp. is Candida albicans; or the Gardnerella sp. strain is Gardnerella vaginalis.
3. The method of claim 1, wherein the Lactobacillus crispatus SNUV 220 deposited under accession No. KCTC18374P Lactobacillus fermentum SNUV 175 deposited under accession No. KCTC18371P, Lactobacillus jensenii SNUV 360 deposited under accession No. KCTC18372P, or Lactobacillus gasseri SNUV 281 deposited under accession No. KCTC18375P has a survival rate of 50% or higher at a concentration of 0.1% (w/v) bile salt.
4. The method of claim 1, wherein the Lactobacillus crispatus SNUV 220 deposited under accession No. KCTC18374P Lactobacillus fermentum SNUV 175 deposited under accession No. KCTC18371P, Lactobacillus jensenii SNUV 360 deposited under accession No. KCTC18372P, or Lactobacillus gasseri SNUV 281 deposited under accession No. KCTC18375P has an activity to inhibit the growth of at least one pathogenic vaginal microorganism selected from the group consisting of a Candida sp., a Sneathia spp., a Gardnerella sp., and human papillomavirus.
5. The method of claim 1, wherein the Lactobacillus crispatus SNUV 220 deposited under accession No. KCTC18374P, Lactobacillus fermentum SNUV 175 deposited under accession No. KCTC18371P, Lactobacillus jensenii SNUV 360 deposited under accession No. KCTC18372P, or Lactobacillus gasseri SNUV 281 deposited under accession No. KCTC18375P is administered by oral, transdermal, intravenous, intramuscular, or subcutaneous routes.
Description
BRIEF DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE EMBODIMENTS
(6) Hereinafter, the present invention will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and the scope of the invention is not intended to be limited by these Examples.
Example 1. Isolation and Identification of Strains
(7) About 190 different Lactobacillus strains were isolated from the vaginal microflora of healthy women. Specifically, the samples for the isolation of vaginal microflora were obtained from nine subjects, including three pairs of identical twins and their mothers who participated in the Korean Twin-Family Cohort Study, and were supplied from Samsung Hospital in the form of mid-vaginal swab samples (IRB No. 144-2011-07-11).
(8) The swab samples were transferred to the present laboratory in the form of being stored in a refrigerator within 4 hours after collection in a modified Liquid Amies solution and immediately used for the isolation of strains. The samples were sequentially diluted from 10-1 times to 10-8 times and spread on three different media of Chocolate agar, Rogosa agar, and Columbia agar, and cultured for 48 hours under anaerobic conditions.
(9) After the culture, purely isolated colonies were randomly selected and subjected to shake culture in brain heart infusion (BHI) broth containing 5% human serum. Genomic DNA was extracted from the cells, and PCR reaction was carried out using UnivFwd (5-AGA GTT TGA TCM TGG CTC AG-3; SEQ ID NO: 5) primer and UnivRev (5-GGY TAC CTT GTT ACG ACT T-3; SEQ ID NO: 6) primer for 16S ribosomal RNA typing. The PCR products were purified using QIAquick PCR purification kit and subjected to sequence analysis using ABI3711 automatic sequencer.
(10) The results are the same as those shown in Tables 1 and 2 below. Using such sequence information, the identification of the strains was finally completed by comparing with BLAST program of Genbank (www.ncbi.nlm.nhi.gov) and ExTaxon database program (www.ezbiocloud.net/eztaxon), together with the identification data of the previous result report.
(11) TABLE-US-00001 TABLE1 SEQ ID Species Name Nucleotidesequence(5.fwdarw.3) NO Lacto- SNUV TTACTTCGGCAATGACGTTAGGAAAGCGAGCGG 1 bacillus 220 CGGATGGGTGAGTAACACGTGGGGAACCTGCCC crispatus CATAGTCTGGGATACCACTTGGAAACAGGTGCT AATACCGGATAAGAAAGCAGATCGCATGATCAG CTTTTAAAAGGCGGCGTAAGCTGTCGCTATGGG ATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGG TAAAGGCTTACCAAGGCGATGATGCATAGCCGA GTTGAGAGACTGATCGGCCACATTGGGACTGAG ACACGGCCCAAACTCCTACGGGAGGCAGCAGTA GGGAATCTTCCACAATGGACGCAAGTCTGATGG AGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGA TCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAG AGGTAGTAACTGGCCTTTATTTGACGGTAATCA ACCAGAAAGTCACGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGTGGCAAGCGTTGTCCG GATTTATTGGGCGTAAAGCGAGCGCAGGCGGAA GAATAAGTCTGATGTGAAAGCCCTCGGCTTAAC CGAGGAACTGCATCGGAAACTGTTTTTCTTGAG TGCAGAAGAGGAGAGTGGAACTCCATGTGTAGC GGTGGAATGCGTAGATATATGGAAGAACACCAG TGGCGAAGGCGGCTCTCTGGTCTGCAACTGACG CTGAGGCTCGAAAGCATGGGTAGCGAACAGGAT TAGATACCCTGGTAGTCCATGCCGTAAACGATG AGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAG TGCTGCAGCTAACGCATTAAGCACTCCGCCTGG GGAGTACGACCGCAAGGTTGAAACTCAAAGGAA TTGACGGGGGCCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGAAGCAACGCGAAGAACCTTACC AGGTCTTGACATCTAGTGCC Lacto- SNUV CTGCCCAGAAGCGGGGGACAACATTTGGAAACA 2 bacillus 175 GATGCTAATACCGCATAACAACGTTGTTCGCAT fermentum GAACAACGCTTAAAAGATGGCTTCTCGCTATCA CTTCTGGATGGACCTGCGGTGCATTAGCTTGTT GGTGGGGTAACGGCCTACCAAGGCGATGATGCA TAGCCGAGTTGAGAGACTGATCGGCCACAATGG GACTGAGACACGGCCCATACTCCTACGGGAGGC AGCAGTAGGGAATCTTCCACAATGGGCGCAAGC CTGATGGAGCAACACCGCGTGAGTGAAGAAGGG TTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGA ACACGTATGAGAGTAACTGTTCATACGTTGACG GTATTTAACCAGAAAGTCACGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCG TTATCCGGATTTATTGGGCGTAAAGAGAGTGCA GGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCG GCTTAACCGGAGAAGTGCATCGGAAACTGGATA ACTTGAGTGCAGAAGAGGGTAGTGGAACTCCAT GTGTAGCGGTGGAATGCGTAGATATATGGAAGA ACACCAGTGGCGAAGGCGGCTACCTGGTCTGCA ACTGACGCTGAGACTCGAAAGCATGGGTAGCGA ACAGGATTAGATACCCTGGTAGTCCATGCCGTA AACGATGAGTGCTAGGTGTTGG
(12) TABLE-US-00002 TABLE2 SEQ ID Species Name Nucleotidesequence(5.fwdarw.3) NO Lacto- SNUV AAAAGCTACTTTCGCATGAAAGAAGTTTAAAAG 3 bacillus 360 GCGGCGTAAGCTGTCGCTAAAGGATGGACCTGC jensenii GATGCATTAGCTAGTTGGTAAGGTAACGGCTTA CCAAGGCGATGATGCATAGCCGAGTTGAGAGAC TGATCGGCCACATTGGGACTGAGACACGGCCCA AACTCCTACGGGAGGCAGCAGTAGGGAATCTTC CACAATGGACGAAAGTCTGATGGAGCAACGCCG CGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCT CTGTTGTTGGTGAAGAAGGATAGAGGTAGTAAC TGGCCTTTATTTGACGGTAATCAACCAGAAAGT CACGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGTGGCAAGCGTTGTCCGGATTTATTGG GCGTAAAGCGAGCGCAGGCGGATTGATAAGTCT GATGTGAAAGCCTTCGGCTCAACCGAAGAACTG CATCAGAAACTGTCAATCTTGAGTGCAGAAGAG GAGAGTGGAACTCCATGTGTAGCGGTGGAATGC GTAGATATATGGAAGAACACCAGTGGCGAAGGC GGCTCTCTGGTCTGTAACTGACGCTGAGGCTCG AAAGCATGGGTAGCGAACAGGATTAGATACCCT GGTAGTCCATGCCGTAAACGATGAGTGCTAAGT GTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCT AACGCATTAAGCACTCCGCCTGGGG Lacto- SNUV CGGATAACAACACTAGACGCATGTCTAGAGTTT 4 bacillus 281 AAAAGATGGTTCTGCTATCACTCTTGGATGGAC gasseri CTGCGGTGCATTAGCTAGTTGGTAAGGCAACGG CTTACCAAGGCAATGATGCATAGCCGAGTTGAG AGACTGATCGGCCACATTGGGACTGAGACACGG CCCAAACTCCTACGGGAGGCAGCAGTAGGGAAT CTTCCACAATGGACGCAAGTCTGATGGAGCAAC GCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAA AGCTCTGTTGGTAGTGAAGAAAGATAGAGGTAG TAACTGGCCTTTATTTGACGGTAATTACTTAGA AAGTCACGGCTAACTACGTGCCAGCAGCCGCGG TAATACGTAGGTGGCAAGCGTTGTCCGGATTTA TTGGGCGTAAAGCGAGTGCAGGCGGTTCAATAA GTCTGATGTGAAAGCCTTCGGCTCAACCGGAGA ATTGCATCAGAAACTGTTGAACTTGAGTGCAGA AGAGGAGAGTGGAACTCCATGTGTAGCGGTGGA ATGCGTAGATATATGGAAGAACACCAGTGGCGA AGGCGGCTCTCTGGTCTGCAACTGACGCTGAGG CTCGAAAGCATGGGTAGCGAACAGGATTAGATA CCCTGGTAGTCCATGCCGTAAACGATGAGTGCT AAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGC AGCTAACGCATTAAGCACTCCGCCTGGGG
(13) TABLE-US-00003 TABLE 3 Pair- wise Com- Similar- Diff/ plete- ity Total ness Name Taxonomy Accession (%) nt (%) SNUV Bacteria; Firmicutes; Bacilli; Y17362 100 0/650 100 220 Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus crispatus; SNUV Bacteria; Firmicutes; Bacilli; 175 Lactobacillales; AP008937 99.86 1/715 100 Lactobacillaceae; Lactobacillus; Lactobacillus fermentum; Bacteria; Firmicutes; Bacilli; AJ575812 99.86 1/715 100 Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus fermentum; SNUV Bacteria; Firmicutes; Bacilli; AF243176 99.86 1/718 98.9 360 Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus jensenii; Bacteria; Firmicutes; Bacilli; Y18654 99.86 1/717 95.5 Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus fornicalis; SNUV Bacteria; Firmicutes; Bacilli; CP000413 99.86 1/722 100 281 Lactobacillales; Lactobacillaceae; Lactobacillus; Lactobacillus gasseri;
(14) As shown in Tables 1 to 3, the results were found to be Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus jensenii, and Lactobacillus gasseri, respectively. Accordingly, they were assigned SNUV 220, SNUV 175, SNUV 360, and SNUV 281, respectively, and deposited with the Korean Collection for Type Cultures (KCTC) located in Yuseong-gu, Daejeon, South Korea and were assigned accession numbers KCTC18374P (SNUV 220, deposited on April 9), KCTC18371P (SNUV 175, deposited on April 7), KCTC18372P (SNUV 360, deposited on April 7) and KCTC18375P (SNUV 281, deposited on April 9).
(15) For pure isolation and long-term storage of the identified strains, glycerol (16% v/v) was added to the culture medium that reached the exponential phase, and stored at ?80? C. as a stock. In order to prepare cell culture medium for the evaluation of inhibitory activity of vaginitis pathogens for each strain, 1% of each strain was inoculated in an anaerobic MRS medium and cultured at 37? C. for 24 hours. Then, the microbial cells were removed by centrifugation at 13000?g for 10 minutes, and the supernatant was passed through a membrane filter with a pore size of 0.22 um and then stored at ?80? C. until they were used in the experiment.
Example 2. Disk Inhibition Assay
(16) Each of Lactobacillus strains isolated in Example 1 was used for the assay by culturing at 37? C. for 20 hours using MRS broth (Difco, USA). The strains of Sneathia spp. and Gardnerella vaginalis were inoculated into New York City III (NYCIII) broth and then anaerobically cultured at 37? C. for 48 hours or 24 hours, respectively, and used in the experiment.
(17) As for the Sneathia spp. strains used in the experiment, two kinds of strains of Sneathia spp., which was isolated in Virginia Commonwealth University School of Medicine and has been reported in the paper, and Sneathia sanguinegens that the present inventors isolated from Korean women were used. As the Gardnerella vaginalis strain used in the experiment, KCTC 5096 strain furnished from the Korean Collection for Type Cultures (KCTC) was used.
(18) After dispensing and solidifying 15 ml of MRS agar medium on a plate, 7 ml of New York City III (NYCIII) soft agar medium (0.75% agar) inoculated with the strains of Sneathia spp. and Gardnerella vaginalis at a density of 5?10.sup.6 CFU/mL was formed as multilayer medium. Once the upper soft agar medium had been solidified, a diffusion paper disc (8 mm in diameter) was placed on the medium, and about 20 ul of culture product of each Lactobacillus strain was absorbed thereto. Then, the plate was added to an anaerobic jar and anaerobically incubated at 37? C. for 48 hours. After the incubation, the growth inhibitory zone of the strains of Sneathia spp. and Gardnerella vaginalis appearing around the disk was measured. The inhibitory activity was expressed as the diameter (mm) of the transparent disk in which the growth of the strains was inhibited from the center of the disk.
(19) The disk inhibition assay was performed against the strains of Sneathia spp. and Gardnerella vaginalis with culture supernatant of isolated Lactobacillus strains, and the representative result are shown in Table 4 below.
(20) TABLE-US-00004 TABLE 4 disk inhibition (diameter, mm) Sn. Sn. G. Species Isolates_No Amnii Sanguinegens vaginalis Lactobacillus SNUV 220 23 46 19 crispatus Lactobacillus SNUV 175 19 fermentum Lactobacillus SNUV 360 60 60 25 jensenii Lactobacillus SNUV 281 23 19 gasseri Lactobacillus SNUV 215 crispatus Lactobacillus SNUV 110 fermentum Lactobacillus SNUV 212 jensenii Lactobacillus SNUV 445 gasseri
(21) As can be confirmed in Table 4, although the size of disk inhibition of the Lactobacillus culture supernatant was slightly different depending on the type of the inhibitory strains, four types of Lactobacillus isolated strains which simultaneously inhibited the growth of Sneathia spp. and Gardnerella vaginalis strains, that is, Lactobacillus crispatus SNUV 220, Lactobacillus fermentum SNUV 175, Lactobacillus jensenii SNUV 360, and Lactobacillus gasseri SNUV 281 were selected. In particular, the Lactobacillus jensenii SNUV 360 strain exhibited a most potent inhibitory activity against both strains of Sneathia spp. and Gardnerella sp.
Example 3. Inhibitory Activity of Lactobacillus Isolates Against Candida albicans
(22) In order to confirm the inhibitory activity against Candida albicans strain, which is a causative yeast of Candidal vaginitis of the four kinds of Lactobacillus strains screened in Example 2 above, Candida albicans ATCC44858 strain (American Type Culture Collection), and MYA4788 strain (American Type Culture Collection), which has been proven to cause vaginitis in animal experiments, were selected as target strains for selection of Lactobacillus isolates having an inhibitory function, and the experiments was conducted accordingly.
(23) Specifically, the Candida albicans strains were evaluated using a 96-well diffusion test. For the experiment, 50 ul of Candida albicans ATCC44858 (or Candida albicans M4788) culture media diluted by adding 100 ul of Yeast Malt (YM) medium and 100 ul of Lactobacillus culture supernatant was added to a well of 96-well plate, and then cultured at 37? C. for 24 hours. Thereafter, the growth of inhibited Candida strains was estimated by measurement of absorbance at 600 nm, and the results are shown in Table 5.
(24) TABLE-US-00005 TABLE 5 optical density after 24 h (O.D. at 600 nm) Candida Candida 44858 MYA4788 Species Isolates_No (co-culture) (co-culture) Negative control group 1 1 Lactobacillus crispatus SNUV 220 0.028 0.007 Lactobacillus SNUV 175 0.003 fermentum Lactobacillus jensenii SNUV 360 0.007 Lactobacillus gasseri SNUV 281 0.012 0.010
(25) As can be confirmed in Table 5, all the selected Lactobacillus crispatus SNUV 220, Lactobacillus fermentum SNUV 175, Lactobacillus jensenii SNUV 360, and Lactobacillus gasseri SNUV 281 had a killing activity against Candida strains close to 100%. Accordingly, all four isolated strains have been shown to have a significant effect on the prevention of vaginitis.
Example 4. Hydrogen Peroxide-Producing Activity of Lactobacillus Isolates
(26) In order to investigate the degree of hydrogen peroxide production, a Tetramethylbenzidine (TMB) agar medium was prepared as shown in Table 6 below (medium composition per 1 L).
(27) TABLE-US-00006 TABLE 6 Difco Lactobacilli MRS medium 55 g TMB 250 mg Starch, soluble 100 g Hemnin solution 10 mL Vitamin K solution 0.2 mL Peroxidase solution (1 mg/mL) 10 mL
(28) Subsequently, each of Lactobacillus crispatus SNUV 220, Lactobacillus fermentum SNUV 175, Lactobacillus jensenii SNUV 360, and Lactobacillus gasseri SNUV 281, which are the four types of Lactobacillus strains screened in Example 2, was cultured in MRS broth at 37? C. for 20 hours, inoculated onto the TMB agar plate, and then anaerobically cultured at 37? C. for 2 days. After the culture, the plate was exposed to the air for 30 minutes and evaluated via a qualitative experiment in which the color of the colonies turns to blue. The extent to which the color of colonies turned to blue was observed with the naked eye, and the results are shown in Table 7 below.
(29) TABLE-US-00007 TABLE 7 Species Isolates_No H.sub.2O.sub.2 production Lactobacillus crispatus SNUV 220 ? Lactobacillus fermentum SNUV 175 +++ Lactobacillus jensenii SNUV 360 +++ Lactobacillus gasseri SNUV 281 ++
(30) As can be confirmed in Table 7, the hydrogen peroxide-producing activity of each strain showed a different pattern, and it was confirmed that the production of hydrogen peroxide actively occurred in Lactobacillus fermentum SNUV 175, Lactobacillus jensenii SNUV 360, and Lactobacillus gasseri SNUV 281. Accordingly, the inhibitory activity of the selected strains is expected to show a difference in the mechanism of action.
Example 5. Evaluation of Acid Resistance of Lactobacillus Isolates
(31) The acid resistance of the strains was determined by comparing the growth rate when cultured at 37? C. for 24 hours in acidic broth prepared by titrating MRS broth (pH 6.7) to pH 2 and pH 3, and the growth rate under basic broth conditions of pH 6.7, and the results are shown in Table 8 below.
(32) TABLE-US-00008 TABLE 8 Species Isolates_No pH 6.7 pH 3 pH 2 Lactobacillus SNUV 220 +++ + ? crispatus Lactobacillus SNUV 175 +++ +++ + fermentum Lactobacillus SNUV 360 +++ + ? jensenii Lactobacillus SNUV 281 ++ + + gasseri
(33) As can be confirmed in Table 8, all four strains showed the growth under the culture conditions of pH 3, and in particular, SNUV 175 strain and SNUV 281 strain showed the growth even under the condition of pH 2, indicating that they have a strong acid resistance.
Example 6. Evaluation of Bile Resistance in Lactobacillus Isolates
(34) For the evaluation of bile resistance, the growth rate of Lactobacillus isolates was measured after culturing in the media containing 0.1% to 4% of bile salt for 24 hours or more, and the results are shown in Table 9 and
(35) TABLE-US-00009 TABLE 9 bile salts bile salts bile salts bile salts bile salts Species 4% 2% 1% 0.5% 0.1% Negative control group 100 100 100 100 100 Lactobacillus crispatus 2.1 13.7 12.1 27.0 81.8 SNUV 220 Lactobacillus fermentum 75.1 88.0 96.5 104.9 127.1 SNUV 175 Lactobacillus jensenii 13.5 12.6 22.8 24.1 59.3 SNUV 360 Lactobacillus gasseri 2.2 16.2 15.3 24.4 76.9 SNUV 281
(36) As can be confirmed in Table 9 and
Example 7. Evaluation of Antibiotic Resistance in Lactobacillus Isolates
(37) In order to confirm the safety when applied to functional food materials, etc., antibiotic resistance of the novel Lactobacillus sp. isolates having an activity to inhibit the growth of pathogenic vaginal microorganisms was evaluated. Currently, codes and standards concerning antibiotic resistance when utilizing Lactobacillus-based lactic acid bacteria in the food were not established, and thus it was evaluated based on the EFSA standard which is the international standard concerning antibiotic resistance of microorganisms added to animal feeds.
(38) Specifically, the evaluation of the antibiotic resistance in Lactobacillus strains was performed according to the European Food Safety Authority (EFSA)'s guidelines for nine antibiotics including ampicillin (AMP), chloramphenicol (CHR), clindamycin (CLM), erythromycin (ERY), gentamycin (GEN), kanamycin (KAN), streptomycin (STR), tetracycline (TET), vancomycin (VAN) and the like. The test method used in the evaluation on the antibiotic resistance was performed according to ISO 10932:2010 (IDF 223: 2010), which is the SOP standard for antibiotic resistance test of lactic acid bacteria. Each Lactobacillus strain was inoculated at a density of ?6?10.sup.6 CFU/mL in LAB susceptibility test medium (LSM)-broth (90% IsoSensitest- and 10% MRS-broth; Oxoid), and then MIC test strip (Liofilchem, Italy) for each antibiotic was placed thereon. The degree of inhibition and MIC were evaluated after anaerobic culture at 37? C. for 48 hours, and the results are shown in Table 10 and
(39) TABLE-US-00010 TABLE 10 L. jensenii EFSA L. fermentum EFSA antibiotics SNUV 360 guideline SNUV 175 guideline AMP 0.094 1 0.125 2 CHL 3 4 2 4 CLM 0.38 1 0.064 1 ERY 0.19 1 0.38 1 GEN 2 16 1 16 KAN 4 16 24 32 STR 3 16 12 64 TET 0.75 4 2 8 VAN 0.5 2 64 Not required
(40) As can be confirmed in Table 10 and
Example 8. Evaluation of Inhibitory Activity of Lactobacillus Isolates on Gardnerella vaginalis Infection
(41) The hormone control and estrous cycle were induced by intraperitoneally injecting 0.5 mg of beta-esteradiol 3-benzonate to female mice (BALB/c mice). After three days, they were directly infected with Gardnerella vaginalis in the vagina at a concentration of 1?10.sup.7 CFU per mouse to establish a vaginitis animal model.
(42) Thereafter, four kinds of Lactobacillus isolates corresponding to 108 to 109 CFU per mouse were vaginally administered (7 mice per group). On day 2, the total bacterial DNA was extracted from the vaginal samples washed with 0.1 mL of PBS (Phosphate Buffered Saline) and microbiome community analysis was performed to measure the relative abundance of Gardnerella vaginalis and other vaginal microbiota. The DNA extraction from the vaginal fluid samples was performed using Mobio PowerSoil DNA extraction kit, and for the community analysis, the DNA was amplified via PCR using a primer corresponding to the V4 region of 16S rDNA, and next-generation sequencing analysis was carried out using Illumina Miseq equipment.
(43) The analyzed sequences were subjected to microbiome analysis including taxon profile, ?-diversity and ?-diversity showing the difference in community structure between groups through Qiime pipeline, and the change in the amount of Gardnerella vaginalis pathogens upon the administration of Lactobacillus isolates was evaluated by calculating relative abundances (/% GV treatment group). The results are shown in Table 11 and
(44) Gardnerella vaginalis treatment group, Lactobacillus crispatus SNUV 220 treatment group after infection with Gardnerella vaginalis, Lactobacillus jensenii SNUV 360 treatment group after infection with Gardnerella vaginalis, Lactobacillus fermentum SNUV 175 treatment group after infection with Gardnerella vaginalis, Lactobacillus gasseri SNUV 281 treatment group after infection with Gardnerella vaginalis, and metronidazole (0.75%) antibiotics treatment group as a positive control group after infection with Gardnerella vaginalis were designated as GV, SNUV 220, SNUV 360, SNUV 175, SNUV 281 and MTZ, respectively.
(45) TABLE-US-00011 TABLE 11 Relative abundance (/% GV group) GV 100 SNUV 220 18.0 SNUV 360 5.3 SNUV 175 2.6 SNUV 281 6.9 MTZ 74.9
(46) As can be confirmed in Table 11 and
(47) As can be confirmed in
(48) In addition to confirming that the microbial community structure in the vagina, significantly changed taxon profile was analyzed by LefSe program after administration of Lactobacillus strains to Gardnerella vaginalis-infected mouse.
(49) The results are as shown in cladogram of