Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods.
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The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases. (+info)
Megasphaera paucivorans sp. nov., Megasphaera sueciensis sp. nov. and Pectinatus haikarae sp. nov., isolated from brewery samples, and emended description of the genus Pectinatus.
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Seven unidentified strictly anaerobic, Gram-negative, non-spore-forming bacteria from spoiled beer or the brewery environment were characterized. Based on 16S rRNA gene sequence analyses, all strains were affiliated to the Sporomusa sub-branch of the class 'Clostridia'. Three of the strains were non-motile cocci, on average 1.5 x 1.2 microm or 1.2 x 1.0 microm, occurring mainly singly or in pairs. They shared nearly identical (>99 %) 16S rRNA gene sequences, being most closely related to the species of the Megasphaera-Anaeroglobus group (< or =93.9 % similarity). According to DNA-DNA hybridization results, the coccoid strains represented two genospecies, neither of which was related to any of the recognized Megasphaera species. Several phenotypic characteristics and/or DNA G+C content also differentiated the strains from each other and from their closest relatives. The other four novel strains were motile, slightly curved to helical rods, 0.6-0.8 x 3-50 microm or more in size. They shared identical 16S rRNA gene sequences and ribofragment patterns. The highest 16S rRNA gene similarity was found between these isolates and Pectinatus cerevisiiphilus ATCC 29359T (95.6 %) and Pectinatus frisingensis ATCC 33332T (93.6 %). The novel strains also differed from recognized Pectinatus species in their sugar utilization, proteolytic activity, catalase activity, antibiotic resistance and temperature tolerance. The results suggest that the bacteria belong to three novel species, for which the names Megasphaera paucivorans sp. nov. (type strain VTT E-032341T = DSM 16981T), Megasphaera sueciensis sp. nov. (type strain VTT E-97791T = DSM 17042T) and Pectinatus haikarae sp. nov. (type strain VTT E-88329T = DSM 16980T) are proposed. (+info)
Association between Lactobacillus species and bacterial vaginosis-related bacteria, and bacterial vaginosis scores in pregnant Japanese women.
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BACKGROUND: Bacterial vaginosis (BV), the etiology of which is still uncertain, increases the risk of preterm birth. Recent PCR-based studies suggested that BV is associated with complex vaginal bacterial communities, including many newly recognized bacterial species in non-pregnant women. METHODS: To examine whether these bacteria are also involved in BV in pregnant Japanese women, vaginal fluid samples were taken from 132 women, classified as normal (n = 98), intermediate (n = 21), or BV (n = 13) using the Nugent gram stain criteria, and studied. DNA extracted from these samples was analyzed for bacterial sequences of any Lactobacillus, four Lactobacillus species, and four BV-related bacteria by PCR with primers for 16S ribosomal DNA including a universal Lactobacillus primer, Lactobacillus species-specific primers for L. crispatus, L. jensenii, L. gasseri, and L. iners, and BV-related bacterium-specific primers for BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium. RESULTS: The prevalences of L. crispatus, L. jensenii, and L. gasseri were significantly higher, while those of BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium were significantly lower in the normal group than in the BV group. Unlike other Lactobacillus species, the prevalence of L. iners did not differ between the three groups and women with L. iners were significantly more likely to have BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium. Linear regression analysis revealed associations of BVAB2 and Megasphaera with Nugent score, and multivariate regression analyses suggested a close relationship between Eggerthella-like bacterium and BV. CONCLUSION: The BV-related bacteria, including BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium, are common in the vagina of pregnant Japanese women with BV. The presence of L. iners may be correlated with vaginal colonization by these BV-related bacteria. (+info)
Prevalence and abundance of uncultivated Megasphaera-like bacteria in the human vaginal environment.
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Relationship of specific vaginal bacteria and bacterial vaginosis treatment failure in women who have sex with women.
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BACKGROUND: Bacterial vaginosis frequently persists after treatment. The role of newly defined bacterial vaginosis-associated bacteria (BVAB), which have a specificity for this condition of 97% or greater, has not been assessed. OBJECTIVE: To define risks for bacterial vaginosis persistence, including pretreatment detection of specific vaginal bacteria, among women reporting sex with women. DESIGN: Observational cohort study. SETTING: University-based research clinic. PATIENTS: 335 women age 16 to 29 years reporting sex with at least 1 woman in the past year. Participants were recruited through advertisements and provider referral. INTERVENTION: Bacterial vaginosis was treated with intravaginal metronidazole gel (0.75%), 37.5 mg nightly for 5 nights. MEASUREMENTS: Species-specific 16S recombinant DNA polymerase chain reaction assays targeting 17 bacterial species were applied to vaginal fluid obtained at baseline. Test of cure by clinical criteria and Gram stain analysis and repeated polymerase chain reaction assays of vaginal fluid were performed 1 month after treatment, and interim behaviors were assessed by using computer-assisted self-interview. RESULTS: Of 335 women, 24% of whom also reported sex with men within 3 months before enrollment, 131 (39%) had bacterial vaginosis. In the 120 (92%) women who returned for follow-up, the incidence of persistent bacterial vaginosis was 26% and was statistically significantly higher in women with baseline detection of 3 Clostridiales bacteria, designated as BVAB1 (risk ratio, 2.0 [95% CI, 1.1 to 4.0]), BVAB2 (risk ratio, 8.7 [CI, 2.5 to infinity]), or BVAB3 (risk ratio, 3.1 [CI, 1.7 to 5.8]); Peptoniphilus lacrimalis (risk ratio, 3.5 [CI, 1.6 to 15.5]); and Megasphaera phylotype 2 (risk ratio, 3.4 [CI, 1.4 to 5.5]). Persistence was lower with treatment adherence (risk ratio, 0.4 [0.2 to 0.9]). Detection of these bacteria at the test-of-cure visit was associated with persistence, whereas posttreatment sexual activity was not. LIMITATIONS: Findings may not be generalizable to women who have sex only with men, or to women whose bacterial vaginosis is treated with oral antibiotics. The study may be too small and may involve a population that is too highly selected to draw definitive conclusions about associations of persistent infection with posttreatment sexual behaviors. CONCLUSION: Persistent bacterial vaginosis is associated with several bacteria in the Clostridiales order, Megasphaera phylotype 2, and P. lacrimalis, suggesting that vaginal microbiology at diagnosis may determine risk for antibiotic failure. (+info)
Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci.
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Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species. (+info)
Effects of dietary changes and yeast culture (Saccharomyces cerevisiae) on rumen microbial fermentation of Holstein heifers.
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Development of a 16S rRNA gene primer and PCR-restriction fragment length polymorphism method for rapid detection of members of the genus Megasphaera and species-level identification.
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