Comparative Microbial Analysis of Paired Amniotic Fluid and Cord Blood from Pregnancies Complicated by Preterm Birth and Early-Onset Neonatal Sepsis Xiaowei Wang 1 , Catalin S. Buhimschi 2 , Stephanie Temoin 1 , Vineet Bhandari 2,3 , Yiping W. Han 1,4,5 *, Irina A. Buhimschi 2 1 Department of Periodontics, Case Western Reserve University, Cleveland, Ohio, United States of America, 2 Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University, New Haven, Connecticut, United States of America, 3 Department of Pediatrics, Yale University, New Haven, Connecticut, United States of America, 4 Department of Pathology, Case Western Reserve University, Cleveland, Ohio, United States of America, 5 Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio, United States of America 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. Citation: Wang X, Buhimschi CS, Temoin S, Bhandari V, Han YW, et al. (2013) Comparative Microbial Analysis of Paired Amniotic Fluid and Cord Blood from Pregnancies Complicated by Preterm Birth and Early-Onset Neonatal Sepsis. PLoS ONE 8(2): e56131. doi:10.1371/journal.pone.0056131 Editor: Adam J. Ratner, Columbia University, United States of America Received September 21, 2012; Accepted January 5, 2013; Published February 20, 2013 Copyright: ß 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants RO1DE014924 (YWH), RO1HD047321 (IAB), and RO1HD062007-01A1 (CSB and IAB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yiping.han@case.edu Introduction Studies of microbes have traditionally focused on bacterial species isolated in culture. Because a large proportion of bacteria are uncultivated, traditional microbiological cultures provide an incomplete picture of the human microbiome [1]. Metagenomic techniques and sequencing technology have revolutionized our understanding of the bacterial diversity [2]. Sequencing of the small-subunit ribosomal RNA (16S rRNA) gene, which is highly conserved among all prokaryotes, yet is variable enough to allow differentiation of species [3], [4], has made it possible to identify microbial species that cannot be grown in culture or recognized based on phenotypic properties alone [5]. We were among the first to employ metagenomic approaches to evaluate amniotic fluid (AF) microbial diversity in pregnancies complicated by preterm birth (PTB) [6], [7]. Applications of 16S rRNA-gene sequencing suggested the existence of a far greater microbial diversity in AF than appreciated based on culture- dependent methods. In pregnancies where there was evidence of an inflammatory host response, approximately two thirds of microbes detected by culture-independent methods were not isolated by cultures. These included both uncultivated and difficult-to-cultivate species, such as Fusobacterium nucleatum, Lepto- trichia (Sneathia), Bergeyella, Peptostreptococcus, Bacteroides and a species of the order Clostridiales. Interestingly, women with positive microbial cultures and/or PCR results often delivered neonates with early-onset neonatal sepsis (EONS) [7]. EONS (sepsis in the first 3 days of life) remains a major cause of neonatal morbidity and mortality [8]. Based on microbial cultures PLOS ONE | www.plosone.org 1 February 2013 | Volume 8 | Issue 2 | e56131