Microbial Diversity in Oral Microbiome of Postpartum Females

Abstract

Pregnancy is a state, which predisposes the female body to various reversible changes including a shift in oral microbial diversity which can be pathogenic. In the present study, salivary culturable and unculturable microbial diversity in early postpartum phase was investigated and compared with nonpregnant females diversity by using standard microbiological methods and by targeting V4 region of 16S rRNA and ITS gene. The association of microbial diversity with oral health status and adverse pregnancy outcomes (APOs) was also assessed. Unstimulated saliva samples were collected from 267 postpartum and 54 nonpregnant healthy females. Oral health problems were present in 47.2% postpartum females. Frequency of low weight birth (LWB) was 22.5%, preterm birth (PTB) 21.7% and preeclampsia was 11.6%. PTB and LWB were not associated with oral disorders. However, preeclampsia had an association with gingivitis (P=0.01). By using culture based methods, various Gram positive and negative species were identified in saliva. Streptococcus mutans were significantly more prevalent (93.2%) in saliva samples of postpartum females, while 53.5% were also culture positive for Streptococcus sobrinus. Colonization of S. mutans was significantly high among females having gingivitis (P=0.007), dental caries (P=0.006), low brushing frequency, high sugary liquid intake, and in those giving birth to a baby of low weight. Colonization of S. sobrinus was significant among females having low brushing frequency and high sugary liquid intake. Its colonization varied with gestational period and preeclampsia. In the postpartum group, 65.1% females were culture positive for Staphylococcus species [Staphylococcus aureus (n=100) Staphylococcus epidermidis (n=78) and Staphylococcus saprophyticus (n=32)]. Postpartum females showed significantly higher S. epidermidis colonization (P=0.005). Staphylococcus species colonization increased approximately one to two-fold risk for oral disorders and APOs. For identification of biofilm former isolates, phenotypic [Congo red assay (CRA) and microtiter plate assay (MTP)] and genotypic methods were used. By CRA method, 15.7% of these isolates showed biofilm forming ability and 40.9% by microtiter plate (MTP) method. By genotypic method of biofilm detection, 64% and 48.3% isolates (S. aureus and S. epidermidis) had presence of Ica A and Ica D gene, respectively. S. aureus was highly resistant to penicillin, quinolones, antibiotics of class cephalosporin and fluoroquinolones, erythromycin and gentamycin. S. epidermidis and S. saprophyticus showed high resistance against antibiotics of class penicillin and cephalosporin. Streptococcus species were detected in 27.3% postpartum females. Prevalence of Streptococcus species was significantly raised in postpartum group (P=0.001) compared to nonpregnant females. Among postpartum group, 12.7% females were culture positive for Lactobacilli, 10.4% for Neisseria meningitides, 6.3% for Klebsiella pneumoniae, 5.6% for Enterobacter species and 2.6% for Escherichia coli. By MTP method 100% E. coli, 80% isolates of Enterobacter species and 58.8% K. pneumoniae displayed biofilm forming ability. K. pneumoniae and E. coli showed high resistance against most of the tested antibiotics. Only detected fungal specie by culture based method was Candida, isolated from 55% postpartum (P<0.001) and 22.2% nonpregnant females. Candida colonization showed one to three-fold risk with oral health and different obstetric factors. Candida isolates from postpartum females expressed various virulence factors such as high esterase, phospholipase, biofilm forming activity and also displayed antifungal resistance. Unculturable bacterial and fungal diversity was also studied among postpartum and nonpregnant healthy female. In total 16 bacterial phyla and 156 genera were observed in all saliva samples tested. Decrease in alpha diversity and high Bray-Curtis dissimilarity was seen in salivary microbiome of postpartum female having oral health issues with preterm low weight birth (PLWB) compared to females with full term birth (FTB). Female with oral health problems and who gave FTB showed predominance of genera Streptococcus followed by Yersinia, Haemophilus, Neisseria, Fusobacterium, Gemella, Prevotella and Aggregatibacter, while female with PLWB was also dominated by Streptococcus, followed by Gemella, Prevotella, Rothia, Veillonella, Haemophilus, Neisseria and Granulicatella. Upon fungal diversity analysis, 55 genera and 92 species were detected in all samples. Postpartum female having oral health issues with PLWB, showed reduced richness, evenness with elevated levels of Saccharomyces, Candida, Hyphodontia and Malassezia compared to those females having FTB. In present work, dual-species biofilm assays for different combinations of Candida and bacterial species were developed as dual-specie biofilm model to study behavior of dual species as consortium in oral niche that can lead to pathogenesis. Analysis showed that all three Candida species with each other and with E. coli in dual-specie biofilms showed antagonistic behavior. Biofilm biomass production was raised for C. albicans and C. glabrata dual-specie biofilm with Staphylococcus species except with clinical isolate of S. aureus and in C. glabrata-S. epidermidis dual-species biofilm. Candida species with clinical isolate of K. pneumoniae in dual-species biofilm assay showed increased in biofilm biomass after 48 h of incubation except for C. glabrata-K. pneumoniae dual-species biofilms. Dual-specie assay showed that in consortium various species change their behavior and display different virulence factors. In consortium, species can be antagonistic or synergistic with other species. In conclusion, findings from present work showed that pregnancy with or without oral health issues is associated with culturable and unculturable oral microbial diversity change. This change in diversity is more towards pathogenic culturable multi-drug resistant isolates with strong biofilms forming ability and enhanced expression of virulence factors. In addition to commonly known fungal and bacterial pathogens, less frequently studied microbes with regard to oral pathologies such as C. glabrata and S. epidermidis also emerged as oral pathogens, their prevalence was high in females with oral disorders and APOs. These isolates also showed either synergistic or antagonistic behavior with each other in consortia when studied in mono and dual-species biofilm model. Females with oral disorders and APOs also showed decrease in species richness and evenness, suggesting the possible role of changing microbial diversity with oral disorders leading to induction of APOs. Overall, this study contributes to literature on existing culturable and unculturable microbial diversity and existence of pathogenic potential for its possible role in APOs. Based on present study findings, it can be easily recommended that there is need for development of guidelines for health practitioners to provide awareness to the females on importance of keeping good oral health and routine dental checkup during pregnancy.