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

A total of 163 pregnant Japanese women were enrolled in this study during routine prenatal visits at Hokkaido University Hospital from May 2005 to February 2006. Informed consent was obtained from all participants in verbal form. Vaginal fluid samples were collected at a mean of 23 weeks of gestation. Estimated date of delivery was determined from the last menstrual period and early gestational fetal ultrasonographic measurements.

A sterile speculum was inserted into the vagina and a specimen of vaginal fluid was obtained by brushing the posterior vaginal fornix with a swab. A vaginal smear was prepared by rolling a swab onto a glass slide, which was then air-dried, heat-fixed, and Gram-stained. The smears were then assessed according to Nugent criteria [32]. The other swab was spread onto Columbia blood agar plates, and incubated at 35°C under aerobic conditions in 5% CO₂ and anaerobic conditions for 48 h. Lactobacilli were identified to the genus level by Gram staining of colonies and from colony morphology on blood agar plates.

Another swab was placed in 1 ml of PBS with subsequent vigorous vortexing to dislodge cells. The cells were centrifuged at 14,000 rpm for 5 min. The pellet was digested with proteinase K at 56°C for 30–60 min and the DNA was extracted and purified with a QIAmp DNA Mini Kit (Qiagen, Germantown, MD) in accordance with the manufacturer's instructions, resulting in 200 μl of DNA solution. PCR mixtures consisted of PCR buffer with 1.5 mM of MgCl₂, 10 pmol of each primer, 2.0 μM of each deoxyribonucleoside triphosphate, 0.1 μl of Taq DNA polymerase, and 1.5 μl of template DNA solution in a final volume of 15 μl.

Sequences and annealing temperatures for the various primer sets are listed in Table 1[19, 33]. All primers were located in the 16S rDNA region. PCR was carried out for 40 cycles. For the Lactobacillus genus and its four species, the denaturation was performed at 95°C for 15 s followed by a 1-min annealing and extension step. For four BV-related bacteria, the denaturation step was set at 94°C for 30 s, followed by the annealing step for 30 s for BVAB2, Megasphaera, and Leptotrichia and for 40 s for Eggerthella-like bacterium, with extension at 72°C for 1 min for all reactions. A final extension step at 72°C for 7 min was added for all reactions. Aliquots of 7 μl of the PCR products were electrophoresed in agarose gels and visualized by ultraviolet transillumination after ethidium bromide staining.

The specificity of the Lactobacillus species-specific PCR for 14 common intestinal Lactobacillus species was evaluated and confirmed using 10⁶ copies of one Lactobacillus species to each reaction as template DNA (Table 2). The universal Lactobacillus primer amplified all of these Lactobacillus species. The specific primers for L. crispatus, L. jensenii, and L. gasseri only amplified the corresponding species and did not amplify 13 other species (Table 2). They also did not amplify a cloned fragment of 16S rDNA region of L. iners. The specific primers for L. iners did not amplify any of 14 Lactobacillus species (Table 2). We analyzed PCR products from several vaginal samples amplified by the specific primers for L. iners and confirmed that the sequences of PCR products were completely consistent with L. iners (GenBank AY526083).

The sensitivities of the species-specific PCR for L. crispatus, L. jensenii, and L. gasseri were measured using serial dilutions of DNA solution of the reference strain. Similarly, for L. iners, serial dilutions of a cloned fragment of 16S rDNA region of L. iners were used instead. The sensitivity of the species-specific PCR for L. crispatus, L. jensenii, and L. gasseri and that of L. iners PCR were 10² to 10³ copies and 10² copies per reaction, respectively.

A total of 163 samples from 163 pregnant women were obtained during the study period and 31 samples were excluded because of lack of information regarding the gestational week at delivery (n = 21) or Gram staining (n = 10). The remaining 132 samples from 132 women were analyzed and classified according to the Nugent criteria. Ninety-eight women (74.2%) were classified as having normal vaginal flora, 21 (15.9%) were intermediate, and 13 (9.8%) were BV. Samples from these women were divided into normal, intermediate, and BV groups, respectively.

The clinical characteristics of the pregnant women are summarized in Table 3. There were no statistically significant differences in the mean maternal age, number of nulliparous women, gestational week at sampling, gestational week at delivery, or birth weight of the neonate among the three groups. Of the total of 132 women, 35 (26.5%) delivered at <37 weeks, 9 (6.8%) at <33 weeks, and 4 (3.0%) at <30 weeks of gestation.

Genus Lactobacillus (any Lactobacillus) was detected in almost all women irrespective of Nugent score (Table 4). The detection rates of L. crispatus, L. jensenii, and L. gasseri were significantly higher in the normal group than in the BV group, while that of L. iners did not differ between the three groups. In contrast, the detection rates of BVAB2, Megasphaera, Leptotrichia, and Eggerthella-like bacterium were significantly lower in the normal group than in the BV group.

Multivariate logistic regression analysis was performed to evaluate the independent contributions of the various bacteria to the abnormal vaginal flora (Tables 5 and 6). Seven bacteria, i.e., L. crispatus,L jensenii,L. gasseri, BVAB2, Megasphaera,Leptotrichia and, Eggerthella-like bacterium, were entered as variates to be analyzed (P < 0.1, Fisher's exact probability test). The absence of L. crispatus and the presence of Megasphaera were selected as two independent risk factors of Nugent score ≥ 4, giving Odds ratios of 0.2 and 13.3, respectively (Table 5). Likewise, the presence of Eggerthella-like bacterium was selected as an independent risk factor of Nugent score ≥ 7, giving an Odds ratio of 6.2 (Table 6). Linear regression analyses revealed that BVAB2 and Megasphaera were associated with Nugent score.

L. iners was detected by PCR in 55 of 132 (41.7%) women, and its prevalence did not differ between the groups classified according to Nugent score (Table 4). However, the presence of L. iners appeared to be positively associated with colonization by BV-related bacteria (Table 7). The detection rates of all BV-related bacteria were significantly higher in samples harboring L. iners. No such association was seen between the presence or absence of L. iners and the detection rate of any other Lactobacillus species.

Lactobacillus was cultured from 91 (92.9%) of 98 samples, 11 (52.4%) of 21 samples, and 2 (15.4%) of 13 samples of the normal, intermediate, and BV groups, respectively (data not shown). These observations conflicted markedly with the results obtained by the PCR method, especially in women with abnormal vaginal flora with respect to detection of Lactobacillus. Of the eleven women with BV from whom Lactobacillus was uncultured but detected by PCR, L. iners was detected in 5 women and L. crispatus was detected in only one woman by PCR, suggesting that L. iners is less likely to be cultured than L. crispatus. To determine which species of Lactobacillus is difficult to culture, the detection rates by PCR of various species of Lactobacillus were compared with those by the conventional culture method (Table 8). Among 130 samples determined to contain any Lactobacillus by the PCR method, 104 (80.0%) were positive for Lactobacillus by the culture method (Table 8). More than 90% of samples determined to contain L. crispatus, L. jensenii, or L. gasseri by the PCR method were determined to have Lactobacillus by the conventional culture method. Among 24 samples in which L. crispatus was the only Lactobacillus species identified by the PCR method, Lactobacillus was cultured from 22 samples (91.7%), while Lactobacillus was cultured only from 47.6% and 27.3% of samples in which L. iners and unspecified Lactobacillus species, respectively, were the only Lactobacillus species identified by the PCR method. Thus, L. iners, and unspecified Lactobacillus species other than L. crispatus, L. jensenii, or L. gasseri appeared to have stringent cultivation requirements.