In the present study, we confirmed that L. crispatus, L. gasseri, and L. jensenii were common species in pregnant Japanese women with normal vaginal flora by species-specific PCR of the 16S rDNA region. These three species were less prevalent in women with BV. In contrast, four BV-related bacteria, i.e., BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium, were detected at higher prevalence in women with BV. As all these results were in accordance with those of Fredricks et al.  who analyzed the vaginal fluid of non-pregnant women with and without BV using the broad-range 16S rDNA PCR and cloning methods, BV is suggested to have remarkably similar microbiological profiles among women with different demographic characteristics, including race and pregnancy, as suggested by the conventional cultivation method.
L. crispatus, L. gasseri, and L. jensenii are common Lactobacillus species found in the vagina [24–28, 31, 34]. L. iners, described recently as a new Lactobacillus species , is one of the common Lactobacillus species of the vaginal microbiota [18, 19, 23, 28, 30, 31], which was also confirmed in the present study. The results showed that L. iners was present in 40% to 50% of women irrespective of Nugent score, as observed in an earlier study . We examined twelve samples positive for L. iners (6 from normal flora and 6 from BV flora) to determine whether the abundance of L. iners was different in the two groups. The species-specific PCR for L. iners using serial dilutions of each sample revealed that both normal and BV flora contained 103 to 105 copies/μl of L. iners and the median concentration was 104 copies/μl for both.
As the presence of H2O2-producing lactobacilli in the vaginal fluid is associated with a reduced risk of BV [15, 24] and because the concentration of H2O2 in the vaginal fluid is low in women with BV as compared with those with normal vaginal flora , the H2O2-producing ability of lactobacilli is thought to play a significant role in protecting the vaginal ecosystem from BV infection, although direct evidence to support this notion is lacking. Nearly all strains of L. crispatus and L. jensenii have been reported to produce H2O2, whereas only 9% of the strains of L. iners produce H2O2 . The prevalences of L. crispatus and L. jensenii were significantly higher in the normal group than in the BV group and the detection rates of all BV-related bacteria were significantly higher in women with than in those without L. iners in this study. Although this observation is consistent with the notion that H2O2-producing ability of lactobacilli is important in protecting the vaginal ecosystem from BV infection, it remains to be determined whether these observations resulted from differences in H2O2-producing ability of these lactobacilli.
The newly proposed "BV-related bacteria," including BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium, were all shown to be associated with BV in the present study, confirming the results of a recent study by Fredricks et al. . However, the detection rates of these bacteria in women with BV were lower, while those in women with normal flora were similar to their results . BVAB2 is cultivation-resistant, one of three bacteria (provisionally named BV-associated bacteria: BVAB1, BVAB2, and BVAB3) newly found to be highly specific for BV in the vagina of non-pregnant women , and not closely related to other bacteria as shown by comparison of 16S rDNA. In the present study, BVAB2 was present in 38.5% (5/13) and 3.1% (3/98) of women with BV and with normal vaginal flora, respectively, while Fredricks et al. reported these rates to be 88.9% (24/27) and 4.3% (2/46), respectively . Similarly, detection rates of Megasphaera (69.2%),Eggerthella-like bacterium (53.8%), and Leptotrichia (53.8%) in women with BV in the present study were lower than those of 96.3%, 92.6%, and 85.2% reported by Fredricks et al. , while detection rates of Megasphaera (11.2%),Eggerthella-like bacterium (7.1%), and Leptotrichia (14.3%) in women with normal vaginal flora were comparable to their values of 8.7%, 8.7%, and 4.3%, respectively .
The results of the present study raised the possibility that the four BV-related bacteria were less prevalent in pregnant Japanese women with BV as compared with non-pregnant American women. However, the number of subjects with BV in the present study was too low to draw definitive conclusions about the prevalence of bacteria in different populations. Further studies using different demographic populations are needed to determine the roles of these BV-related bacteria in the pathogenesis of BV.
Twelve (92%) of 13 women with BV were positive for genus Lactobacillus by 16S rDNA PCR using the universal Lactobacillus primer, including 5 women with L. iners, one with both L. iners and L. crispatus, one with L. crispatus, and 5 with unspecified Lactobacillus. Of these 13 women, only one with L. iners was positive for Lactobacillus by general cultivation methods and positive for Gram-positive rods on Gram staining. These results suggested that many women with BV harbor genus Lactobacillus in the vagina and that the number of these lactobacilli colonizing the vagina is small. Further, as L. iners has been reported to require specialized blood agar media for isolation , the conventional culture method used in this study may have failed to reveal its colonization in the vagina.