Screening And Biological Characterization Of Probiotic Lactobacilli From Women's Vagina

Predominated lactobacilli in vagina had been proved to be of utrnost importantfor prevention of various urogenital infections and maintenance-of the balance ofvaginal microecosystem. The putative mechanisms include: 1) steric exclusion for theadhesion of pathogens; 2) secretion of organic acids, mainly lactic acid from thehydrolysis of mucosal glycogen; 3)production of bacteriocin, hydrogen peroxide andbiosurfactant etc; 4) stimulation of the host's local immune system. Among thoselactobacilli H₂O₂-producing lactobacilli might be the potential probioticmicroorganisms using in the prevention and curing of bacterial vaginosis (BV). Up tonow there were some commercial lactobacilli coming into being, which had beenproved to be efficiency in the treatment of BV.In our studies a modified MRS agar was used to screen out acidophilic strainsfrom 162 vaginal samples of healthy pregnant women. The isolates werecharacterized preliminarily with regard to gram-staining, catalase reaction andmorphology. TMB-HRP-MRS agar was used for semi-quatitively analyzing theH₂O₂-production ability of isolates. Totally 326 strains of lactobacilli were obtained,among them 244 were hydrogen peroxide producing strains, including 89 weaklyproducing strains, 116 standard producing strains and 39 strongly producing strains.The recovery of hydrogen peroxide producing strains was as high as 87.59% inpregnant women. This recovery was found to decrease with the increase of thepregnant age and increase with prolonging of pregnant weeks, which indicated thatpregnancy in the younger age resulted in lower infection rate of BV.Currently the genus of lactobacilli covers 102 species and some of them wereused as probiotics in foods and pharmaceutics e.g. Lactobacillus plantarum, L.delbruechii, L. salivarius and L. acidophilus. Traditionally for identification ofspecies of lactobacilli morphological and biochemical characteristics were routinelyused. In our studies, API system, the golden standard of bacterial identification, wasused. Moreover, group PCR and species-specific PCR were performed with the aimto identify the isolates on species level. It had been found that L. acidophilus and L. crispatus were the dominant lactobacilli in women's vagina, and presented potentialyielding high amount of hydrogen peroxide.Antagonistics and adherence are the most important properties for screening ofvaginal probiotic microorganisms. For that purpose, Hela cell line was used as amodel for adherence and double-layer agar plates for detection of the antagonisticsagainst pathogens. It had been demonstrated that the adherence ability was relevantwith species, however, on the other hand, even the same species presented differentialadherence with different strains. It had been shown that 53.24 % of the lactobacilliisolates inhibited Candida albicans CMCC 98001, 13.65% presented stronger effect.The presence of lactobacilli on Hela cell line prevented the adherence of C. albicanssubsequently.Eleven strains were screened out of 326 lactobaciUi mainly by their antagonisticpotential against C. albicans, and partially by the antagonistics against Escherichiacoli, Staphylococcus aureus and Pseudomonas aeruginosa and their 16S rDNA wereamplified and sequenced. Their taxonomy status, probiotic and biologicalcharacteristics were studied thoroughly. The phylogenetic tree was constructed withthe aid of blasting the sequence in the sources of GeneBank (GenBank, EMBL,DDBJ and PDB), and using the type strain of lactobacilli as references (similarity＞99%), and thus their taxonomy status were confirmed. 11 lactobacilli strains had beendemonstrated strong antagonistic effects on pathogenic microorganisms and theirsensitivity patterns on antibiotics were determined also. The growth curves weredepicted out. Partial of the metabolic substances of strains and the inhibitionsubstances were ascertained preliminarily.In summary, 11 indigenous vaginal lactobacilli producing hydrogen peroxidedemonstrated stronger adherence and/or inhibitory function against pathogenicmicroorganisms, and could be used for candidate strains for development of vaginalprobiotics. Further studies should be performed to identify the function mechanismand probiotic characteristics.