1 Department of Biological Sciences, University of Idaho, Moscow, ID, USA
2 Procter and Gamble, Cincinnati, OH, USA
Correspondence
Larry J. Forney
lforney{at}uidaho.edu
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ABSTRACT |
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INTRODUCTION |
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The vagina and its unique microflora form a finely balanced ecosystem, with the vaginal environment controlling the microbial types present and the microflora in turn controlling the vaginal environment (Pybus & Onderdonk, 1999). This ecosystem is dynamic with changes in structure and composition being influenced by age, menarche, time in the menstrual cycle, pregnancy, infections, methods of birth control, frequency of sex, number of sexual partners, as well as various habits and practices such as douching (Burton & Reid, 2002
; Clarke et al., 2002
; Eschenbach et al., 2000
; Ness et al., 2002
; Schwebke et al., 1999
) and sexual behaviours (Schwebke et al., 1999
). In the past 100 years since the first microbiological study of the human vagina (Döderlein, 1892
), lactobacilli have been thought to be the predominant members of normal postpubertal vaginal microflora (Antonio et al., 1999
). A diverse array of other bacteria such as Staphylococcus, Ureaplasma, Corynebacterium, Streptococcus, Peptostreptococcus, Gardnerella, Bacteroides, Mycoplasma, Enterococcus, Escherichia, Veillonella, Bifidobacterium and Candida (Larsen & Monif, 2001
; Marrazzo et al., 2002
; Redondo-Lopez et al., 1990
) can be present but in much lower numbers. It has been postulated that lactobacilli play a critical role in maintaining the normal vaginal ecosystem by preventing overgrowth by pathogens and other opportunistic organisms by producing lactic acid, hydrogen peroxide (H2O2), bacteriocins and other antimicrobial substances (Hillier, 1998
). Given this, it is not surprising that various efforts are being made to promote the maintenance of normal flora (Hughes & Hillier, 1990
; McLean & Rosenstein, 2000
; Reid & Burton, 2002
). Unfortunately, these have not proven to be very successful (Nyirjesy et al., 1997
). This could be because about 1042 % of women whose vaginal microbial communities lack appreciable numbers of lactobacilli apparently maintain normal vaginal ecosystems (Hillier, 1998
, 1999
; Larsen & Monif, 2001
; Marrazzo et al., 2002
; Redondo-Lopez et al., 1990
). Obviously microbial populations other than lactobacilli are dominant in a rather large proportion of normal vagina microbial communities, and alone or in some combination work to suppress the growth of pathogens. However, the identity and diversity of these populations remain largely obscure and the complex interactions of the various members of the vaginal flora are still poorly understood.
Prior efforts to characterize microbial populations found in the vagina have largely employed methods commonly used in clinical microbiology laboratories that involved plating of samples on selective media, semi-quantitative estimates of their abundance and classification based on phenetic criteria into broad taxonomic groups. While these studies have provided insight into the composition of these communities, they suffer from incompleteness, often lack statistical rigour and do not provide sufficiently detailed information. Studies on many habitats have demonstrated the limitations of cultivation-dependent methods to assess microbial community composition. In most instances, this is because readily cultivated populations represent a small fraction of the extant community (McCaig et al., 1999). In recent years, culture-independent methods based on the analysis of 16S and 18S rRNA gene sequences have been used to overcome many of these limitations (Ward et al., 1998
). These molecular techniques provide the most powerful tools currently available to reveal the phylogenetic diversity of micro-organisms found within complex ecosystems and are widely employed to explore microbial diversity and understand community dynamics. These studies have often included construction and analysis of 16S rRNA gene clone libraries to provide precise information as to the phylogeny of the constituent populations. In addition to being widely used for studies on the ecology of terrestrial and aquatic habitats (Dunbar, 1999
; Eilers et al., 2000
; McCaig et al., 1999
), they are increasingly being used to study human and animal flora, including that of the colon and subgingival crevice (Burton & Reid, 2002
; Hold et al., 2002
; Kazor et al., 2003
; Kroes et al., 1999
; Paster et al., 2001
; Suau et al., 1999
).
The aim of this study was to characterize the structure of microbial communities found in five normal, healthy women of reproductive age using culture-independent methods. 16S rRNA gene libraries were prepared from total community DNA and phylogenetic analyses of 16S rRNA gene sequences were done. To our knowledge, this is the first report describing the use these approaches to characterize the composition and diversity of normal vaginal communities. The results showed that heretofore unknown populations are abundant in certain women, that Lactobacillus iners may be more common than previously thought and that the within-species diversity of lactobacilli in the vagina can vary significantly between individuals.
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METHODS |
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The bacterial cells retrieved on swabs were resuspended in 3 ml liquid dental transport medium (LDTM; Anaerobe Systems) and stored either on dry ice or at 80 °C until the samples were thawed and analysed. Genomic DNA was isolated from 0·5 ml aliquots of the cell suspensions using a two-step cell lysis procedure. First, bacterial cell walls were disrupted enzymically by the addition of mutanolysin (50 µg) and lysozyme (500 µg) followed by incubation for 1 h at 37 °C. Second, the cells were mechanically disrupted by six freezethaw cycles. Each cycle consisted of 2 min incubation at 100 °C that was immediately followed by 2 min in liquid nitrogen. Between each freezethaw cycle, the cell suspensions were incubated for 1 min in an ultrasonic bath. Proteins in the disrupted cell suspension were digested with proteinase K (Qiagen) during a 1 h incubation at 55 °C. Further isolation and purification of total DNA extract were performed using the Wizard DNA purification kit (Promega).
PCR amplification.
Universal bacterial primers 8f and 926r (based on Escherichia coli positions) were used to amplify internal fragments of 16S rRNA genes in the genomic DNA obtained from samples. Amplification was performed in 100 µl (total volume) reaction mixtures that contained 100 ng (1 µl) vaginal sample DNA, 2 U AmpliTaq DNA polymerase (Roche), 1x AmpliTaq reaction buffer, 3 mM MgCl2, 200 µM each deoxyribonucleotide triphosphate, 5 % DMSO and 0·1 µM each primer. Initial DNA denaturation was performed at 94 °C in a PTC-100 programmable thermal controller (MJ Research) for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and elongation at 72 °C for 2 min, which was followed by a final extension at 72 °C for 10 min. To confirm amplicon production, the mixture was analysed by electrophoresis in 1·5 % agarose gel followed by staining with ethidium bromide and visualization under ultraviolet light.
16S rRNA gene clone library construction.
16S rDNA genes were cloned into pCR2.1-TOPO (Invitrogen) using a vector/insert ratio of 1 : 1 and procedures recommended by the manufacturer. Ligation mixtures were used to transform E. coli TOP 10 cells (Invitrogen) that were subsequently plated onto LuriaBertani agar plates containing 100 µg kanamycin ml1 and incubated overnight at 37 °C. Approximately 200 white, well-isolated colonies were randomly selected from each of library and grown in 200 µl LuriaBertani broth containing 100 µg kanamycin ml1 in 96-well microtitre plates for 2448 h. These cultures were reinoculated into Hogness buffer containing 3 % glycerol and cultured overnight. The cells from 800 µl of culture were harvested and the plasmid DNAs were isolated using QIAprep96 Turbo Miniprep Kits (Qiagen) using standard operation procedures by a Qiagen BioRobot 3000 workstation. The remainder of the cultures was stored at 80 °C.
Sequencing and sequence analysis.
Approximately 1200 isolated plasmids with cloned inserts (approx. 920 bp length for all libraries) were sequenced with both M13R and M13F primers. The sequences of the inserts were determined using Big Dye version 3 cycle sequencing reactions (Applied Biosystems) and resolved on an automatic sequencer (3100 PRISM Genetic Analyser; Applied Biosystems). Sequences were edited to exclude the PCR primer binding sites and manually corrected with Chromas 2 (Chromas Version 2.22; www.technelysium.com.au/chromas.html). For identification of closest relatives, newly determined sequences were compared to those available in the Ribosomal Database Project (RDP) (Maidak et al., 2001) and GenBank (www.ncbi.nlm.nih.gov) databases using the standard nucleotidenucleotide BLAST program (BLASTN; www.ncbi.nlm.nih.gov) to ascertain their closest relatives.
Phylogenetic analysis.
Sequence data were edited and combined with ContigExpress from InforMax Vector NTI Suite 8 (www.informaxinc.com). The sequence data for reference strains were obtained from the GenBank and RDP databases (Maidak et al., 2001). Similar sequences were aligned by using the CLUSTAL X program (version 1.81; www-igbmc.u-strasbg.fr/BioInfo/ClustalX/Top.html/) and ALIGNX from the InforMax Vector NTI Suite 8 (www.informaxinc.com). These alignments were manually adjusted to reduce errors before the sequences were used further. Phylogenetic trees were reconstructed using neighbour-joining/minimum evolution, maximum-parsimony and maximum-likelihood algorithms using the PAUP program. TREEVIEW 1.6.6 (Win32) (http://taxonomy.zoology.gla.ac.uk/rod/rod.html), a software package for the Microsoft Windows environment, was used to graphically represent the phylogenetic trees. The trees calculated with these three different algorithms were almost identical in topology. Only representative sequences and sequences that were at least 90 % complete were used for tree construction. Bootstrap analyses for 500 resamplings were performed to provide confidence estimates for tree topologies. Alignment positions at which less than 50 % of sequences of the entire set of data had the same residues were excluded from the calculations to prevent uncertain alignment within highly variable positions of 16S rRNA genes to avoid errors in tree topology.
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RESULTS |
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Phylogenetic analyses
The phylogenies of populations in the vaginas of the women sampled were determined by comparing the 16S rRNA gene sequences from this study to those of previously described species (Fig. 1 and Fig. 2
). The diversity of populations was greatest in W-1 and W-5. Clones from W-1 (Fig. 1
) belonged to five clades, including A. vaginae (n=175), L. iners (n=8), Megasphaera sp. (n=6), Aerococcus sp. (n=2) and Peptostreptococcus sp. (n=1), while clones from W-5 (Fig. 2
) were related to L. iners (n=177), Megasphaera sp. (n=51), A. vaginae (n=13) and Leptotrichia sp. (n=6). Interestingly, clones of Megasphaera sp. and A. vaginae were coincident, with one being found only when the other was also present.
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DISCUSSION |
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Consistent with previous studies (Antonio et al., 1999; Hillier et al., 1993
), the data showed that lactobacilli were probably the most abundant organisms in the vaginal communities in most of the women. However, it is noteworthy that two of these women were colonized only with L. crispatus, while two others were only colonized with L. iners. Antonio et al. (1999)
surveyed 215 sexually active women and reported that most were colonized with L. crispatus (32 %), Lactobacillus jensenii (23 %), Lactobacillus sp. 1085V (15 %) or Lactobacillus gasseri (5 %). Rarely (1·5 %) were other species of Lactobacillus isolated, while 29 % of the women apparently lacked lactobacilli. Recently, the occurrence of L. iners as a member of vaginal communities has also been demonstrated by others (Burton et al., 2003
; Burton & Reid, 2002
; Vasquez et al. 2002
). For example, Vasquez et al. (2002)
used temperature gradient gel electrophoresis (TTGE) of 16S rRNA genes to show that L. iners was commonly found in healthy Swedish women. Although L. crispatus, L. jensenii, L. iners and L. gasseri are phylogenetically closely related to one another (Ennahar et al., 2003
), they differ in ways that may be important to the microbial ecology of the human vagina. For example, while H2O2 is commonly produced by strains of L. crispatus and L. jensenii (95 and 94 % of strains tested, respectively; Antonio et al., 1999
), it is an uncommon characteristic among strains of L. iners and L. gasseri (9 and 7 % of strains tested, respectively; Antonio et al., 1999
).
Interestingly, two of the women sampled had high numbers of Atopobium sp., organisms that also produce lactic acid (Dewhirst et al., 2001; Downes et al., 2001
). The production of lactic acid is also characteristic of Megasphaera sp. and Leptotrichia sp., organisms that were found to be numerically dominant in two of the five women in this study. Although species of Atopobium have been found in human faeces and subgingival plaque (Harmsen et al., 2000
; Paster et al., 2001
), there is only one previous report of their isolation from the human vagina (Jovita et al., 1999
). This is the first time that a Megasphaera species has been reported as a member of the vaginal microbial community. The production of lactic acid by members of the normal flora and maintenance of vaginal acidity is widely recognized as important to sustaining an environment that is inhospitable to many bacteria (Schwebke, 2001
), and it is negatively correlated with the incidence of bacterial vaginosis among women (Cu-Uvin et al., 1999
; Skarin & Sylwan, 1986
; Taha et al., 1998
) and risk of acquiring HIV. The findings of this study indicate that although the structure of vaginal microbial communities varied between the women, the function of these communities (lactic acid production) was apparently conserved.
Our observation that the women were colonized by a single species (or group of closely related strains) of Lactobacillus is consistent with the findings of other studies (Antonio et al., 1999; Hillier et al., 1993
; Reid et al., 2003
). For example, of the 215 women sampled by Antonio et al. (1999)
, only 8 % were found to have more than one species of Lactobacillus present in the vaginal community. The rare coexistence of multiple species of lactobacilli in vaginal communities could be caused by competitive exclusion of one species by another, pre-emptive colonization by a particular species or host factors that strongly influence which species are able to colonize the environment. Support for the latter notion can be inferred from the observation that white women are more likely to be colonized by L. crispatus and/or L. jensenii than by other species of lactobacilli (Antonio et al., 1999
), and similar findings that the composition of the vaginal flora differs among racial groups (Pavlova et al., 2002
). Any of the three mechanisms, either alone or in some combination, would account for the lack of Lactobacillus species diversity found in vaginal communities. It seems unwise to presume that differences between species of Lactobacillus are inconsequential or ecologically irrelevant since there is a near complete lack of information on the nature of possible hostbacterium interactions in the vagina and the ecology of the microbial community. Future efforts to develop probiotics should take these differences in the species composition of the vaginal community into account.
In the present study three taxa, namely A. vaginae, Megasphaera sp. and Leptotrichia sp., were found to be constituents of the normal flora of some women. While A. vaginae has rarely been isolated from any environment, the species has been isolated from the vagina of a healthy individual in Sweden (Jovita et al., 1999), and species of Atopobium have also been implicated in halitosis (Kazor et al., 2003
). The clones of Megasphaera sp. from the vagina had modest similarity (8995 %) to Megasphaera cerevisiae a Gram-negative, obligate anaerobe that is associated with beer spoilage by causing turbidity, off-flavours and off-odours (Doyle et al., 1995
; Ziola et al., 1999
). Leptotrichia sp., an anaerobic Gram-negative rod, is reportedly part of the normal oral flora and has rarely been isolated from clinical material (Konomen et al., 1994
; Kroes et al., 1999
; Tee et al., 2001
). However, there are reports of Leptotrichia spp. associated with infections, and the organism has been isolated from a neutropenic patient with bacteraemia and from the amniotic fluid of a woman after intrauterine fetal demise (Midolo & Kerr, 2001
; Patel et al., 1999
; Shukla et al., 2002
). Little is known about the ecology of Leptotrichia sp., but they do produce lactic acid as the primary fermentation product from glucose (Tee et al., 2001
) and may represent opportunistic pathogens.
The occurrence of two genera that have previously been linked to the production of malodorous metabolites (Kazor et al., 2003; Ziola et al., 1999
) in normal vaginal communities could mean that certain normal flora could be responsible for vaginal odour that is not indicative of bacterial vaginosis or any other disease condition. Amsel et al. (1983)
proposed criteria for the clinical diagnosis of bacterial vaginosis that are, in part, based on the strong correlation that exists between bacterial vaginosis and malodour (fishy odour). Moreover, new diagnostic tests based on amine production and odour formation have been developed to help clinic doctors to quickly diagnosis bacterial vaginosis (O'Dowd et al., 1996
; Wolrath et al., 2001
). These tests can result in false-positives, since in two studies (Chen et al., 1982
; Kubota et al., 1995
) amines were found in samples from women without bacterial vaginosis. If future studies show that normal flora may cause odour, then diagnostic criteria should be amended to take this into account lest the condition be misdiagnosed and antibiotics be unnecessarily prescribed.
Several bacterial populations recovered in 16S rRNA clone libraries prepared in this study are not readily cultivated and may have been overlooked in previous studies. L. iners does not grow on certain selective media commonly used for the isolation of Lactobacillus, namely MRS and Rogosa media (Falsen et al., 1999). Likewise, A. vaginae, Megasphaera sp. and Leptotrichia are strict anaerobes, require specialized media and often grow slowly. The finding of these organisms as members of normal vaginal flora illustrates how cultivation-based studies can be misleading. Further studies are needed to develop detection methods and approaches to determine the prevalence of these organisms and to recover them from clinical samples.
In summary, data in this study suggest that the structure of vaginal microbial communities varies between women with respect to number as well as kinds of numerically prominent populations. Despite these differences, all communities were dominated by species of either Lactobacillus or Atopobium that produce lactic acid. Thus, the ecological function of the flora maintenance of a low pH environment that precludes the colonization and growth of pathogens and other undesirable organisms may be conserved despite differences in community structure.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Antonio, M. A., Hawes, S. E. & Hillier, S. L. (1999). The identification of vaginal Lactobacillus species and the demographic and microbiologic characteristics of women colonized by these species. J Infect Dis 180, 19501956.[CrossRef][Medline]
Burton, J. P. & Reid, G. (2002). Evaluation of the bacterial vaginal flora of 20 postmenopausal women by direct (nugent score) and molecular (polymerase chain reaction and denaturing gradient gel electrophoresis) techniques. J Infect Dis 186, 17701780.[CrossRef][Medline]
Burton, J. P., Cadieux, P. A. & Reid, G. (2003). Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Appl Environ Microbiol 69, 97101.
Chen, K. C. S., Amsel, R., Escenbach, D. A. & Holmes, K. K. (1982). Biochemical diagnosis of vaginitis: detection of diamines in vaginal fluid. J Infect Dis 145, 337345.[Medline]
Chow, A. W. & Barlett, (1989). Sequential assessment of vaginal microflora in healthy women randomly assigned to tampon or napkin use. Rev Infect Dis 11, S68.[Medline]
Clarke, J. G., Peipert, J. F., Hillier, S. L., Heber, W., Boardman, L., Moench, T. R. & Mayer, K. (2002). Microflora changes with the use of a vaginal microbicide. Sex Transm Dis 29, 288293.[Medline]
Cohen, C. R., Duerr, A., Pruithithada, N., Rugpao, S., Hiller, S., Garcia, P. & Nelson, K. (1995). Bacterial vaginosis and HIV seroprevalance among female commercial sex workers in Chiang Mai, Thailand. AIDS 9, 10931097.[Medline]
Cu-Uvin, S., Hogan, J. W., Warren, D. & 8 other authors (1999). Prevalence of lower genital tract infections among human immunodeficiency virus (HIV)-seropositive and high-risk HIV-seronegative women. HIV epidemiology research study group. Clin Infect Dis 29, 11451150.[CrossRef][Medline]
Dewhirst, F. E., Paster, B. J., Tzellas, N., Coleman, B., Downes, J., Spratt, D. A. & Wade, W. G. (2001). Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of Olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profusa sp. nov. Int J Syst Evol Microbiol 51, 17971804.
Döderlein, A. (1892). Das scheidensekret und seine bedeutung fur puerperalfieber. Zbl Bakteriol 11, 699.
Donders, G. G. G., Bosmans, E., Dekkersmaecker, A., Verecken, A., Bulck, B. V. & Spitz, B. (2000). Pathogenesis of abnormal vaginal bacterial flora. Am J Obstet Gynecol 182, 872878.[Medline]
Downes, J., Munson, M. A., Spratt, D. A., Kononen, F., Trakka, E., Jousimies-Somer, H. & Wade, W. G. (2001). Characterization of Eubacterium-like strains isolated from oral infections. J Med Microbiol 50, 947951.
Doyle, L., Mclnerney, J., Mooney, J., Powell, R., Haikara, A. & Moran, A. (1995). Sequence of the gene encoding the 16S rDNA of the beer spoilage organism Megasphaera cerevisiae. J Indust Microbiol 15, 6770.[Medline]
Dunbar, J. (1999). Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning. Appl Environ Microbiol 65, 16621669.
Eilers, H., Pernthaler, J., Glockner, F. O. & Amann, R. (2000). Culturability and in situ abundance of pelagic bacteria from the north sea. Appl Environ Microbiol 66, 30443051.
Ennahar, S., Cai, Y. & Fujita, Y. (2003). Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl Environ Microbiol 69, 444451.
Eschenbach, D. A., Thwin, S. S., Patton, D. L., Hooton, T. M., Stapleton, A. E., Agnew, K., Winter, C., Meier, A. & Stamm, W. E. (2000). Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora. Clin Infect Dis 30, 901907.[CrossRef][Medline]
Falsen, E., Pauscual, C., Sjoden, B., Ohlen, M. & Collins, M. D. (1999). Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human source: description of lactobacillus iners sp. nov. Int J Syst Bacteriol 49, 217221.[Abstract]
Gupta, K., Stapleton, A. E. & Hooton, T. M. (1998). Inverse association of H2O2-producing lactobacilli and vaginal E. coli colonization in women with recurrent urinary tract infections. J Infect Dis 178, 446450.[Medline]
Harmsen, H. J. M., Wildeboer-veloo, A. C. M., Grijpstra, J., Knol, J., Degener, J. E. & Welling, G. W. (2000). Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their amplification for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups. Appl Environ Microbiol 66, 45234527.
Hillier, S. L. (1998). The vaginal microbial ecosystem and resistance to HIV. AIDS Res Hum Retroviruses 14 Suppl. 1, S17S21.[Medline]
Hillier, S. L. (1999). Normal vaginal flora. In Sexually Transmitted Diseases, pp. 191203. Edited by K. K. Holmes, P. F. Sparling, P.-A. Mårdh, S. M. Lemon, W. E. Stamm, P. Piot & J. M. Wasserheit. New York: McGraw-Hill.
Hillier, S. L., Krohn, M. A., Rabe, L. K., Klebanoff, S. J. & Eschenbach, D. A. (1993). The normal vaginal flora, H2O2-producing lactobacilli, and bacterial vaginosis in pregnant women. Clin Infect Dis 16, S273S821.[Medline]
Hold, G. L., Pryde, S. E., Russell, V. J., Furrie, E. & Flint, H. J. (2002). Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. FEMS Microbiol Ecol 39, 3339.[CrossRef]
Hughes, V. L. & Hillier, S. L. (1990). Microbiological characteristics of Lactobacillus products used for colonization of the vagina. Obstet Gynecol 75, 244248.[Abstract]
Johnson, S. R., Petzold, C. R. & Galask, R. P. (1985). Qualitative and quantitative changes of the vaginal microbial flora during the menstrual cycle. J Reprod Immunol Microbiol 9, 15.
Jovita, M. R., Collins, M. D., Sjoden, B. & Falsen, E. (1999). Characterization of a novel Atopobium isolate from the human vagina: description of Atopobium vaginae sp. nov. Int J Syst Bacteriol 49, 15731576.[Abstract]
Kazor, C. E., Mitchell, P. M., Lee, A. M., Stokes, L. N., Loesche, W. J., Dewhirst, F. E. & Paster, B. J. (2003). Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J Clin Immunol 41, 558563.
Konomen, E., Jousimies-Somer, H. & Asikainen, S. (1994). The most frequently isolated gram-negative anaerobes in saliva and subgingival samples taken from young women. Oral Microbiol Immunol 9, 126128.[Medline]
Kroes, I., Lepp, P. W. & Relman, D. A. (1999). Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci U S A 96, 1454714552.
Kubota, T., Sakae, U., Takeuchi, H. & Usui, M. (1995). Detection and identification of amines in bacterial vaginosis. J Obstet Gynecol 76, 727730.
Larsen, B. & Monif, G. R. (2001). Understanding the bacterial flora of the female genital tract. Clin Infect Dis 32, e69e77.[CrossRef][Medline]
Maidak, B. L., Cole, J. R., Lilburn, T. G. & 7 other authors (2001). The RDP-II (Ribosomal Database Project). Nucleic Acids Res 29, 173174.
Marrazzo, J. M., Koutsky, L. A., Eschenbach, D. A., Agnew, K., Stine, K. & Hillier, S. L. (2002). Characterization of vaginal flora and bacterial vaginosis in women who have sex with women. J Infect Dis 185, 13071313.[CrossRef][Medline]
Martin, H. L., Richardson, B. A., Nyange, P. M. & 7 other authors (1999). Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition. J Infect Dis 180, 18631868.[CrossRef][Medline]
McCaig, A. E., Glover, L. A. & Prosser, J. I. (1999). Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures. Appl Environ Microbiol 65, 17211730.
McLean, N. W. & Rosenstein, I. J. (2000). Characterization and selection of a Lactobacillus species to re-colonize the vagina of women with recurrent bacterial vaginosis. J Med Microbiol 49, 543552.
Midolo, W. T. P. & Kerr, P. H. J. T. (2001). Bacteremia due to Leptotrichia trevisanii sp. nov. Eur J Clin Microbiol Infect Dis 20, 765769.[CrossRef][Medline]
Ness, R. B., Hillier, S. L., Richter, H. E., Soper, D. E., Stamm, C., McGregor, J., Bass, D. C., Sweet, R. L. & Rice, P. (2002). Douching in relation to bacterial vaginosis, lactobacilli, and facultative bacteria in the vagina. Obstet Gynecol 100, 765.
Nyirjesy, P., Weitz, M. V., Grody, M. H. & Lorber, B. (1997). Over-the-counter and alternative medicines in the treatment of chronic vaginal symptoms. Obstet Gynecol 90, 5053.
O'Dowd, T. C., West, R. R., Winterburn, P. J. & Hewlins, M. J. (1996). Evaluation of a rapid diagnostic test for bacterial vaginosis. Br J Obstet Gynaecol 103, 366370.[Medline]
Paster, B. J., Boches, S. K., Galvin, J. L., Ericson, R. E., Lau, C. N., Levanos, V. A., Sahasrabudhe, A. & Dewhirst, F. E. (2001). Bacterial diversity in human subgingival plaque. J Bacteriol 183, 37703783.
Patel, J. B., Clarridge, J., Schuster, M. S., Waddington, M., Osborne, J. & Nachamkin, I. (1999). Bacteremia caused by a novel isolate resembling Leptotrichia species in a neutropenic patient. J Clin Microbiol 37, 20642067.
Pavlova, S. I., Kilic, A. O., Kilic, S. S., So, J.-S., Nader-Macias, M. E. & Simoes, J. A. (2002). Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences. J Appl Microbiol 92, 451459.[CrossRef][Medline]
Pybus, V. & Onderdonk, A. B. (1999). Microbial interactions in the vaginal ecosystem, with emphasis on the pathogenesis of bacterial vaginosis. Microbes Infect 1, 285292.[CrossRef][Medline]
Redondo-Lopez, V., Cook, R. L. & Sobel, J. D. (1990). Emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora. Rev Infect Dis 12, 856872.[Medline]
Reid, G. & Burton, J. (2002). Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4, 319324.[CrossRef][Medline]
Reid, G., Charbonneau, D., Erb, J., Kochanowski, B., Beuerman, D., Poehner, R. & Bruce, A. W. (2003). Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 singnificantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 35, 131134.[CrossRef][Medline]
Schwebke, J. R. (2001). Role of vaginal flora as a barrier to HIV acquisition. Curr Infect Dis Rep 3, 152155.[Medline]
Schwebke, J. R., Richey, C. M. & Weiss, H. L. (1999). Correlation of behaviors with microbiological changes in vaginal flora. J Infect Dis 180, 16321636.[CrossRef][Medline]
Sewankambo, N., Gray, R. H., Wawer, M. J. & 11 other authors (1997). HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet 350, 546550.[CrossRef][Medline]
Shukla, S. K., Meier, P. R., Mitchell, P. D., Frank, D. N. & Reed, K. D. (2002). Leptotrichia amnionii sp. nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise. J Clin Microbiol 40, 33463349.
Skarin, A. & Sylwan, J. (1986). Vaginal lactobacilli inhibiting growth of Gardnerella vaginalis, Mobiluncas and other bacterial species cultured from vaginal content of women with bacterial vaginosis. Acta Pathol Microbiol Immunol Scand Sect B 94, 399403.[Medline]
Sobel, J. D. (1999). Is there a protective role for vaginal flora? Curr Infect Dis Rep 1, 379383.[Medline]
Stahl, C. E. & Hill, G. B. (1986). Microflora of the female genital tract. In Infectious Diseases in the Female Patient. The Clinical Perspectives in Obstretrics and Gynecology, pp. 1642. Edited by L. B. R. P. Galask. New York: Springer.
Suau, A., Bonnet, R., Sutren, M., Godon, J.-J., Gibson, G. R., Collins, M. D. & Dore, J. (1999). Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 65, 47994807.
Taha, T. E., Hoover, D. R., Dallabetta, G. A. & 7 other authors (1998). Bacterial vaginosis and disturbances of vaginal flora: association with increased acquisition of HIV. AIDS 12, 16991706.[CrossRef][Medline]
Tee, W., Midolo, P., Janssen, P. H., Kerr, T. & Dyall-Smith, M. L. (2001). Bacteremia due to Leptotrichia trevisanii sp. nov. Eur J Clin Microbiol Infect Dis 20, 765769.[CrossRef][Medline]
van De Wijgert, J. H., Mason, P. R., Gwanzura, L., Mbizvo, M. T., Chirenje, Z. M., Iliff, V., Shiboski, S. & Padian, N. S. (2000). Intravaginal practices, vaginal flora disturbances, and acquisition of sexually transmitted diseases in Zimbabwean women. J Infect Dis 181, 587594.[CrossRef][Medline]
Vasquez, A., Jakobsson, T., Ahrne, S., Forsum, U. & Molin, G. (2002). Vaginal Lactobacillus flora of healthy Swedish women. J Clin Microbiol 40, 27462749.
Ward, D. M., Ferris, S. C., Nold, S. C. & Bateson, M. M. (1998). A nature view of microbial biodiversity within hot spring cyanobacterial mat communities. Microbiol Mol Biol Rev 62, 13531370.
Wolrath, H., Forsum, U., Larsson, P. G. & Boren, H. (2001). Analysis of bacterial vaginosis-related amines in vaginal fluid by gas chromatography and mass spectrometry. J Clin Microbiol 39, 40264031.
Yen, S., Shafer, M. A., Moncada, J., Campbell, C. J., Flinn, S. D. & Boyer, C. B. (2003). Bacterial vaginosis in sexually experienced and non-sexually experienced young women entering the military. Obstet Gynecol 102, 927933.
Ziola, B., Gee, L., Berg, N. N. & Lee, S. Y. (1999). Serogroups of the beer spoilage bacterium Megasphaera cerevisiae with the molecular weight of the major EDTA-extractable surface protein. Can J Microbiol 46, 95100.[CrossRef]
Received 12 November 2003;
revised 5 March 2004;
accepted 10 May 2004.
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