Vaginal dysbiosis increases risk of preterm fetal membrane rupture, neonatal sepsis and is exacerbated by erythromycin

Abstract

Background: Preterm prelabour rupture of the fetal membranes (PPROM) precedes 30% of preterm births and is a risk factor for early onset neonatal sepsis. As PPROM is strongly associated with ascending vaginal infection, prophylactic antibiotics are widely used. The evolution of vaginal microbiota compositions associated with PPROM and the impact of antibiotics on bacterial compositions are unknown.

Methods: We prospectively assessed vaginal microbiota prior to and following PPROM using MiSeq-based sequencing of 16S rRNA gene amplicons and examined the impact of erythromycin prophylaxis on bacterial load and community structures.

Results: In contrast to pregnancies delivering at term, vaginal dysbiosis characterised by Lactobacillus spp. depletion was present prior to the rupture of fetal membranes in approximately a third of cases (0% vs. 27%, P = 0.026) and persisted following membrane rupture (31%, P = 0.005). Vaginal dysbiosis was exacerbated by erythromycin treatment (47%, P = 0.00009) particularly in women initially colonised by Lactobacillus spp. Lactobacillus depletion and increased relative abundance of Sneathia spp. were associated with subsequent funisitis and early onset neonatal sepsis.

Conclusions: Our data show that vaginal microbiota composition is a risk factor for subsequent PPROM and is associated with adverse short-term maternal and neonatal outcomes. This highlights vaginal microbiota as a potentially modifiable antenatal risk factor for PPROM and suggests that routine use of erythromycin for PPROM be re-examined.

Keywords: Antibiotics; Erythromycin; Neonatal sepsis; Pregnancy; Preterm birth; Preterm prelabour rupture of membranes; Vaginal microbiota.

Fig. 1

Decreased vaginal Lactobacillus spp. abundance occurs prior to PPROM and is further exacerbated by membrane rupture and erythromycin treatment. a Ward’s linkage hierarchical clustering analysis of vaginal bacterial species data from cervical vaginal fluid samples (n = 165) collected from women with uncomplicated term delivery, sampled at 28 weeks (n = 20), pre-PPROM (n = 15), following PPROM before erythromycin (n = 39), after 48 hours of erythromycin (n = 43), 48 hours to 1 week of erythromycin (n = 22) and >1 week of erythromycin treatment (n = 26). Vaginal bacterial communities were classified based on Lactobacillus spp. abundance into dominant, intermediate and depleted, and further into eight vaginal microbiota groups: VMG 1: L. iners dominant, VMG 2: L. iners high diversity, VMG 3: L. crispatus dominant, VMG 4: L. gasseri dominant, VMG 5: L. jensenii dominant, VMG 6: L. crispatus high diversity, VMG 7: Lactobacillus spp. depleted and low diversity, VMG 8: Lactobacillus spp. depleted and high diversity. b Relative Lactobacillus spp. abundance is significantly lower in the pre-PPROM and membrane rupture groups compared to gestation age matched and normal pregnancy controls (P = 0.011). Erythromycin treatment exacerbates Lactobacillus spp. depletion and expansion of dysbiotic vaginal communities (P = 0.001). Reduced Lactobacillus spp. abundance is accompanied by a reciprocal increase in c richness and d diversity. e Bacterial load is significantly higher pre-membrane rupture in comparison to post-membrane rupture (P = 9.37 × 10^-8) but remains stable thereafter, despite ongoing erythromycin treatment. PPROM preterm prelabour rupture of the fetal membranes

Fig. 2

Bacterial taxonomic groups discriminate between normal-term delivery and women destined to experience PPROM. a Cladogram describing differentially abundant vaginal microbial clades and nodes observed between women subsequently experiencing normal-term delivery or PPROM as identified using LEfSe analysis. b The effect size for each differentially abundant species was estimated using LDA. Vaginal microbiota of patients prior to PPROM was enriched with Bacteroidales, Fusobacteriales and Clostridiales whereas those with a term delivery were comparatively enriched in Lactobacillales. c Stacked bar charts of relative abundance for each individual sampled highlight the emergence of a high-diversity microbial profile and reduced dominance of the Lactobacillus genus. d Comparison of relative abundance across the four differentially expressed bacterial genera showing reduced Lactobacillales (P = 0.0172) and increased Fusobacteriales (P = 0.0035), Clostridiales (P = 0.0356) and Bacteroidales (P = 0.009, Mann–Whitney U, two-tailed) in women prior to membrane rupture compared to controls. LDA latent discriminatory analysis, LEfSe linear discriminant analysis with effect size, PPROM preterm prelabour rupture of the fetal membranes

Fig. 3

Erythromycin treatment promotes vaginal dysbiosis. a Analysis of vaginal microbiota communities from paired samples before and following 48 hours of erythromycin treatment demonstrates transition from Lactobacillus spp. dominance (n =  = 10, 63%) towards intermediate (n = 4) or complete Lactobacillus spp. depletion (n = 2) whilst communities initially low in Lactobacillus spp. remain so. Erythromycin treatment did not reduce b richness, c diversity or d bacterial load. A sub-analysis showed that erythromycin treatment was associated with decreased e richness (P = 0.03) and f diversity (P = 0.03) in communities initially depleted in Lactobacillus spp., treatment but was associated with a significant increase in diversity (P = 0.01) in communities initially dominated by Lactobacillus spp. g Bacterial load was similar between subgroups. h Lactobacillus spp. abundant communities experienced a 40% reduction in Lactobacillus spp. post treatment (P = 0.004, Wilcoxon signed rank test). Inv. inverse, VM vaginal microbiota

Fig. 4

Vaginal dysbiosis is associated with increased risk of chorioamnionitis with funisitis following PPROM. a Differentially abundant vaginal taxa detected using LEfSe analysis in samples taken prior to delivery in women with normal histology or chorioamnionitis with funisitis. b The vaginal microbiome of patients with normal histology was enriched with Lactobacillus spp. whereas those with chorioamnionitis with funisitis were enriched with Fusobacteriales including Sneathia, Bacteroidales, Peptostreptococcus and Catonella species. c Increased prevalence of Lactobacillus depleted/dysbiotic communities with significantly reduced Lactobacillus abundance was observed in the chorioamnionitis with funisitis cohort (P = 0.0025, Fisher’s exact). d No difference was detected in vaginal microbial richness between groups. However, e diversity was increased in cases complicated by chorioamnionitis with funisitis. f Bacterial load was comparable between histological groups. g, h Serum markers of maternal infection and inflammation were significantly elevated in cases of chorioamnionitis, +/- funisitis (CRP: P = 0.000016, WCC: P = 0.0016). i, j Both WCC (rho = 0.54, P = 0.0001) and CRP (rho = 0.45, P =0.0013) were positively correlated with vaginal bacterial alpha diversity. CRP C-reactive protein, LDA latent discriminatory analysis, LEfSe linear discriminant analysis with effect size; WCC white cell count

Fig. 5

Vaginal dysbiosis is a risk factor for early onset neonatal sepsis (EONS) following PPROM. a Cladogram and b LDA of differentially abundant species detected in vaginal swab samples collected just prior to delivery in cases who did or did not subsequently develop EONS. Lactobacillus crispatus was void from EONS samples, which were enriched with bacteria from genera Sneathia and Catonella. c Distribution of the 21 most abundant bacterial species across the two patient groups. Lactobacillus crispatus was present only in mothers whose babies did not develop EONS. d Relative abundance of differentially expressed genera: Lactobacillus (P = 0.001), Leptotrichiaceae (P = 0.0291) and Lachnospiraceae (P = 0.0114). EONS early onset neonatal sepsis, LDA latent discriminatory analysis, PPROM preterm prelabour rupture of the fetal membranes