Microbial prevalence, diversity and abundance in amniotic fluid during preterm labor: a molecular and culture-based investigation

Abstract

Background: Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking.

Methods and findings: In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r(2) = 0.42; P<0.002).

Conclusions: The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship.

Figure 1. Gestational Outcomes and Amniotic Fluid Findings.

Panel A shows outcomes of enrolled subjects. Panel B summarizes results of culture and PCR analysis of amniotic fluid. PCR refers to end-point PCR for bacteria, fungi and archaea. Culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi. Circle areas are not to scale.

Figure 2. Microbial Diversity.

Phylogeny of the 17 bacterial taxa identified in this study, based on a maximum likelihood algorithm. Colored boxes indicate the number of subjects who were positive for a given taxon by culture (gray) or PCR (blue) (some samples were polymicrobial). For most individual taxa, the larger of the two numbers in the corresponding gray or blue box represents the total number of positive subjects; for taxa where neither method detected all positive subjects, the total number is shown in the white box. A 99% sequence similarity cutoff threshold was used for phylotype assignment, which was based on 621 unambiguous nucleotide positions. Bergeyella sp. (bracketed and in gray type) is included as a reference species only and was not detected in the study population. A single fungal species, Candida albicans, was detected by culture in 1 subject and by PCR in 2 (data not shown). GenBank accession numbers of bacterial and fungal rDNA sequences from this study appear in Supporting Table S2.

Figure 3. Correlation of PCR and Culture Results with Markers of Infection in Amniotic Fluid.

Amniotic fluid concentrations of white blood cells (WBCs) (Panel A) and interleukin-6 (IL-6) (Panel B) as a function of PCR and culture results. Each circle represents one subject; each horizontal bar and adjacent number indicates the median value for that group. P values were calculated by means of the Mann-Whitney U test. PCR refers to end-point PCR for bacteria, fungi and archaea. Culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi.

Figure 4. Gestational Outcomes According to Results of Culture and PCR.

Panel A shows the proportion of subjects from each gestational age cohort who were positive by culture or PCR. The total of number of subjects in each cohort appears across the top of the graph, and the number of positive subjects appears above each bar. Panel B is a Kaplan-Meier analysis of amniocentesis-to-delivery interval according to culture and PCR results. Subjects in whom labor was augmented were censored and are represented by crosses. P values were calculated by means of the Mantel-Haenszel log-rank test. The inset table shows the median interval for each group. For both panels, PCR results refer to end-point PCR for bacteria, fungi and archaea, and culture refers to the use of routine cultivation methods for bacteria (aerobic, anaerobic and genital mycoplasmas) and fungi.

Figure 5. Gestational Age at Delivery as a Function of Bacterial rDNA Concentration.

Data are for samples yielding detectable results within the sensitivity and dynamic range of the real-time PCR assay (∼250–1×10⁸ genes/µl amniotic fluid).