Association between Lactobacillus species and bacterial vaginosis-related bacteria, and bacterial vaginosis scores in pregnant Japanese women

Bacterial vaginosis (BV) is the disturbed vaginal flora, in which normal lactobacilli are replaced by an overgrowth of various anaerobic bacteria [1]. This condition is common in women of reproductive age [1, 2] and may cause malodorous vaginal discharge, although in many women it is asymptomatic [3]. In pregnant women, bacterial vaginosis has been suggested to be a risk factor of perinatal complications, including preterm birth [1, 4–12] and chorioamnionitis [4, 13]. These complications are closely associated with neonatal morbidity and mortality worldwide.

Bacteria detected in BV flora include Gardnerella vaginalis, Mycoplasma hominis, Mobiluncus species (sp.), and other anaerobic bacteria, i.e., Peptostreptococcus sp., Prevotella sp., and Bacteroides sp. [1, 14–16]. Recently, bacteria such as Atopobium vaginae, Megasphaera sp., Leptotrichia sp., and Eggerthella-like bacterium have been reported as microorganisms related to this condition by molecular analyses [17–19]. Fredricks et al. identified three phylogenetically distinct bacterial DNA sequences in human vaginal samples highly specific for this condition and designated them BV-associated bacteria (BVAB) 1~3 [19]. They showed that BVABs, Megasphaera, Leptotrichia, and Eggerthella-like bacterium are more specific for BV than Gardnerella and Atopobium [19]. Among BVABs, BVAB2 was shown to be more sensitive for BV than BVAB1 and BVAB3, while the specificities of three BVABs were similar [19]. We use the term "BV-related bacteria" for bacteria including BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium in this manuscript. However, it is important to note that these organisms have not been proven to be causative agents of BV.

The healthy human vaginal flora in reproductive age is usually predominated by Lactobacillus species. Their metabolic products, such as hydrogen peroxide (H₂O₂), lactic acid, and bacteriocin are believed to play an important role in maintenance of the normal vaginal flora by inhibiting colonization by other pathogens [20–23]. The predominant Lactobacillus species in the normal lactobacillary flora were shown by molecular biological analyses to be L. crispatus, L. gasseri, and L. jensenii [23–28]. In recent studies, L. iners described as L. 1086V by Antonio et al. [24] was identified as one of the common Lactobacillus species colonizing the human vagina [18, 28–31]. Only 9% of the strains of this species produce H₂O₂, whereas almost all strains of L. crispatus and L. jensenii produce H₂O₂ [24].

To date, there have been few studies regarding the frequencies of the BV-related bacteria described above and Lactobacillus species in healthy and abnormal vaginal flora in pregnant women. The present study was performed to evaluate the prevalence of the BV-related bacteria and the common Lactobacillus species in normal and BV flora in pregnant Japanese women. We used a specific PCR method targeting the bacterial 16S ribosomal DNA (rDNA) region for this purpose.

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. [19] 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 [29], 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 [19]. 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 [21], 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 [24]. 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. [19]. 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 [19]. 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 [19], 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 [19]. 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. [19], 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 [19].

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 [29], the conventional culture method used in this study may have failed to reveal its colonization in the vagina.

Our results suggested that BV-related bacteria, including BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium, were associated with BV in pregnant Japanese women. The presence of L. iners, one of the common Lactobacillus species in the vagina, may be correlated with vaginal colonization by these BV-related bacteria. It remains to be determined whether BV-related bacteria cause BV or are common and abundant as a consequence of BV.