Uncultivated bacteria as etiologic agents of intra-amniotic inflammation leading to preterm birth

Abstract

Intra-amniotic infection and inflammation are major causes of preterm birth (PTB). However, intra-amniotic inflammation is often detected in the absence of infection. This may partly be due to the culturing methods employed in hospital laboratories, which are unable to detect the uncultivated species. In this study, intra-amniotic microbial infections associated with PTB were examined by both culture and 16S rRNA-based culture-independent methods and were corroborated by the presence of intra-amniotic inflammation. Amniotic fluid (AF) specimens from 46 pregnancies complicated by PTB and 16 asymptomatic women were analyzed. No bacterial DNA was amplified in AF collected from the asymptomatic women. Among the 46 samples associated with PTB, bacterial DNA was amplified from all (16/16) of the culture-positive samples and 17% (5/30) of the culture-negative samples. In the culture-positive group, additional species were detected in more than half (9/16) of the cases by PCR and clone analysis. Altogether, approximately two- thirds of the species detected by the culture-independent methods were not isolated by culture. They included both uncultivated and difficult-to-cultivate species, such as Fusobacterium nucleatum, Leptotrichia (Sneathia) spp., a Bergeyella sp., a Peptostreptococcus sp., Bacteroides spp., and a species of the order Clostridiales. To examine intra-amniotic inflammation, an AF proteomic fingerprint (mass-restricted score) was determined by surface-enhanced laser desorption ionization-time-of-flight mass spectrometry. Inflammation was detected in all five samples which were culture negative but PCR positive. Women who were PCR positive more often had elevated interleukin-6 levels in their AF, histological chorioamnionitis, and funisitis and delivered neonates with early-onset neonatal sepsis. Previously unrecognized, uncultivated, or difficult-to-cultivate species may play a key role in the initiation of PTB.

FIG. 1.

Flowchart of patients in the study and control groups, results of culture-dependent and culture-independent methods, and pregnancy outcome.

FIG. 2.

PCR examination of AF samples with universal primers A17F and 1512R. The PCR products from representative samples of AF were examined on 1% agarose gels. Lanes + and −, AF samples with positive and negative microbial culture results, respectively; lanes M, DNA size markers; lanes N, negative control consisting of phosphate-buffered saline; lanes F, Fusobacterium nucleatum 12230; lanes K, Klebsiella pneumoniae NCIMB8267; lane E, Eikenella corrodens ATCC 23834.

FIG. 3.

Phylogenic analysis of total 16S rRNA gene sequences amplified from AF samples. The 16S rRNA sequences were aligned by using the ClustalX1.8 program and were edited manually to remove ambiguities. Molecular Evolutionary Genetics Analysis (version 4) software was used to estimate the evolutionary distance (the number of nucleotide substitutions per site). The statistical robustness of the neighbor-joining tree was confirmed by bootstrapping (1,000 replicates). The reference strains are shown in gray. The prevalence of each species in the 21 positive samples is shown in parentheses as a percentage.

FIG. 4.

Biological relevance of intra-amniotic infection as assessed by culture-dependent and culture-independent methods. The glucose (A) and interleukin-6 (B) concentrations in AF samples positive by culture and/or by culture-independent methods are presented as percentiles with medians. The ends of the boxes define the 25th and 75th percentiles, the line inside the box defines the median, and the whiskers show the highest and the lowest values. Comparisons among groups were done by Kruskal-Wallis analysis of variance, followed by post-hoc Dunn's tests. AFC, AF culture; PCR, analysis of AF for 16S rRNA by PCR; Neg, negative result; Pos, positive result; ns, not significant.