Study of the vaginal and rectal microflora in pregnant women, with emphasis on Group B streptococci

In a first part of the PhD research we established the degree of correspondence between the vaginal and the rectal microflora. The composition of the human vaginal microflora is affected by several host factors, including, among others, age, menarche, hormonal changes, sexual activity, pregnancy and the use of contraceptives or spermicides, as well as individual habits such as douching (405). Studies of vaginal lactobacilli have demonstrated that Lactobacillus crispatus, L. jensenii, L. gasseri and L. vaginalis are the most commonly recovered species of H2O2-producing lactobacilli (5, 172, 287, 380) and the absence of H2O2-producing lactobacilli from the vagina has been associated with an increased risk for bacterial vaginosis (BV) (161, 241). BV is a condition whereby the lactobacilli are overgrown by anaerobic bacteria, such as Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp. , Mycoplasma spp. , Peptostreptococcus spp. and Prevotella spp. BV has been linked to increased shedding of HIV in the female genital tract (63), increased acquisition of HIV (161) and herpes simplex virus type 2 (63, 161) and with preterm birth (174). Several bacterial species are known to colonize both the gastrointestinal and the reproductive tract, and the rectum may play an important role as a source or reservoir for organisms that colonize the vagina (6, 238). The question remains whether these vaginal organisms are endogeneous or originate from the gastrointestinal tract. To establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimen from pregnant women at 35-37 weeks of gestation as part of the group B streptococci (GBS) screening program to prevent GBS neonatal disease, we identified the isolates to the species level with tRNA intergenic length polymorphism analysis (tDNA-PCR) and genotyped the isolates for those subjects from which the same species was isolated simultaneously vaginally and rectally, by RAPD-analysis (100). Also we compared the genotype of GBS isolates between the vagina and rectum (99). To further establish the potential role of the rectum as a reservoir for the vaginal microflora, we quantified the bacterial loads of six of the vaginally most important species by quantitative real-time PCR (qPCR), for those women where these species were present simultaneously in both niches (96). In summary, the species composition and the genotype similarity for those species simultaneously present in both niches, were unexpectedly high, indicating that indeed the rectum is probably the most important source of the vaginal microflora, or that strong dynamic exchange between both niches exist. This was further confirmed by the finding that for five of the six species tested (except for G. vaginalis), the vaginal and rectal bacterial load were in strong correspondence. A second part of this PhD research regarded improving the prepartum detection of group B streptococci. Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Early neonatal GBS infection can be prevented by identifying high-risk pregnancies and administering intrapartum antibiotics. Two different strategies, the screening-based and the risk-based approach, are used today in order to decrease the incidence of early-onset neonatal infection. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim Broth (Todd–Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar (57). However, this procedure may require 48 h to complete and its sensitivity might be improved. We compared different sampling and culture techniques for the detection of group B streptococci (98) and found that rectovaginal sampling was the most sensitive method for sampling and that Granada agar and Chromagar were the most sensitive culture techniques, only slightly improved by Lim broth enrichment. Also we compared two different targets (sip and cfb genes), using two different real-time PCR (qPCR) approaches, i.e. the hydrolysis probe (Taqman, Roche) and hybridisation probe (Hybprobe, Roche) techniques, directly on the clinical sample and after Lim broth enrichment, with the previously optimized culture approach (97), i.e. Lim broth enrichment, followed by subculture on Chromagar (97). We could establish that the sensitivity of these molecular techniques is significantly higher than that of the CDC recommended culture approach and also than that of our improved culture approach. Rather surprisingly, the sensitivity of these molecular techniques is further significantly improved by Lim broth enrichment compared to direct application on the clinical sample. The consequences of this observation for the application of the rapid combined DNA extraction/qPCR techniques, as they are now available for intrapartum screening, are still unclear, and will be the basis for future research in the rapidly evolving GBS diagnostics.