Meconium microbiome analysis identifies bacteria correlated with premature birth

Abstract

Background: Preterm birth is the second leading cause of death in children under the age of five years worldwide, but the etiology of many cases remains enigmatic. The dogma that the fetus resides in a sterile environment is being challenged by recent findings and the question has arisen whether microbes that colonize the fetus may be related to preterm birth. It has been posited that meconium reflects the in-utero microbial environment. In this study, correlations between fetal intestinal bacteria from meconium and gestational age were examined in order to suggest underlying mechanisms that may contribute to preterm birth.

Methods: Meconium from 52 infants ranging in gestational age from 23 to 41 weeks was collected, the DNA extracted, and 16S rRNA analysis performed. Resulting taxa of microbes were correlated to clinical variables and also compared to previous studies of amniotic fluid and other human microbiome niches.

Findings: Increased detection of bacterial 16S rRNA in meconium of infants of <33 weeks gestational age was observed. Approximately 61·1% of reads sequenced were classified to genera that have been reported in amniotic fluid. Gestational age had the largest influence on microbial community structure (R = 0·161; p = 0·029), while mode of delivery (C-section versus vaginal delivery) had an effect as well (R = 0·100; p = 0·044). Enterobacter, Enterococcus, Lactobacillus, Photorhabdus, and Tannerella, were negatively correlated with gestational age and have been reported to incite inflammatory responses, suggesting a causative role in premature birth.

Interpretation: This provides the first evidence to support the hypothesis that the fetal intestinal microbiome derived from swallowed amniotic fluid may be involved in the inflammatory response that leads to premature birth.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflict: Dr. Josef Neu is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Gestational age and bacterial colonization of meconium.

(A) A larger percentage of meconium samples from infants 33 weeks gestational age (52·9%; N = 17). (B) The bacterial composition of meconium is dominated by few genera; on average the most abundant genera in any given sample comprised 57·3±22·5% of reads; 33 weeks is displayed by the black, dashed line. (C)Dominant genera contribute to low diversity, measured by Shannon index; this is indicative of a founding population. Furthermore, gestational age was not correlated with Shannon diversity index (Spearman: rho = 0·03, p = 0·85).

Figure 2. Meconium microbiome is most suggestive of amniotic fluid origin.

(A) The average percent relative abundance in meconium samples of this study for genera reported in amniotic fluid, and the oral and vaginal cavities of pregnant women6,7,27 are displayed by the Venn diagram which distinguishes unique and shared maternal environments of genera. (B) The potential total mean contribution and standard deviation of any particular maternal locale (amniotic fluid5,6, oral21, or vaginal21), and the phyletic distribution of contributing genera is shown in the stacked bar plot. The color assignment is as follows: Actinobacteria  =  purple; Bacteroidetes  =  green; Firmicutes  =  blue; Fusobacteria  =  orange; Proteobacteria  =  red; Tenericutes  =  aquamarine.

Figure 3. Inflammatory marker S100A12 was correlated with gestational age.

(A) Non-metric multidimensional scaling ordination plot depicting the relatedness of the bacterial communities from all meconium samples; communities from >33 week infants (blue) clustered more closely than those from 33 weeks) had the largest effect on meconium microbial structure (R = 0·16; p-value = 0·03). (B) Of the four predominant phyla, the relative abundance of Firmicutes and Actinobacteria was correlated with low gestational age (**p