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

Prevalence of Group B Streptococcus Colonization among Pregnant Women in Gaza strip, Palestine

Streptococcus agalactiae (group B Streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. The aim of this study was to evaluate the prevalence of GBS colonization among pregnant women in Gaza strip. A total of 200 rectovaginal swabs collected from pregnant women from Al Shifa hospital were screened for GBS colonization. Standard microbiological methods according to the Centers for Disease Control and Prevention (CDC) recommendations were used to isolate and identify GBS. Selective and chromogenic culture in addition to PCR were employed for the detection of GBS. Antimicrobial susceptibility testing (AST) was performed according to CLSI guidelines. Out of 200 pregnant women, 42 (21%) were colonized by GBS. The sensitivity, specificity, positive predictive value and negative predictive value of PCR were 54%, 88%, 76%, and 72%, respectively. Of the GBS isolates examined 76%, 57%, 50%, 48% and 31% were susceptible to vancomycin, penicillin, erythromycin, tetracycline and clindamycin, respectively. There was no statistically significant association between GBS colonization and chronic diseases, complications (previous abortion, delivery at <37 weeks gestation, premature birth, intrauterine death and endometrtitis), and previous antibiotic intake (p>0.05). In conclusion, this study showed high prevalence of GBS colonization among pregnant women in Gaza strip. Despite the fact that PCR is well known for its high sensitivity, low sensitivity was obtained in this study which may be due to the collection methods. Vancomycin was the most effective antibiotic against GBS isolates. We recommend a screening-based strategy to detect GBS in Palestinian pregnant women

Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women

Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. 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. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS.status: publishe

Antibiotic resistance and mecA gene characterization of Staphylococcus epidermidis isolated from some hospitals in Gaza strip

Antibiotic resistance of S. epidermidis isolated from biological specimens is a global problem to public health. In this study a total of 256 S. epidermidis isolates (128 clinical isolates and 128 nasal isolates) from Gaza strip, Palestine were investigated. All isolates were tested for its antimicrobial susceptibilities and carriage of the mecA gene. Out of the 256 isolates, 184 (71.9%) were resistant to multiple antibiotics with all displaying increased susceptibility toward rifampicin (100%), doxycycline (98.4%) and vancomycin (98%). Ninety-six isolates (37.5%) were multidrug resistant (MDR) while, 99 isolates (38.7%) were mecA positive. A significant difference was demonstrated between clinical and nasal isolates. Clinical isolates were significantly more resistant for 8/12 tested antibiotics including resistance to cefoxitin (30μg) (p=0.000) and significantly (p=0.000) represents the MDR isolates while nasal isolates were significantly (p=0.000) sensitive for all tested antibiotics. No significant difference between the two groups in carrying mecA. We find that clinical isolates gain an extra-feature that qualify it to cause a disease and methicillin resistance (MR) was not mecA dependent in all MR isolates

Longitudinal Study of the Dynamics of Vaginal Microflora during Two Consecutive Menstrual Cycles

Although the vaginal microflora (VMF) has been well studied, information on the fluctuation of the different bacterial species throughout the menstrual cycle and the information on events preceding the presence of disturbed VMF is still very limited. Documenting the dynamics of the VMF during the menstrual cycle might provide better insights. In this study, we assessed the presence of different Lactobacillus species in relation to the BV associated species during the menstrual cycle, assessed the influence of the menstrual cycle on the different categories of vaginal microflora and assessed possible causes, such as menstruation and sexual intercourse, of VMF disturbance. To our knowledge, this is the first longitudinal study in which swabs and Gram stains were available for each day of two consecutive menstrual cycles, whereby 8 grades of VMF were distinguished by Gram stain analysis, and whereby the swabs were cultured every 7(th) day and identification of the bacterial isolates was carried out with a molecular technique.status: publishe

The development of a 16S rRNA gene based PCR for the identification of Streptococcus pneumoniae and comparison with four other species specific PCR assays

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is one of the most frequently encountered pathogens in humans but its differentiation from closely related but less pathogenic streptococci remains a challenge.</p> <p>Methods</p> <p>This report describes a newly-developed PCR assay (Spne-PCR), amplifying a 217 bp product of the 16S rRNA gene of <it>S. pneumoniae</it>, and its performance compared to other genotypic and phenotypic tests.</p> <p>Results</p> <p>The new PCR assay designed in this study, proved to be specific at 57°C for <it>S. pneumoniae</it>, not amplifying <it>S. pseudopneumoniae </it>or any other streptococcal strain or any strains from other upper airway pathogenic species. PCR assays (psaA, LytA, ply, spn9802-PCR) were previously described for the specific amplification of <it>S. pneumoniae</it>, but <it>psaA</it>-PCR was the only one found not to cross-react with <it>S. pseudopneumoniae</it>.</p> <p>Conclusion</p> <p>Spne-PCR, developed for this study, and psaA-PCR were the only two assays which did not mis-identify <it>S. pseudopneumoniae </it>as <it>S. pneumoniae</it>. Four other PCR assays and the AccuProbe assay were unable to distinguish between these species.</p

Identification and genotyping of bacteria from paired vaginal and rectal samples from pregnant women indicates similarity between vaginal and rectal microflora

Background: The vaginal microflora is important for maintaining vaginal health and preventing infections of the reproductive tract. The rectum has been suggested as the major source for the colonisation of the vaginal econiche. Methods: To establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimens from pregnant women at 35-37 weeks of gestation, 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. One vaginal and one rectal swab were collected from a total of each of 132 pregnant women at 35-37 weeks of gestation. Swabs were cultured on Columbia CNA agar and MRS agar. For each subject 4 colonies were selected for each of both sites, i.e. 8 colonies in total. Results: Among the 844 isolates that could be identified by tDNA-PCR, a total of 63 bacterial species were present, 9 (14%) only vaginally, 26 (41%) only rectally, and 28 (44%) in both vagina and rectum. A total of 121 (91.6%) of 132 vaginal samples and 51 (38.6%) of 132 rectal samples were positive for lactobacilli. L. crispatus was the most frequently isolated Lactobacillus species from the vagina (40% of the subjects were positive), followed by L. jensenii (32%), L. gasseri (30%) and L. iners (11%). L. gasseri was the most frequently isolated Lactobacillus species from the rectum (15%), followed by L. jensenii (12%), L. crispatus (11%) and L. iners (2%). A total of 47 pregnant women carried the same species vaginally and rectally. This resulted in 50 vaginal/rectal pairs of the same species, for a total of eight different species. For 34 of the 50 species pairs (68%), isolates with the same genotype were present vaginally and rectally and a high level of genotypic diversity within species per subject was also established. Conclusion: It can be concluded that there is a certain degree of correspondence between the vaginal and rectal microflora, not only with regard to species composition but also with regard to strain identity between vaginal and rectal isolates. These results support the hypothesis that the rectal microflora serves as a reservoir for colonisation of the vaginal econiche

Genotyping of Streptococcus agalactiae (group B streptococci) isolated from vaginal and rectal swabs of women at 35-37 weeks of pregnancy

<p>Abstract</p> <p>Background</p> <p>Group B streptococci (GBS), or <it>Streptococcus agalactiae</it>, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Here we compared different culture media for GBS detection and we compared the occurrence of different genotypes and serotypes of GBS isolates from the vagina and rectum.</p> <p>Methods</p> <p><it>Streptococcus agalactiae </it>was cultured separately from both rectum and vagina, for a total of 150 pregnant women, i) directly onto Columbia CNA agar, or indirectly onto ii) Granada agar resp. iii) Columbia CNA agar, after overnight incubation in Lim broth.</p> <p>Results</p> <p>Thirty six women (24%) were colonized by GBS. Of these, 19 harbored GBS in both rectum and vagina, 9 only in the vagina and 8 exclusively in the rectum. The combination of Lim broth and subculture on Granada agar was the only culture method that detected all GBS positive women. Using RAPD-analysis, a total of 66 genotypes could be established among the 118 isolates from 32 women for which fingerprinting was carried out. Up to 4 different genotypes in total (rectal + vaginal) were found for 4 women, one woman carried 3 different genotypes vaginally and 14 women carried two 2 different genotypes vaginally. Only two subjects were found to carry strains with the same genotype, although the serotype of both of these strains was different.</p> <p>Eighteen of the 19 subjects with GBS at both sites had at least one vaginal and one rectal isolate with the same genotype.</p> <p>We report the presence of two to four different genotypes in 22 (61%) of the 36 GBS positive women and the presence of identical genotypes in both sites for all women but one.</p> <p>Conclusion</p> <p>The combination of Lim broth and subculture on Granada medium provide high sensitivity for GBS detection from vaginal and rectal swabs from pregnant women. We established a higher genotypic diversity per individual than other studies, with up to four different genotypes among a maximum of 6 isolates per individual picked. Still, 18 of the 19 women with GBS from both rectum and vagina had at least one isolate from each sampling site with the same genotype.</p

Fecal carriage of extended-spectrum β-lactamase-producing enterobacterales from hospitals and community settings in Gaza Strip, Palestine

Abstract Background The fecal carriage of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) is a major driver of the global spread of these antibiotic resistance determinants. Here we determined the rate of fecal ESBL-PE carriage in pediatric hospitals and community-serving healthcare centers serving adults and children in the Gaza Strip, Palestine. Methods A total of 373 fecal and rectal samples were collected from different hospitals and clinics in Gaza. The antibiotic susceptibility was determined using the disk diffusion method and interpreted according to CLSI guidelines. The bacterial isolates were tested for ESBL production using phenotypic methods (double disk synergy test and growth on selective chromogenic media). Bla CTX−M, bla SHV, and bla TEM genes were sought by PCR. Results Out of the 373 isolates tested, 138 (37%) were considered ESBL positive as revealed by phenotypic tests. The prevalence of ESBLs among hospitalized patients was 39.1% (hospital setting) whereas, among outpatients attending community healthcare centers, it was 35.1% (community setting). ESBL production among Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, and Klebsiella aerogenes isolates was 52.8%, 39.1%, 26.7%, 2.8%, and 2.1% respectively. Meropenem and amikacin were the most effective antibiotics against ESBL producers (68.9% and 73.6% susceptibility, respectively), while only 15.2%, 22.5%, and 24.6% remained susceptible to ceftazidime, cefotaxime, and ceftriaxone, respectively. Out of 138 phenotypically ESBL-positive isolates, 98 randomly chosen were screened for bla CTX−M, bla TEM, and bla SHV genes. The prevalence rate of bla CTX−M was 45.9%, while bla TEM and bla SHV genes were detected in 16.8% and 5.2% of CTX-M-negative isolates (corresponding mostly for K. pneumoniae isolates in the case of SHV-PCR), respectively. Conclusions The study revealed an alarmingly high prevalence of fecal carriage of ESBL-producing Enterobacterales among hospitalized children but also in the community of the Gaza Strip. In addition, 30% of ESBL-producers were already resistant to carbapenems, the treatment of choice of infections with ESBL-producers