Species diversity and relative abundance of vaginal lactic acid bacteria from women in Uganda and Korea

AIMS: Lactobacilli play an important role in maintaining vaginal health of women. The aim of this study was to compare the species richness and relative abundance of Lactobacillus and other lactic acid bacteria in women of two geographically distant countries, Uganda and Korea. METHODS AND RESULTS: Vaginal samples were obtained from two women populations in Uganda and Korea. The Lactobacillus Rogosa SL agar was used for initial isolation of lactic acid bacteria. After phenotypic analyses, the 16S rRNA gene was amplified by polymerase-chain reaction and analysed by the BLAST program and phylogenetic tree construction. A total of 338 (128 Korean and 210 Ugandan) vaginal lactic acid bacterial strains were isolated, including five genera: Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Weissella. While Lactobacillus crispatus was common in both populations, Lactobacillus fermentum was common only in Korean women, and Lactobacillus gasseri, Lactobacillus reuteri and Lactobacillus vaginalis only in Ugandan women. Among other lactic acid bacteria, Weissella was more common in Ugandan, and Pediococcus in Korean women. All Weissella strains produced hydrogen peroxide, and all Pediococcus strains inhibited Candida species. CONCLUSION: Although many lactic acid bacteria colonize women, their species distributions may be different in women of geographically separated communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of species richness and relative abundance of vaginal lactic acid bacteria, including Lactobacillus, Pediococcus and Weissella, may lead to the design of better probiotic products as bacterial replacement therapy.     

The correction of dysbiotic disorders of the vaginal microflora by using a preparation made from highly adhesive lactobacteria

The effectiveness of a new bacterial preparation obtained from highly adhesive lactobacteria and intended for the correction of dysbiotic disturbances of vaginal microflora was studied in the treatment of 60 pregnant women with dysbacteriosis of the maternal passages. 30 pregnant women were simultaneously treated by the vaginal application of Lactobacterin. The study showed that the use of the preparation of highly adhesive lactobacteria caused the pronounced and stable correction of the microflora of the maternal passages. This correction was manifested by the domination of lactic acid bacterial flora and a decrease in the number of opportunistic microorganisms.     

Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (Gadus morhua)

A growing concern for the high consumption of antibiotics in aquaculture has initiated a search for alternative methods of disease control. Improved resistance against infectious diseases can be achieved by the use of probiotics. Probiotics are live microorganisms supplemented in food or feed which give beneficial effects on the intestinal microbial balance. In the present study a dry feed containing lactic acid bacteria (Carnobacterium divergens) isolated from Atlantic cod (Gadus morhua)intestines was given to cod fry. After three weeks of feeding the fry was exposed to a virulent strain of Vibrio anguillarum. The death rate was recorded during further three weeks of feeding with lactic acid bacteria supplemented feed. A certain improvement of disease resistance was obtained, and at the end of the experiment lactic acid bacteria dominated the intestinal flora in surviving fish given feed supplemented with lactic acid bacteria. No obvious growth inhibition of V. anguillarum was observed in an in vitro mixed culture of this bacterium and the C. divergens isolated from cod intestines. This revised version was published online in August 2006 with corrections to the Cover Date.     

Vaginal pH and Microbicidal Lactic Acid When Lactobacilli Dominate the Microbiota

Lactic acid at sufficiently acidic pH is a potent microbicide, and lactic acid produced by vaginal lactobacilli may help protect against reproductive tract infections. However, previous observations likely underestimated healthy vaginal acidity and total lactate concentration since they failed to exclude women without a lactobacillus-dominated vaginal microbiota, and also did not account for the high carbon dioxide, low oxygen environment of the vagina. Fifty-six women with low (0-3) Nugent scores (indicating a lactobacillus-dominated vaginal microbiota) and no symptoms of reproductive tract disease or infection, provided a total of 64 cervicovaginal fluid samples using a collection method that avoided the need for sample dilution and rigorously minimized aerobic exposure. The pH of samples was measured by microelectrode immediately after collection and under a physiological vaginal concentration of CO2. Commercial enzymatic assays of total lactate and total acetate concentrations were validated for use in CVF, and compared to the more usual HPLC method. The average pH of the CVF samples was 3.5 ± 0.3 (mean ± SD), range 2.8-4.2, and the average total lactate was 1.0% ± 0.2% w/v; this is a five-fold higher average hydrogen ion concentration (lower pH) and a fivefold higher total lactate concentration than in the prior literature. The microbicidal form of lactic acid (protonated lactic acid) was therefore eleven-fold more concentrated, and a markedly more potent microbicide, than indicated by prior research. This suggests that when lactobacilli dominate the vaginal microbiota, women have significantly more lactic acid-mediated protection against infections than currently believed. Our results invite further evaluations of the prophylactic and therapeutic actions of vaginal lactic acid, whether provided in situ by endogenous lactobacilli, by probiotic lactobacilli, or by products that reinforce vaginal lactic acid.     

细菌性阴道病微生态与益生菌

细菌性阴道病(BV)是育龄妇女阴道炎的最常见原因,其特征是栖居在阴道内的乳酸杆菌减少导致阴道菌群平衡失调的复杂变化。用乳酸杆菌替代抗生素是治疗BV的一种有效的治疗措施。乳酸杆菌产生过氧化氢、乳酸、杆菌素能抑制引起BV的细菌生长。多胺在BV的病理机制中有重要作用。本研究拟综述BV患者阴道菌群的变化,免疫防御反应,阴道乳酸杆菌对BV的影响,多胺的意义,益生菌在治疗选择中的作用。     

Influence of manufacturing processes on in vitro properties of the probiotic strain Lactobacillus rhamnosus Lcr35®

Probiotics are administered as complex manufactured products and yet most studies on probiotic bacterial strains have been performed with native culture strains. Little is known about the influence of industrial processes on the properties of the microorganisms. In this study, we comparatively assessed the characteristics of the probiotic bacterial strain Lactobacillus rhamnosus (Lcr35(?)) together with four of its commercial formulations, including three intestinal formulas (BACILOR with Lcr Restituo(?) packet and capsule and FLOREA Lcr Lenio(?)) and one vaginal formula (GYNOPHILUS Lcr Regenerans(?)). Lcr35(?) grown from the intestinal formulas displayed increased resistance to acidic pH and bile stress, especially FLOREA (Lcr Lenio(?)), which showed a 4.5log higher number of viable bacteria compared to the results obtained with the control native Lcr35(?) strain. Adhesion to intestinal cells was significantly higher with Lcr Restituo(?) packet and Lcr Restituo(?) capsule vs Lcr35(?). Bacteria from the vaginal formulation GYNOPHILUS had increased ability to metabolize glycogen thereby increasing lactic acid production. In vitro growth inhibition of the pathogen Candida albicans was significantly higher with bacteria from the vaginal formulation (4.5 log difference) and in the presence of vaginal epithelial cells than with the native strain. Our results show that the manufacturing process influences strain properties and should therefore be adapted according to the strain and the therapeutic indication.     

Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers

Metabolic acidosis can result from accumulation of organic acids in the blood due to anaerobic metabolism or intestinal bacterial fermentation of undigested substrate under certain conditions. These conditions include short-bowel syndrome, grain overfeeding of ruminants and, as recently reported, severe gastroenteritis. Measuring fermentation products such as short-chain fatty acids (SCFAs) and lactic acid in various biological samples is integral to the diagnosis of bacterial overgrowth. Stereospecific measurement of D- and L-lactic acid is necessary for confirmation of the origin and nature of metabolic acidosis. In this paper, methods for the separation of SCFAs and lactic acid are reviewed. Analysis of the organic acids involved in carbohydrate metabolism has been achieved by enzymatic methods, gas chromatography, high-performance liquid chromatography and capillary electrophoresis. Sample preparation techniques developed for these analytes are also discussed.     

Lactic acid bacteria colonization and clinical outcome after probiotic supplementation in conventionally treated bacterial vaginosis and vulvovaginal candidiasis

This randomized double-blind placebo controlled study assessed the vaginal colonization of lactic acid bacteria and clinical outcome. Vaginal capsules containing L gasseri LN40, Lactobacillus fermentum LN99, L. casei subsp. rhamnosus LN113 and P. acidilactici LN23, or placebos were administered for five days to 95 women after conventional treatment of bacterial vaginosis and/or vulvovaginal candidiasis. Vulvovaginal examinations and vaginal samplings were performed before and after administration, after the first and second menstruation, and after six months. Presence of LN strains was assessed using RAPD analysis. LN strains were present 2–3 days after administration in 89% of the women receiving LN strains (placebo: 0%, p < 0.0001). After one menstruation 53% were colonized by at least one LN strain. Nine percent were still colonized six months after administration. Ninety-three percent of the women receiving LN strains were cured 2–3 days after administration (placebo: 83%), and 78% after one menstruation (placebo: 71%) (ns). The intervention group experienced less malodorous discharge 2–3 days after administration (p = 0.03) and after the second menstruation (p = 0.04), compared with placebo. In summary, five days of vaginal administration of LN strains after conventional treatment of bacterial vaginosis and/or vulvovaginal candidiasis lead to vaginal colonization, somewhat fewer recurrences and less malodorous discharge.     

健康妊娠妇女阴道乳酸杆菌及pH变化的研究

目的研究健康妊娠妇女阴道乳酸菌数量及pH的变化与妊娠时间的关系。方法选择20例健康非妊娠妇女,60例健康妊娠妇女(其中早期妊娠、中期妊娠及晚期妊娠各20例)阴道分泌物进行乳酸杆菌数量检测及pH测定,并对乳酸杆菌产生过氧化氢的情况进行检测。结果随着妊娠时间的增加,乳酸杆菌的数量明显增高,伴随阴道分泌物的pH逐渐降低。结论乳酸杆菌与健康妊娠妇女阴道的生物屏障和酸性环境的维持有重要关系。     

多菌种酸奶中活性乳酸菌的计数方法研究

利用4种选择性培养基MRS、MRS-山梨醇、MRS-5．2、Elliker,采用平板涂布法和倾注接种法。在蜡烛缸法（缺氧）、封口膜法（微氧）和供氧条件下对4种市售多菌种酸奶中保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌进行选择性计数方法研究。结果表明：蜡烛缸法较能反映乳酸菌活菌数量的实际情况,封口膜法次之;在不同培养条件下4种选择性培养基对于乳酸菌活菌的计数都是灵敏的;而涂布法和倾注法接种活菌数有显著差异,倾注法要优于涂布法。     

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.     

Lactobacilli Inactivate Chlamydia trachomatis through Lactic Acid but Not H2O2

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H₂O₂, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H₂O₂ concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H₂O₂ production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H₂O₂, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection.     

Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces

In this study, a total of 94 lactic acid bacterial (LAB) isolates of porcine small intestinal and fecal origin were screened for their probiotic properties. The aim was to evaluate whether their isolation site and putative species identity play a role in these characteristics and whether either of these can be used as a predictive factor for the probiotic potential of bacterial isolates. The isolates were preliminarily identified by partial 16S rRNA gene sequencing and characterized in vitro for their pH and bile tolerance, adhesion capacity towards porcine enterocytes isolated from five intestinal sites and for antimicrobial activity towards five indicator pathogens. The interdependence of these characteristics was statistically evaluated. The isolates tolerated low pH and bile well. Adherence to the enterocytes of different origins did not correlate with the strain isolation site. In general, higher adherence was observed to colon cells in comparison to the small intestinal enterocytes. Culture filtrates of the isolates caused a decrease of up to three orders of magnitude in the intestinal pathogen cell numbers. The inhibition was mostly due to lactic and other organic acids. The predominating phylotypes identified were Lactobacillus reuteri and Lactobacillus salivarius, of which the former generally had the best adhesion capacity, whereas the latter appeared to be the best inhibitor. Based on the results, several strains of the pig Lactobacillus isolates tested may function as promising candidates for use in probiotic products. However, it was not possible to use the isolation site or the species identity of the isolates as reliable preliminary screening factors.     

Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin

Metabolic and functional properties of probiotic lactic acid bacteria (LAB) in the human gastro-intestinal ecosystem may be related to certain beneficial health effects. In this study, lactobacilli of either intestinal or fermented food origin were compared in their capability to survive low pH and bile, in their metabolic activity in the presence of bile salts and mucins, as well as in their potential to attach to enterocyte-like CaCO-2 cells. Food fermenting bacteria especially strains of the species Lactobacillus plantarum showed high tolerance to the consecutive exposure to hydrochloric acid (pH 1.5-2.5) and cholic acid (10 mM). Growth in and deconjugation of glycocholic (5 mM) and taurocholic acids (5 mM), as demonstrated for all lactobacilli of intestinal origin, was detected for food fermenting strains of the species L. plantarum, but not L. paracasei and L. sakei. Degradation of mucins was not observed for lactobacilli. Adhesion to the intestinal epithelial cell line CaCO-2 was demonstrated for several food fermenting bacterial strains in vitro. Soluble factors in the spent culture supernatants from intestinal and fermented food lactobacilli but not staphylococci cross reacted and synergized with cell wall components to promote adhesion to CaCO-2 cells. A competitive role of fecal bacteria on the adhesion of lactobacilli to CaCO-2 cells was demonstrated. In conclusion we have shown that metabolic and functional properties of intestinal lactobacilli are also found in certain bacteria of fermented food origin.     

Inhibitory activity of vaginal Lactobacillus bacteria on yeasts causing vulvovaginal candidiasis

Growing frequency of therapeutical failures of vulvovaginal candidiasis, resulting from resistance of certain species of Candida to imidazole agents, raises interest in the use of probiotics from Lactobacillus genera as prophylaxis. Unfortunately, little is known about inhibitory mechanisms of Lactobacillus on Candida. The aim of this study was to compare the activity of selected Lactobacillus species, representing the physiological vaginal flora, against Candida as well as investigation whether their inhibitory activity against Candida is related strictly to hydrogen peroxide and lactic acid production. 125 strains from vaginal smears of healthy women were classified by making use of phenotypic and genotypic methods. The majority of strains belonged to L. acidophilus: L. acidophilus sensu stricto, L. crispatus, L. gasseri and L. johnsonii as well as L. fermentum and L. plantarum species. Culture supernatants of selected 25 strains representing the isolated species were examined for their inhibitory activity against the growth of Candida albicans and C. glabrata. The results showed that the strongest and the fastest activity against C. albicans was demonstrated by L. delbrueckii strains, producing the largest quantities of hydrogen peroxide. On the other hand, extended activity, demonstrable after 24 hours, was shown by non-H2O2 producing L. plantarum supernatants. Growth of C. glabrata was not inhibited by any of the examined strains of Lactobacillus. Comparison of activity of live active cultures of Lactobacillus strains and their mixtures with this of pure H2O2 and lactic acid has shown that pure chemical compounds were less active than the cultures. This suggests that mixtures of Lactobacillus strains are in cooperation with each other using many different metabolites.     

Intestinal Microbiota is Different in Women with Preterm Birth: Results from Terminal Restriction Fragment Length Polymorphism Analysis

Preterm birth is a leading cause of perinatal morbidity and mortality. Studies using a cultivation method or molecular identification have shown that bacterial vaginosis is one of the risk factors for preterm birth. However, an association between preterm birth and intestinal microbiota has not been reported using molecular techniques, although the vaginal microbiota changes during pregnancy. Our aim here was to clarify the difference in intestinal and vaginal microbiota between women with preterm birth and women without preterm labor. 16S ribosomal ribonucleic acid genes were amplified from fecal and vaginal DNA by polymerase chain reaction. Using terminal restriction fragment length polymorphism (T-RFLP), we compared the levels of operational taxonomic units of both intestinal and vaginal flora among three groups: pregnant women who delivered term babies without preterm labor (non-PTL group) (n = 20), those who had preterm labor but delivered term babies (PTL group) (n = 11), and those who had preterm birth (PTB group) (n = 10). Significantly low levels of Clostridium subcluster XVIII, Clostridium cluster IV, Clostridium subcluster XIVa, and Bacteroides, and a significantly high level of Lactobacillales were observed in the intestinal microbiota in the PTB group compared with those in the non-PTL group. The levels of Clostridium subcluster XVIII and Clostridium subcluster XIVa in the PTB group were significantly lower than those in the PTL group, and these levels in the PTL group were significantly lower than those in non-PTL group. However, there were no significant differences in vaginal microbiota among the three groups. Intestinal microbiota in the PTB group was found to differ from that in the non-PTL group using the T-RFLP method.     

The Effects of Dietary Ferric Iron and Iron Deprivation on the Bacterial Composition of the Mouse Intestine

The influence of dietary ferric iron on the intestinal microbiota of mice was investigated with a view to promoting benign lactic acid bacteria (which have minimal iron requirements) in order to enhance colonization-resistance potential. Three groups of eight mice received a diet differing only in iron content, for a period of 12 weeks. Dietary iron deprivation resulted in overall increased small intestinal bacterial populations, including lactic acid bacteria, but these differences were generally not significant (p > 0.05). With the exception of coliforms, all examined bacterial groups (anaerobes, micro-aerophiles, lactobacilli, and enterococci) were significantly (p < 0.05) elevated in the colons of iron-deprived mice. The relatively low numbers of total anaerobes in the colons of iron-replete and iron-overloaded mice suggested that, as well as promotion of bacteria under iron-deprived condition, provision of ferric iron suppressed bacteria, probably by oxidation of normally reduced environments. Received: 13 October 2000 / Accepted: 20 December 2000     

Chitosan gels for the vaginal delivery of lactic acid: Relevance of formulation parameters to mucoadhesion and release mechanisms

The aim of this work was to assess the effect of formulation parameters of a mucoadhesive vaginal gel based on chitosan and lactic acid, and to highlight its release mechanisms. Two molecular weight chitosans were used to prepare gels with 2 lactic acid concentrations. Both chitosan molecular weight and lactic acid concentration had a significant and mutually dependent influence on mucoadhesion, measured on pig vaginal mucosa. Similarly, the lactate release profiles were found to be dependent on lactic acid content and polymer molecular weight. One gel formulation based on the stoichiometric lactate to chitosan ratio was subjected to release test in media with 2 different counterions and increasing ionic strength. This test demonstrated that the lactate release is mainly due to ionic displacement.