Abstract

To determine whether different racial groups shared common types of vaginal microbiota, we characterized the composition and structure of vaginal bacterial communities in asymptomatic and apparently healthy Japanese women in Tokyo, Japan, and compared them with those of White and Black women from North America. The composition of vaginal communities was compared based on community profiles of terminal restriction fragments of 16S rRNA genes and phylogenetic analysis of cloned 16S rRNA gene sequences of the numerically dominant bacterial populations. The types of vaginal communities found in Japanese women were similar to those of Black and White women. As with White and Black women, most vaginal communities were dominated by lactobacilli, and only four species of Lactobacillus (Lactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri) were commonly found. Communities dominated by multiple species of lactobacilli were common in Japanese and White women, but rare in Black women. The incidence, in Japanese women, of vaginal communities with several non-Lactobacillus species at moderately high frequencies was intermediate between Black women and White women. The limited number of community types found among women in different ethnic groups suggests that host genetic factors, including the innate and adaptive immune systems, may be more important in determining the species composition of vaginal bacterial communities than are cultural and behavioral differences.

Introduction

Each area of the human body has a unique collection of microorganisms. Although previous studies have tended to focus on understanding the etiological agents of infectious disease, there has recently been an increased emphasis on the role of indigenous microbiota in the maintenance of human health. The notion of a mutualistic relationship between the human host and its microbiome is being increasingly appreciated (Backhed et al., 2005; Dethlefsen et al., 2007). As a partner in mutualism, the host exerts strong selective pressures on determining the composition of microbiota found at different anatomical sites (Backhed et al., 2005), and in turn, the microbiota of humans are essential to many aspects of normal human physiology, ranging from nutrition, metabolic activities, homeostasis of the immune system (Backhed et al., 2004; Kelly et al., 2004; Rakoff-Nahoum et al., 2004; Cash et al., 2006) to competitive exclusion of pathogens. Moreover, the microbiota in the human body may reduce the risk of cancer, diabetes, obesity, arthritic diseases, cardiovascular disease, and other serious ailments (Naruszewicz et al., 2002; Ohashi et al., 2002; Rayes et al., 2002; Rozanova et al., 2002; Tannock, 2002). Hence, the benefits of accurately defining the human microbiota and understanding their ecological function and role in human health may be crucially important for understanding differential risk to disease, and ultimately in disease prevention and treatment.

The human vagina is a dynamic and complex microbial ecosystem in which the symbiotic vaginal microbiota play an important protective role in maintaining the health of women. Disrupting these communities may increase susceptibility to various urogenital infections including sexually transmitted diseases and HIV in women (Schwebke, 2005; McClelland et al., 2008). Recently, several studies have characterized the vaginal microbial communities using cultivation-independent methods, and tested whether differences in the species composition of vaginal bacterial communities may predispose certain individuals to bacterial vaginosis and various infectious diseases (Verhelst et al., 2004; Fredricks et al., 2005; Hyman et al., 2005; Vitali et al., 2007; Shi et al., 2009). Previously, we have reported that several kinds of vaginal microbial communities were found in healthy North American White and Black women. The community types in White women were similar to those of Black women, but the relative frequencies of the types differed (Zhou et al., 2004, 2007). In this study, we extended our previous work by determining the bacterial community types in vaginas of apparently healthy Asian women from Tokyo, Japan, and compared them with those of White and Black women. The aim of this study was to determine whether the vaginal communities were similar types in all three racial groups.

Materials and methods

Clinical study

The samples used in this study were collected as part of a study described previously by Parsonnet (2008). Vaginal samples were obtained from 73 Japanese women from the Tokyo area of Japan. The subjects were evenly distributed among three age groups: 18–25, 26–34, and 35–45 years old. For inclusion in this study, the women enrolled must self-declare that they were born in Japan and be of Japanese ancestry. The criteria for enrollment of healthy and asymptomatic Japanese women were essentially the same as those used for a previously reported study of White and Black women (Zhou et al., 2007). Women of Japanese descent were eligible for enrollment if they had a history of regular menstrual cycles for the past 2 years and agreed to refrain from the use of mouth wash, mouth rinse or medicated drop or sprays, douching substances, vaginal medications, suppositories, feminine sprays, genital wipes, or contraceptive spermicides, and from sexual intercourse for 48 h before sample collection. Subjects were also required to refrain from bathing, showering, or swimming within 2 h before sample collection. Women were excluded if they worked in healthcare settings; had been hospitalized in the past 6 weeks; had experienced a genital or a sexually transmitted infection within the past 6 weeks; were pregnant, actively trying to become pregnant, or suspected they were pregnant; had been diagnosed as having diabetes, kidney failure, hepatitis, HIV infection, or toxic shock syndrome; were currently suffering from sinus infection or pharyngitis (self-declared); or had taken immunosuppressive drugs, chemotherapy, or systemic or topical antimicrobial drugs within the past 30 days. The subjects enrolled did not have vaginal symptoms associated with bacterial vaginosis during the 6 weeks before enrollment. When the vaginal samples were collected, the attending health care practitioner noted any signs of possible genital infections (e.g. abnormal discharge, cervicitis, or foul odor). None of the subjects in this study had signs of vaginal infection. These were samples of convenience and Nugent scores (Nugent et al., 1991) were not available.

Written informed consent was obtained from the subjects before the collection of any information or clinical samples. Subjects were removed from the study if they failed to meet the inclusion criteria or satisfied any of the exclusion criteria any time during the study. The study protocol and informed consent document were reviewed and approved by the Ethics Committee-Sogo Clinical Pharmacology Co. Ltd.

Each vaginal sample was collected by a physician by inserting a swab into the vagina (without using a speculum) and swabbing the midupper vaginal walls approximately 5 cm past the introitus. The labia were spread during this procedure to minimize the potential for contamination by perineal flora (Parsonnet et al., 2008). The swab was placed in a sterile cryovial and stored at −70 °C until analysis.

Extraction of genomic DNA

The bacterial cells retrieved on swabs were resuspended in 2 mL cell lysis solution from the Wizard DNA purification kit (Promega, Madison, WI). Genomic DNA was isolated from 0.5-mL aliquots of the cell suspensions using a two-step cell lysis procedure as described previously (Zhou et al., 2007). Briefly, bacterial cell walls were disrupted enzymatically by mixing with mutanolysin (50 µg) and lysozyme (500 µg), followed by incubation for 1 h at 37 °C. The cells were then mechanically disrupted by six freeze–thaw cycles. Each cycle consisted of 2 min of incubation at 100 °C, which was immediately followed by 2 min in a dry-ice/ethanol bath. Between each freeze–thaw cycle, the cell suspensions were incubated for 1 min in an Ultrasonic Cleaning bath. Proteins in the disrupted cell suspension were digested with proteinase K (Qiagen, Hilden, Germany) during a 1-h incubation at 55 °C. Further purification of the total DNA extract was performed using the Wizard DNA purification kit (Promega).

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes

For analysis of T-RFLP of 16S rRNA genes, internal regions of 16S rRNA genes in each sample were amplified in two separate reactions using fluorescently labeled primer pairs, 8fm–926r and 49f–926r (based on Escherichia coli sequence). Primers 8fm, 49f, and 926r were labeled with VIC, NED, and 6-carboxy-fluorescein (6-FAM), respectively (Applied Biosystems, Foster City, CA). PCR was performed as described previously (Zhou et al., 2004).

The profiles of T-RFs from the vaginal microbial communities were determined as follows: a mixture of the two fluorescently labeled amplicons was equally divided and separately digested with MspI or HaeIII, and the digested products were recombined. The resulting mixture had six fluorescently labeled T-RFs from the use of three fluorophores and two restriction enzymes. This allowed for high resolution of the microbial communities. T-RFLP profiles were determined using an ABI PRISM 3100 DNA Analyzer, genescan software (Applied Biosystems), and CST ROX 25-1000 (BioVentures Inc., Murfreesboro, TN) as an internal standard.

Cluster analysis of T-RFLP data

Cluster analysis of the T-RFLP community profiles for Japanese women from this study and White and Black women from our previous study was performed to identify similar communities and the number of clusters. The data were first standardized by defining a threshold (baseline) and identifying the true peaks (Abdo et al., 2006). The fragments were then binned based on length; those fragments within 2 bp in length were binned together and represented by their average length. The peak areas of binned fragments from the same samples were summed. Second, the Pearson correlation distances between T-RFLP profiles were calculated; these were hierarchically clustered based on Ward's linkage method and a dendrogram was constructed. Third, the number of clusters was determined using the methods described previously (Zhou et al., 2007).

Clone library construction and 16S rRNA gene sequence analysis

The samples used to construct clone libraries were chosen using a ‘coverage sampling approach’ (Abdo et al., 2006). This approach provided a way to identify the fewest samples necessary to describe 85% of the phylotype diversity within each cluster. The 16S rRNA genes in each sample identified using this approach were amplified using primers 8fm–926r without fluorescent labels and cloned as described previously (Zhou et al., 2004, 2007). Approximately 100 clones of each sample were randomly chosen from each library and the cloned DNA fragments were partially sequenced using an ABI 3730 Prism DNA Analyzer. Phylogenetic analysis of cloned 16S rRNA gene sequences from the numerically dominant microbial populations was performed to determine the composition of bacterial communities in each sample. High-quality sequences with <3% uncalled bases and >500 bp long were analyzed using high-throughput methods (Brown et al., 2007) to identify similar sequences among the eubacterial type strains found in the Ribosomal Database Project and GenBank databases.

Differences in the distribution of vaginal community among the different racial groups

Two methods were chosen to show the distribution of vaginal microbial communities among the various racial groups, and both of them were based on the T-RFLP community profiles. First, the distance matrix based on the Pearson correlation distance analysis was constructed, and subjected to multidimensional-scaling (MDS) to graphically present the similarity among the bacterial communities for each sample and each racial group. Second, we evaluated differences in the species rank abundance of microbial communities of women in each racial group. Pearson's χ2-test and Fisher's exact test were used to assess whether differences in the distribution of microbial community types among Japanese, White, and Black women were statistically significant.

Results

Classification of vaginal microbial communities based on T-RFLP and sequence data

The number of different kinds of bacterial communities in the vaginas of Japanese women was determined by comparing T-RFLP profiles and by sequence analysis of cloned 16S rRNA genes. We performed cluster analysis on the profiles of T-RFLPs and constructed a dendrogram in which the neighboring communities had similar species composition and structure (Fig. 1). We categorized the communities into nine clusters (designated as C1–9). Community types were further defined by sequence analysis of the 16S rRNA genes in samples that were representative of each cluster. Clusters that had similar species composition were combined and assigned to a single group. For example, both clusters C2 and C4 (Fig. 1) were dominated by Lactobacillus crispatus and were combined into a single group, namely group II (Tables 1 and 2). Likewise, the communities with multiple non-Lactobacillus species found in C8 and C9 were combined into group III. After combining communities that were similar in terms of species composition, there were seven different groups of communities (groups I–VII). These communities were also found in our previous study of vaginal microbiota in White and Black women (Zhou et al., 2007).

Figure 1

Dendrogram constructed by cluster analysis (Ward's method) based on similarity in the T-RFLP profiles among vaginal microbial communities in Japanese, White, and Black women. The samples from White, Black, and Japanese women are designated with an open circle, a closed circle, and a red triangle, respectively. The red line shows the grouping baseline. Large triangles labeled with various colors indicate clusters. The clusters are designated with a ‘C,’ followed by a number. *The samples from each group used to construct clone libraries of 16S rRNA genes.

Figure 1

Dendrogram constructed by cluster analysis (Ward's method) based on similarity in the T-RFLP profiles among vaginal microbial communities in Japanese, White, and Black women. The samples from White, Black, and Japanese women are designated with an open circle, a closed circle, and a red triangle, respectively. The red line shows the grouping baseline. Large triangles labeled with various colors indicate clusters. The clusters are designated with a ‘C,’ followed by a number. *The samples from each group used to construct clone libraries of 16S rRNA genes.

Table 1

Species composition of vaginal communities in healthy Japanese women

 Group (% clones) 
 II III IV VI VII 
 J30 J35 J40 J45 J13 J17 J37 J25 J63 J65 J47 J79 J1 J5 J46 J38 J67 J77 J81 J4 J20 J83 
Phylotype n=96 n=96 n=85 n=96 n=67 n=64 n=96 n=75 n=92 n=94 n=94 n=90 n=90 n=91 n=80 n=94 n=89 n=96 n=92 n=63 n=91 n=82 
Lactobacillus iners 98 100.0 82.4 92.7  20.3       3.3 8.8 30.0     39.7 80.2  
L. crispatus   17.6 7.3 100.0 78.0 98.9 98.7 100.0 100.0  10.0     1.1   60.3 4.4  
L. jensenii      1.7  1.3             6.6 100.0 
L. gasseri                98.0 82.0 44.0 98.9    
L. vaginalis       1.1                
L. aviarius 2.0                      
Aerococcus sp.              3.2 10.0        
Anaerobranca sp.                 2.2      
Anaerococcus sp.            2.2           
Atopobium vaginae             41.1 48.4 36.2 1.0       
Bergeyella sp.                 3.3      
Bifidobacterium breve           1.0            
Bradyrhizobium sp.            1.1           
Chryseobacterium sp.                 1.1      
Clostridium sp.             13.3 2.2         
Dialister sp.           2.0 3.3 4.4   1.0       
Enterococcus faecalis                   1.1    
Finegoldia magna                 1.1      
Gardnerella vaginalis            15.6           
Gemella palaticanis            4.4  1.1         
Lachnospiraceae sp.            1.1           
Lachnospira sp.            57.9     1.1      
Leptotrichia sp.               2.5        
Megasphaera sp.             1.1 4.4 6.2        
Mobiluncus mulieris             18.9          
Neisseria sp.            1.1           
Peptoniphilus sp.             1.1          
Peptostreptococcus sp.             1.1 7.7         
Prevotella sp.             15.7 11.0 7.5  2.2      
Staphylococcus sp.           2.0            
Streptococcus sp.           95.0 3.3     5.9 56.0   8.8  
Veillonella sp.               1.2        
Novel              13.2 6.4        
 Group (% clones) 
 II III IV VI VII 
 J30 J35 J40 J45 J13 J17 J37 J25 J63 J65 J47 J79 J1 J5 J46 J38 J67 J77 J81 J4 J20 J83 
Phylotype n=96 n=96 n=85 n=96 n=67 n=64 n=96 n=75 n=92 n=94 n=94 n=90 n=90 n=91 n=80 n=94 n=89 n=96 n=92 n=63 n=91 n=82 
Lactobacillus iners 98 100.0 82.4 92.7  20.3       3.3 8.8 30.0     39.7 80.2  
L. crispatus   17.6 7.3 100.0 78.0 98.9 98.7 100.0 100.0  10.0     1.1   60.3 4.4  
L. jensenii      1.7  1.3             6.6 100.0 
L. gasseri                98.0 82.0 44.0 98.9    
L. vaginalis       1.1                
L. aviarius 2.0                      
Aerococcus sp.              3.2 10.0        
Anaerobranca sp.                 2.2      
Anaerococcus sp.            2.2           
Atopobium vaginae             41.1 48.4 36.2 1.0       
Bergeyella sp.                 3.3      
Bifidobacterium breve           1.0            
Bradyrhizobium sp.            1.1           
Chryseobacterium sp.                 1.1      
Clostridium sp.             13.3 2.2         
Dialister sp.           2.0 3.3 4.4   1.0       
Enterococcus faecalis                   1.1    
Finegoldia magna                 1.1      
Gardnerella vaginalis            15.6           
Gemella palaticanis            4.4  1.1         
Lachnospiraceae sp.            1.1           
Lachnospira sp.            57.9     1.1      
Leptotrichia sp.               2.5        
Megasphaera sp.             1.1 4.4 6.2        
Mobiluncus mulieris             18.9          
Neisseria sp.            1.1           
Peptoniphilus sp.             1.1          
Peptostreptococcus sp.             1.1 7.7         
Prevotella sp.             15.7 11.0 7.5  2.2      
Staphylococcus sp.           2.0            
Streptococcus sp.           95.0 3.3     5.9 56.0   8.8  
Veillonella sp.               1.2        
Novel              13.2 6.4        

Clones were assigned to phylotypes by comparing their 16S rRNA gene sequences to those of known organisms. The species name was used if the sequence similarity to a type species was >97%; the genus only was used if the sequence similarity was <97%, but >90%. The clones were described as novel if the sequence similarity of clones to known organisms was <90%.

Groups were defined based on T-RFLP profiles and phylogenetic analysis of partial 16S rRNA gene sequences from clone libraries prepared from samples representative of each cluster. Groups I, IV, V, VI, and VII designate C6, C7, C3, C1, and C5, respectively (see Fig. 1). Group II merges C2 and C4 and Group III merges C8 and C9.

The number of clones analyzed per woman.

Relative abundances of populations in each clone library.

‘Novel’ includes various phylotypes within the phylum Firmicutes.

Table 2

Comparison of vaginal microbiota in different racial groups

 Group (% clones) 
 II III IV VI VII 
Phylotype 
Lactobacillus iners 84.4 90.1 93.3 0.8 0.3 3.4 4.4 4.0 0.0 2.7 1.7 14.0  1.3  38.5 59.4 60.0  25.5  
L. crispatus 0.4 1.1 6.2 97.4 86.4 95.9   5.0  0.5   25.4 0.3 13.8 25.8 32.4  40.1  
L. jensenii 0.2 0.6  1.8 6.2 0.5       3.6 0.4   2.9 3.3 80.9 34.4 100.0 
L. gasseri 0.7 0.1   1.4      21.2  66.7 70.3 80.7  8.9     
L. vaginalis      0.2        0.5        
L. coleohominis 0.4    0.2        0.9         
L. salivarius 0.1                     
L. aviarius   0.5                   
Actinobaculum sp. 0.2                     
Aerococcus sp. 1.5 0.6     0.5 0.3  0.4 1.5 4.4     0.6     
Anaerobranca sp. 0.2      1.4 0.7       0.6       
Anaerococcus sp. 0.3      0.0 10.6 1.1 2.3 0.5 0.0          
Atopobium vaginae 3.2 1.6     2.6 1.4 0.0 25.7 30.7 41.9 0.6  0.3       
Bergeyella sp.               0.8       
Bifidobacterium breve         0.5             
Bradyrhizobium sp.         0.6             
Catonella sp.         0.0          2.3   
Chryseobacterium sp.         0.0      0.3       
Clostridium sp.  0.2     0.2     5.2          
Corynebacterium sp.             1.8         
Dialister sp.  0.3     2.3 1.0 2.7 3.8 1.4 1.5   0.3       
Eggerthella hongkongensis          0.8            
Enterococcus faecalis     0.5   0.4       0.3       
Escherichia coli       3.5 0.0              
Finegoldia magna  0.1      0.7  0.7     0.3    1.2   
Garanulicatella elegans        0.4              
Gardnerella vaginalis         7.8  0.5  4.5 0.5        
Gemella palaticanis 0.4 0.3     2.8 7.1 2.2 2.6  0.4          
Lachnospiraceae sp.  0.7      4.8 0.6 0.8 3.7           
Lachnospira sp.         29.0      0.3       
Leptotrichia sp.           9.7 0.8          
Megasphaera sp. 1.6 1.8     6.7 0.5  4.8 6.4 3.9          
Micromonas sp. 0.8 0.4     5.0 1.4  11.4 1.1           
Mobiluncus mulieris 0.2      0.3     6.3          
Mycoplasma sp.       9.6               
Neisseria sp.         0.6             
Peptococcus niger        1.4  0.4            
Peptoniphilus sp. 0.7      3.1   0.7  0.4          
Peptostreptococcus sp.       8.0   5.7  2.9          
Prevotella sp.       0.3    1.1 11.4   0.6    2.5   
Pseudomonas sp.                1.5      
Shigella sp.       2.5               
Staphylococcus sp.     2.6    1.0             
Streptococcus sp. 0.3 1.0   2.1   36.2 49.2    17.2 0.4 15.5 46.2 0.6 4.4 11.9   
Veillonella sp. 0.2 0.1     0.1 19.3  0.8 2.5 0.4          
Novel 4.4 0.9   0.5  46.8 9.8  36.6 17.6 6.5 4.8 1.1   2.0  1.2   
Number of women (per race) 22 26 14 12 23 23 21 10 
Number of women (per group) 62 58 37 19 17 13 11 
 Group (% clones) 
 II III IV VI VII 
Phylotype 
Lactobacillus iners 84.4 90.1 93.3 0.8 0.3 3.4 4.4 4.0 0.0 2.7 1.7 14.0  1.3  38.5 59.4 60.0  25.5  
L. crispatus 0.4 1.1 6.2 97.4 86.4 95.9   5.0  0.5   25.4 0.3 13.8 25.8 32.4  40.1  
L. jensenii 0.2 0.6  1.8 6.2 0.5       3.6 0.4   2.9 3.3 80.9 34.4 100.0 
L. gasseri 0.7 0.1   1.4      21.2  66.7 70.3 80.7  8.9     
L. vaginalis      0.2        0.5        
L. coleohominis 0.4    0.2        0.9         
L. salivarius 0.1                     
L. aviarius   0.5                   
Actinobaculum sp. 0.2                     
Aerococcus sp. 1.5 0.6     0.5 0.3  0.4 1.5 4.4     0.6     
Anaerobranca sp. 0.2      1.4 0.7       0.6       
Anaerococcus sp. 0.3      0.0 10.6 1.1 2.3 0.5 0.0          
Atopobium vaginae 3.2 1.6     2.6 1.4 0.0 25.7 30.7 41.9 0.6  0.3       
Bergeyella sp.               0.8       
Bifidobacterium breve         0.5             
Bradyrhizobium sp.         0.6             
Catonella sp.         0.0          2.3   
Chryseobacterium sp.         0.0      0.3       
Clostridium sp.  0.2     0.2     5.2          
Corynebacterium sp.             1.8         
Dialister sp.  0.3     2.3 1.0 2.7 3.8 1.4 1.5   0.3       
Eggerthella hongkongensis          0.8            
Enterococcus faecalis     0.5   0.4       0.3       
Escherichia coli       3.5 0.0              
Finegoldia magna  0.1      0.7  0.7     0.3    1.2   
Garanulicatella elegans        0.4              
Gardnerella vaginalis         7.8  0.5  4.5 0.5        
Gemella palaticanis 0.4 0.3     2.8 7.1 2.2 2.6  0.4          
Lachnospiraceae sp.  0.7      4.8 0.6 0.8 3.7           
Lachnospira sp.         29.0      0.3       
Leptotrichia sp.           9.7 0.8          
Megasphaera sp. 1.6 1.8     6.7 0.5  4.8 6.4 3.9          
Micromonas sp. 0.8 0.4     5.0 1.4  11.4 1.1           
Mobiluncus mulieris 0.2      0.3     6.3          
Mycoplasma sp.       9.6               
Neisseria sp.         0.6             
Peptococcus niger        1.4  0.4            
Peptoniphilus sp. 0.7      3.1   0.7  0.4          
Peptostreptococcus sp.       8.0   5.7  2.9          
Prevotella sp.       0.3    1.1 11.4   0.6    2.5   
Pseudomonas sp.                1.5      
Shigella sp.       2.5               
Staphylococcus sp.     2.6    1.0             
Streptococcus sp. 0.3 1.0   2.1   36.2 49.2    17.2 0.4 15.5 46.2 0.6 4.4 11.9   
Veillonella sp. 0.2 0.1     0.1 19.3  0.8 2.5 0.4          
Novel 4.4 0.9   0.5  46.8 9.8  36.6 17.6 6.5 4.8 1.1   2.0  1.2   
Number of women (per race) 22 26 14 12 23 23 21 10 
Number of women (per group) 62 58 37 19 17 13 11 

Clones were assigned to phylotypes by comparing their 16S rRNA gene sequences to those of known organisms. The species name was used if the sequence similarity to a type species was >97%; the genus only was used if the sequence similarity was <97%, but >90%. The clones were described as novel if the sequence similarity of clones to known organisms was <90%.

Groups were defined based on T-RFLP profiles and phylogenetic analysis of partial 16S rRNA gene sequences from clone libraries prepared from samples representative of each cluster. Groups I, IV, V, VI, and VII designate C6, C7, C3, C1, and C5, respectively (see Fig. 1). Group II merges C2 and C4 and Group III merges C8 and C9.

‘B,’‘W,’ and ‘J’ represent Black, White, and Japanese women.

Mean relative abundances of populations in each clone library.

‘Novel’ includes various phylotypes within the phylum Firmicutes, most of them belonging to novel phylotype of Clostridiales.

Species composition of vaginal communities

The 16S rRNA genes sequences from 19 clone libraries representing each of the major groups of Japanese women were identified and their relative abundances were calculated. The numerically important bacterial phylotypes that constituted >1% of a community in Japanese women are summarized in Table 1.

These data were compared with those obtained from the analysis of vaginal communities in White and Black women (57 clone libraries; Zhou et al., 2007). The mean relative abundances of populations in vaginal communities of women in all three racial groups are presented in Table 2. Among the Japanese women, most of the vaginal communities were dominated by species of Lactobacillus (group I, II, and V–VII), and these communities were found in about 75.3% (54/72) of the women. Only four species of Lactobacillus (Lactobacillus iners, L. crispatus, Lactobacillus gasseri, and Lactobacillus jensenii) were found to be common in Japanese women, which was similar to the findings for White and Black women. Of the Lactobacillus-dominated communities of Japanese women, L. crispatus was the most common dominant species. The data showed that 40.2% (29/72) of the Japanese women had microbial communities with high numbers of L. crispatus (groups II and VI). In most cases, L. crispatus constituted from 78% to 100% of the clones (group II), and in a few cases, L. crispatus represented <50% of the sequenced clones (group VI, mean values). The second most common Lactobacillus species in Japanese women was L. iners, which occurred at a high frequency in 27.8% (20/72) of the women sampled (groups I and VI). In Group I, L. iners was the predominant species, but group VI also contained high proportions of several other Lactobacillus species including L. crispatus and L. jensenii. Vaginal communities of groups III and IV found in Japanese, White, and Black women exhibited greater species evenness and lower numbers of lactobacilli (Tables 1 and 3). Group IV was characterized by communities with high frequencies of Atopobium (20–50%), and included various other species of strict or facultative anaerobes, for example, lactobacilli, Clostridium sp., Dialister sp., Gemella sp., Lachnospiraceae sp., Leptotrichia sp., Megasphaera sp., Micromonas sp., Mobiluncus mulieris, Peptinophilus sp., Prevotella sp., Veillonella sp., and some novel microorganisms that belong to the order Clostridiales. In contrast, group III contained diverse species of anaerobes other than lactobacilli and Atopobium. The dominant members included Streptococcus sp., novel microorganisms, Veillonella sp., Lachnospira sp., Gardnerella vaginalis, Anaerococuus sp., Peptostreptococcus sp., Gemella sp., Lachnospiraceae sp., Megasphaera sp., Micromonas sp., Mycoplasma sp., Peptostreptococcus sp., Prevotella sp., E. coli, and Shigella sp.

Table 3

Composition of vaginal microbiota in Groups III and IV

 Group (% clones) 
 III IV 
 B8 B14 B20 B37 B45 B49 B50 B56 W13 W27 W68 J47 J79 B19 B38 B48 W39 W63 W69 J1 J5 J46 
Phylotype n=83 n=88 n=84 n=94 n=87 n=89 n=41 n=86 n=69 n=90 n=83 n=94 n=90 n=87 n=90 n=88 n=61 n=66 n=94 n=90 n=91 n=80 
Lactobacillus iners  31.8 1.2    2.4  2.9 5.5 3.6   4.6  3.4  3.0 2.0 3.3 8.8 30.0 
L. crispatus             10.0     1.5     
L. gasseri                  63.6     
Aerococcus sp.  1.2     2.4   1.0     1.1   4.5   3.2 10.0 
Anaerobranca sp.   1.2    9.8   2.2             
Anaerococcus sp.         31.8    2.2  1.1 5.7  1.5     
Atopobium vaginae 1.2 1.2 2.4  3.4 6.7 4.9 1.2 4.3     29.9 27.8 19.3 31.0 15.4 45.7 41.1 48.4 36.2 
Bifidobacterium breve            1.0           
Bradyrhizobium sp.             1.1          
Clostridium sp.      1.2              13.3 2.2  
Corynebacterium sp.                       
Dialister sp. 4.8  3.6    4.9 4.7 2.9   2.0 3.3 6.9 3.3 1.1 1.6 1.5 1.0 4.4   
Eggerthella hongkongensis                2.3       
Enterococcus faecalis          1.1             
Escherichia coli    27.6      0.0             
Finegoldia magna          2.2     2.2        
Garanulicatella elegans          1.1             
Gardnerella vaginalis             15.6     1.5     
Gemella palaticanis 15.7     6.7   4.3 12.2 4.8  4.4  7.8      1.1  
Lachnospiraceae sp.         14.4    1.1   2.3 2.6  8.5    
Lachnospira sp.             57.9          
Leptotrichia sp.                 29.0     2.5 
Megasphaera sp.  5.7 4.8  10.3  9.8 23.3 1.4      12.2 2.3 9.8  9.5 1.1 4.4 6.2 
Micromonas sp. 1.2  2.4  4.5 30.3  1.2 4.3     26.4 6.7 1.1 3.2      
Mobiluncus mulieris     2.3               18.9   
Mycoplasma sp. 74.7      2.4                
Neisseria sp.             1.1          
Peptococcus niger         4.3       1.1       
Peptoniphilus sp.  1.1 10.7  3.4  9.8        1.1 1.1    1.1   
Peptostreptococcus sp. 2.4     30.3 29.3 2.3      1.2  15.9    1.1 7.7  
Prevotella sp.      2.2           3.2   15.7 11.0 7.5 
Shigella sp.    20.2                   
Staphylococcus sp.            2.0           
Streptococcus sp.          16.9 91.6 95.0 3.3          
Veillonella sp.  1.1        57.8      2.3  7.5    1.2 
Novel  57.9 73.7 52.2 76.1 22.6 24.3 67.3 29.4     31.0 36.7 42.1 19.6  33.3  13.2 6.4 
 Group (% clones) 
 III IV 
 B8 B14 B20 B37 B45 B49 B50 B56 W13 W27 W68 J47 J79 B19 B38 B48 W39 W63 W69 J1 J5 J46 
Phylotype n=83 n=88 n=84 n=94 n=87 n=89 n=41 n=86 n=69 n=90 n=83 n=94 n=90 n=87 n=90 n=88 n=61 n=66 n=94 n=90 n=91 n=80 
Lactobacillus iners  31.8 1.2    2.4  2.9 5.5 3.6   4.6  3.4  3.0 2.0 3.3 8.8 30.0 
L. crispatus             10.0     1.5     
L. gasseri                  63.6     
Aerococcus sp.  1.2     2.4   1.0     1.1   4.5   3.2 10.0 
Anaerobranca sp.   1.2    9.8   2.2             
Anaerococcus sp.         31.8    2.2  1.1 5.7  1.5     
Atopobium vaginae 1.2 1.2 2.4  3.4 6.7 4.9 1.2 4.3     29.9 27.8 19.3 31.0 15.4 45.7 41.1 48.4 36.2 
Bifidobacterium breve            1.0           
Bradyrhizobium sp.             1.1          
Clostridium sp.      1.2              13.3 2.2  
Corynebacterium sp.                       
Dialister sp. 4.8  3.6    4.9 4.7 2.9   2.0 3.3 6.9 3.3 1.1 1.6 1.5 1.0 4.4   
Eggerthella hongkongensis                2.3       
Enterococcus faecalis          1.1             
Escherichia coli    27.6      0.0             
Finegoldia magna          2.2     2.2        
Garanulicatella elegans          1.1             
Gardnerella vaginalis             15.6     1.5     
Gemella palaticanis 15.7     6.7   4.3 12.2 4.8  4.4  7.8      1.1  
Lachnospiraceae sp.         14.4    1.1   2.3 2.6  8.5    
Lachnospira sp.             57.9          
Leptotrichia sp.                 29.0     2.5 
Megasphaera sp.  5.7 4.8  10.3  9.8 23.3 1.4      12.2 2.3 9.8  9.5 1.1 4.4 6.2 
Micromonas sp. 1.2  2.4  4.5 30.3  1.2 4.3     26.4 6.7 1.1 3.2      
Mobiluncus mulieris     2.3               18.9   
Mycoplasma sp. 74.7      2.4                
Neisseria sp.             1.1          
Peptococcus niger         4.3       1.1       
Peptoniphilus sp.  1.1 10.7  3.4  9.8        1.1 1.1    1.1   
Peptostreptococcus sp. 2.4     30.3 29.3 2.3      1.2  15.9    1.1 7.7  
Prevotella sp.      2.2           3.2   15.7 11.0 7.5 
Shigella sp.    20.2                   
Staphylococcus sp.            2.0           
Streptococcus sp.          16.9 91.6 95.0 3.3          
Veillonella sp.  1.1        57.8      2.3  7.5    1.2 
Novel  57.9 73.7 52.2 76.1 22.6 24.3 67.3 29.4     31.0 36.7 42.1 19.6  33.3  13.2 6.4 

Clones were assigned to phylotypes by comparing their 16S rRNA gene sequences to those of known organisms. The species name was used if the sequence similarity to a type species was >97%; the genus only was used if the sequence similarity was <97%, but >90%. The clones were described as novel if the sequence similarity of clones to known organisms was <90%.

‘B,’‘W,’ and ‘J’ represent Black, White, and Japanese women.

Number of clones analyzed per woman.

Relative abundance of population in clone library.

‘Novel’ includes various phylotypes within the phylum Firmicutes, most of them belonging to novel phylotype of Clostridiales.

Distribution of vaginal community among the different racial groups

An MDS analysis of T-RFLP data was used to assess the variation of vaginal microbial communities among the different women and racial groups (Fig. 2). Some communities were very similar and clustered together in the figure, while others showed little similarity to any other communities and were scattered across the graph. A striking finding was the distribution of communities and the observation that, for all clustered data, women of all three racial groups were represented. There were no isolated communities, nor any clusters of communities that included only a specific racial group. Thus, we can conclude that all three racial groups share common vaginal community types.

Figure 2

Two-dimensional scatter plot constructed by MDS analysis based on the similarity in T-RFLP profiles among vaginal microbial communities in Japanese, White, and Black women. The samples from White, Black, and Japanese women are designated with green, black, and red dots, respectively.

Figure 2

Two-dimensional scatter plot constructed by MDS analysis based on the similarity in T-RFLP profiles among vaginal microbial communities in Japanese, White, and Black women. The samples from White, Black, and Japanese women are designated with green, black, and red dots, respectively.

The rank abundances of vaginal community types in Japanese, White, and Black women are shown in Fig. 3. Although the types of vaginal microbial communities found in healthy Japanese women were similar to those of the other two racial groups, there were significant differences in the frequencies of communities in these groups (P<0.01). Within groups III and IV, the incidence of Japanese women (24.7%) was lower than that in Black women (40.5%), but higher than that in White women (13.3%). These communities were not dominated by lactobacilli, but had comparatively equal proportions of other dominant species. On the other hand, communities dominated by multiple species of lactobacilli (group VI) were common in both Japanese and White women, but were rare in Black women.

Figure 3

Rank abundances of vaginal communities found in White, Black, and Japanese women. Each column was designated with each group (community types; see Tables 1–3). The proportion of Black and White women of North America, and Japanese women are shown as dark gray, gray, and light gray in each column, respectively.

Figure 3

Rank abundances of vaginal communities found in White, Black, and Japanese women. Each column was designated with each group (community types; see Tables 1–3). The proportion of Black and White women of North America, and Japanese women are shown as dark gray, gray, and light gray in each column, respectively.

It should be noted that the age range in the Japanese women differed slightly from those of White and Black women. The White and Black women sampled were on average younger than the Japanese women. However, our previous studies have shown that there are no significant differences in the distribution of community types between young (13–18 years) and older age groups (19–40 years) (Yamamoto et al., 2009).

Discussion

In this study, we found that the types of vaginal microbial communities found in apparently healthy Japanese women resembled those of White and Black women from North America. In total, there were seven major kinds of microbial communities identified in Japanese women, and the numbers of community types were the same as those in Black and White women. As with White and Black women, most vaginal communities were dominated by lactobacilli, and only four species of Lactobacillus (L. iners, L. crispatus, L. jensenii, and L. gasseri) were commonly found. Comparable results were reported by Shi (2009), who analyzed the vaginal community composition of a limited number of Chinese women. Taken together, these findings suggest that there may be limited differences among women in different racial/ethnic groups in terms of the bacterial species found in the vagina. Moreover, it suggests that host-determined factors, but not race per se, exert selective pressures that allow persistent colonization of the vagina by only a limited number of different kinds of bacterial species. Studies could be performed in the future to directly test whether there are differences in the vaginal communities in women of the same ethnic group who reside in two widely separate geographical regions.

How host factors might govern the composition of vaginal bacterial communities is not well understood. However, recent work has shown that the host immune system may influence human–microbial symbioses, and differences in cell surface receptors may dictate adhesion of beneficial microorganisms in the human gastrointestinal tract (Mazmanian et al., 2005; Dethlefsen et al., 2007). It is thought that specialized tissues and cells actively sample the intestinal content and initiate local immune responses that help to confine and shape the microbial diversity of the human intestine (Macpherson et al., 2005). McFall-Ngai (2007) recently proposed that adaptive immunity plays a role in recognizing and managing the complex community composition of beneficial microorganisms in vertebrates. Similar selectivity may occur in the human urogenital tract, in which case the local vaginal immune system may play an important role in determining the composition of vaginal microbial communities.

Complex food webs exist in most microbial communities in which populations occupy different trophic levels (Azam & Malfatti, 2007), and this is likely to be true for the vagina as well. This means that one or more populations are primary consumers, while others consume their metabolites, and so on. This results in a ‘network,’ wherein the number and strength of these ecological interactions determine the stability and resilience of the community (Azam & Malfatti, 2007). Different lactic acid bacteria such as L. crispatus, L. iners, Streptococcus sp., and Atopobium sp. are known to have different nutritional needs, and thus ecological interactions, competition for resources, and cross-feeding among species may determine whether the physiological needs of a given species can be met (Falsen et al., 1999; Rodriguez Jovita et al., 1999). Conceptually, this may be important because vaginal communities that differ in species composition may be more or less resilient to various kinds of disturbances (Peterson et al., 1998) such as sexual intercourse, douching, spermicides, and (in women of reproductive age) changes in the environment due to menses. If these communities differ in their responses to disturbance, it may also affect an individual's ability to resist invasive infectious agents.

Adhesins expressed by different strains of lactobacilli exhibit specificity in their ability to mediate adhesion to host epithelial cells, mucus, and extracellular matrices (Velez et al., 2007). These proteins constitute a diverse group of molecules with important functions related to adherence, signaling, and interaction with the host immune system or environment (Dramsi et al., 2005). To date, several kinds of specific mucus adhesion proteins (extracellular mucus-binding proteins) have been identified and functionally characterized in lactobacilli (Velez et al., 2007). In addition, several Lactobacillus proteins belonging to the sortase-dependent protein family have been functionally characterized (Dramsi et al., 2005). These are all involved in the binding of a particular strain to human epithelial cells and mucus, and may play a role in mediating colonization of the human vagina.

The vaginal bacterial communities of healthy women are not always dominated by Lactobacillus species. The results from this study on Japanese women confirm our previous findings that a fair proportion of women have vaginal communities dominated by lactic acid bacteria other than Lactobacillus such as Atopobium and Streptococcus, in addition to a number of populations of the order Clostridiales. This suggests that these various microbial species are naturally selected by their host and constitute bacteria normally found in the human vagina. Most of the communities that were dominated by these kinds of bacteria appear to have greater species diversity than those dominated by Lactobacillus species; thus, we postulate that the ecology of these communities may differ from that of other kinds of vaginal communities.

Statement

Study protocol and informed consent document were reviewed and approved by the Ethics Committee-Sogo Clinical Pharmacology Co. Ltd. Documented informed consent was obtained from all subjects before participation in this study.

Acknowledgements

We would like to thank Zaid Abdo, Christopher Williams, and James Foster for their suggestions for data analysis, Sanqing Yuan, Maria G. Schneider, Hyun-Joon La, and Hyo-Jin Ahn for their technical assistance, and Linda Rogers and Ivan Kluetz for editing the manuscript. Dr Forney has served as a consultant to the Procter & Gamble Co. on issues unrelated to this article. The project described was supported by grants P20RR16448 and P20RR016454 from the National Center for Research Resources of the National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. Financial support from the Procter & Gamble Company, Cincinnati, OH, was used to design and conduct the study.

References

Abdo
Z.
Schuette
U.
Bent
S.
Williams
C.
Forney
L.J.
Joyce
P.
(
2006
)
Statistical methods for characterizing diversity in microbial communities by analysis of terminal restriction fragment length polymorphism of 16S rRNA
.
Environ Microbiol
 
8
:
929
938
.
Azam
F.
Malfatti
F.
(
2007
)
Microbial structuring of marine ecosystems
.
Nat Rev Microbiol
 
5
:
782
791
.
Backhed
F.
Ding
H.
Wang
T.
Hooper
L.V.
Koh
G.Y.
Nagy
A.
Semenkovich
C.F.
Gordon
J.I.
(
2004
)
The gut microbiota as an environmental factor that regulates fat storage
.
P Natl Acad Sci USA
 
101
:
15718
15723
.
Backhed
F.
Ley
R.E.
Sonnenburg
J.L.
Peterson
D.A.
Gordon
J.I.
(
2005
)
Host–bacterial mutualism in the human intestine
.
Science
 
307
:
1915
1920
.
Brown
C.J.
Wong
M.
Davis
C.C.
Kanti
A.
Zhou
X.
Forney
L.J.
(
2007
)
Preliminary characterization of the normal microbiota of the human vulva using cultivation-independent methods
.
J Med Microbiol
 
56
:
271
276
.
Cash
H.L.
Whitham
C.V.
Behrendt
C.L.
Hooper
L.V.
(
2006
)
Symbiotic bacteria direct expression of an intestinal bactericidal lectin
.
Science
 
313
:
1126
1130
.
Dethlefsen
L.
McFall-Ngai
M.
Relman
D.A.
(
2007
)
An ecological and evolutionary perspective on human-microbe mutualism and disease
.
Nature
 
449
:
811
818
.
Dramsi
S.
Trieu-Cuot
P.
Bierne
H.
(
2005
)
Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria
.
Res Microbiol
 
156
:
289
297
.
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
:
217
221
.
Fredricks
D.N.
Fiedler
T.L.
Marrazzo
J.M.
(
2005
)
Molecular identification of bacterial vaginosis
.
New Engl J Med
 
353
:
1899
1911
.
Hyman
R.W.
Fukushima
M.
Diamond
L.
Kumm
J.
Giudice
L.C.
Davis
R.W.
(
2005
)
Microbes on the human vaginal epithelium
.
P Natl Acad Sci USA
 
102
:
7952
7957
.
Kelly
D.
Campbell
J.I.
King
T.P.
Grant
G.
Jansson
E.A.
Coutts
A.G.
Pettersson
S.
Conway
S.
(
2004
)
Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear–cytoplasmic shuttling of PPAR-gamma and RelA
.
Nat Immunol
 
5
:
104
112
.
Macpherson
A.J.
Geuking
M.B.
McCoy
K.D.
(
2005
)
Immune responses that adapt the intestinal mucosa to commensal intestinal bacteria
.
Immunology
 
115
:
153
162
.
Mazmanian
S.K.
Liu
C.H.
Tzianabos
A.O.
Kasper
D.L.
(
2005
)
An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system
.
Cell
 
122
:
107
118
.
McClelland
R.S.
Richardson
B.A.
Graham
S.M.
Masese
L.N.
Gitau
R.
Lavreys
L.
Mandaliya
K.
Jaoko
W.
Baeten
J.M.
Ndinya-Achola
J.O.
(
2008
)
A prospective study of risk factors for bacterial vaginosis in HIV-1-seronegative African women
.
Sex Transm Dis
 
35
:
617
623
.
McFall-Ngai
M.
(
2007
)
Adaptive immunity: care for the community
.
Nature
 
445
:
153
.
Naruszewicz
M.
Johansson
M.L.
Zapolska-Downar
D.
Bukowska
H.
(
2002
)
Effect of Lactobacillus plantarum 299v on cardiovascular disease risk factors in smokers
.
Am J Clin Nutr
 
76
:
1249
1255
.
Nugent
R.P.
Krohn
M.A.
Hillier
S.L.
(
1991
)
Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation
.
J Clin Microbiol
 
29
:
297
301
.
Ohashi
Y.
Nakai
S.
Tsukamoto
T.
et al
. (
2002
)
Habitual intake of lactic acid bacteria and risk reduction of bladder cancer
.
Urol Int
 
68
:
273
280
.
Parsonnet
J.
Goering
R.V.
Hansmann
M.A.
Jones
M.B.
Ohtagaki
K.
Davis
C.C.
Totsuka
K.
(
2008
)
Prevalence of toxic shock syndrome toxin 1 (TSST-1)-producing strains of Staphylococcus aureus and antibody to TSST-1 among healthy Japanese women
.
J Clin Microbiol
 
46
:
2731
2738
.
Peterson
G.
Allen
C.R.
Holling
C.S.
(
1998
)
Ecological resilience, biodiversity, and scale
.
Ecosystems
 
1
:
6
18
.
Rakoff-Nahoum
S.
Paglino
J.
Eslami-Varzaneh
F.
Edberg
S.
Medzhitov
R.
(
2004
)
Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis
.
Cell
 
118
:
229
241
.
Rayes
N.
Hansen
S.
Seehofer
D.
Muller
A.R.
Serke
S.
Bengmark
S.
Neuhaus
P.
(
2002
)
Early enteral supply of fiber and lactobacilli versus conventional nutrition: a controlled trial in patients with major abdominal surgery
.
Nutrition
 
18
:
609
615
.
Rodriguez Jovita
M.
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
:
1573
1576
.
Rozanova
G.N.
Voevodin
D.A.
Stenina
M.A.
Kushnareva
M.V.
(
2002
)
Pathogenetic role of dysbacteriosis in the development of complications of type 1 diabetes mellitus in children
.
B Exp Biol Med
 
133
:
164
166
.
Schwebke
J.R.
(
2005
)
Abnormal vaginal flora as a biological risk factor for acquisition of HIV infection and sexually transmitted diseases
.
J Infect Dis
 
192
:
1315
1317
.
Shi
Y.
Chen
L.
Tong
J.
Xu
C.
(
2009
)
Preliminary characterization of vaginal microbiota in healthy Chinese women using cultivation-independent methods
.
J Obstet Gynaecol Re
 
35
:
525
532
.
Tannock
G.W.
(
2002
)
Exploring the relationships between intestinal microflora and inflammatory conditions of the human bowel and spine
.
Antonie van Leeuwenhoek
 
81
:
529
535
.
Velez
M.P.
De Keersmaecker
S.C.
Vanderleyden
J.
(
2007
)
Adherence factors of Lactobacillus in the human gastrointestinal tract
.
FEMS Microbiol Lett
 
276
:
140
148
.
Verhelst
R.
Verstraelen
H.
Claeys
G.
Verscharaegen
G.
Delanghe
J.
Simaey
L.V.
Ganck
C.D.
Temmerman
M.
Vaneechoutte
M.
(
2004
)
Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis
.
BMC Microbiol
 
4
:
1
11
.
Vitali
B.
Pugliese
C.
Biagi
E.
Candela
M.
Turroni
S.
Bellen
G.
Donders
G.G.
Brigidi
P.
(
2007
)
Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR
.
Appl Environ Microb
 
73
:
5731
5741
.
Yamamoto
T.
Zhou
X.
Williams
C.J.
Hochwalt
A.
Forney
L.J.
(
2009
)
Bacterial populations in the vaginas of healthy adolescent women
.
J Pediatr Adolesc Gynecol
 
22
:
11
18
.
Zhou
X.
Bent
S.J.
Schneider
M.G.
Davis
C.C.
Islam
M.R.
Forney
L.J.
(
2004
)
Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods
.
Microbiology
 
150
:
2565
2573
.
Zhou
X.
Brown
C.G.
Abdo
Z.
Davis
C.C.
Hansmann
M.A.
Joyce
P.
Foster
J.A.
Forney
L.J.
(
2007
)
Difference in the composition of vaginal microbial communities found in healthy Caucasian and Black women
.
ISME J
 
1
:
121
133
.

Author notes

Editor: Patrik Bavoil