Comparative microbial analysis of paired amniotic fluid and cord blood from pregnancies complicated by preterm birth and early-onset neonatal sepsis

Xiaowei Wang, Catalin S Buhimschi, Stephanie Temoin, Vineet Bhandari, Yiping W Han, Irina A Buhimschi, Xiaowei Wang, Catalin S Buhimschi, Stephanie Temoin, Vineet Bhandari, Yiping W Han, Irina A Buhimschi

Abstract

Background: 16S rRNA-based genomic analyses have revolutionized our understanding of infectious diseases. Many cases which were recognized as "idiopathic" are now known to have an infectious etiology. Here, we present a proof-of-concept study to examine the microbial link between intra-amniotic infection (IAI) and early-onset neonatal sepsis (EONS).

Results: Using culture independent methods, we analyzed paired amniotic fluid (AF) and cord blood (CB) samples from 36 singleton pregnancies complicated by preterm birth (PTB), IAI, and/or EONS. PTB cases were grouped as 1) Group 1- neonatal blood culture-positive EONS (n=6). 2) Group 2- neonatal blood culture-negative presumed EONS with positive IAI (n=16). 3) Group 3- neonatal blood culture-negative presumed EONS with no IAI (n=7); 4) Group 4- no EONS or IAI (n=7). In addition, samples from term healthy deliveries (n=8) served as technical controls. A total of 31 species (15 non-redundant) were identified in AF, of which only 1/3 were cultivated. Significantly fewer microorganisms were detected in CB, with a total of 18 species (7 non-redundant) identified, of which only 2 (Escherichia coli, Streptococcus agalactiae) were cultivated. Of those, Bergeyella, Fusobacterium nucleatum, and Sneathia sanguinegens had not been detected in EONS before. The novel species identified in AF by PCR include Peptoniphilus harei and Lachnospiraceae sp. The majority (72%) of CB species were also detected in the matching AF, with E. coli and F. nucleatum as the most prevalent. The 16S rRNA sequences of paired AF and CB were 99.9-100% identical, while no identical sequences were found between different pregnancies.

Conclusions: Previously unrecognized, uncultivated or difficult-to-cultivate species are implicated in EONS. Microbial species in paired AF and CB likely share the same infectious origin. Given its prevalence in EONS, F. nucleatum should be placed on the same importance scale as E. coli.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Proportion of cases found either…
Figure 1. Proportion of cases found either sterile (open portion of the bar), mono- or poly-microbial (shaded portions of the bars).
From left to right, the 4 compartments are: amniotic fluid (AF) and neonatal blood (NB) analyzed by culture, followed by AF and cord blood (CB) analyzed by the culture-independent methods of PCR and clone analysis.
Figure 2. Euler-Venn diagrams showing the number…
Figure 2. Euler-Venn diagrams showing the number of species shared among compartments of each case (co-occurrences).
Compartments of each case were analyzed by either the culture-dependent (A) or culture-independent methods (B). The size of each space (ellipse) is proportional to the number of species in each compartment. 4-way intersecting diagrams of the 4 compartments showing either non-null spaces that are proportional to the number of species (Euler-like diagram) (C) or the complete non-scaled design which includes spaces with no overlapping species (Venn diagram) (D).

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