First-Pass Meconium Samples from Healthy Term Vaginally-Delivered Neonates: An Analysis of the Microbiota

Abstract

BackgroundConsiderable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility.MethodsHealthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples.ResultsThe mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2 - 41.8 x 104 cells/g with a mean of 15.4 x 104 cells/g. (The limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab.ConclusionThis study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.

Bronchopulmonary dysplasia: A review of pathogenesis and pathophysiology

“…Contrary to the traditional belief that the human neonatal respiratory tract is sterile at birth, current studies indicate that the human respiratory tract microbial colonization begins in utero 100,101 or shortly after birth 102,103 . A recent study demonstrated that the lung microbiome, including the relative abundance of bacterial phyla and diversity index of the preterm and term neonates were similar at birth irrespective of the gestational age and the mode of delivery.…”

Gut Microbiota Composition in Healthy Japanese Infants and Young Adults Born by C-Section

“…In our quantitative investigation of the gut microbiota composition in Japanese infants, we detected several bacterial clades, albeit with varying detection rates and numbers, in the firstpass meconium of 95% infants [17], which suggests that several bacterial communities inhabit the intrauterine environment and may provide the pioneer inoculum for gut microbiota colonization. Notably, previous studies on fullterm babies reported the detection of bacteria in about 65-75% of total babies studied [18,19]. This difference may be ascribed to differences in the detection sensitivity of the methods used and hence support the advantage of the high sensitivity of our real-time quantitative (RT-qPCR)-based approach.…”

Sensitive Quantitative Analysis of the Meconium Bacterial Microbiota in Healthy Term Infants Born Vaginally or by Cesarean Section

“…We detected bacteria in the meconium of 95% babies while rest 5% samples appeared to be sterile (i.e., no bacteria detected). However, previous studies on term infants reported the presence of bacteria in 66% (using 16S rRNA gene-targeted FISH method) (Hansen et al, 2015) and 77% (using 16S/23S rRNA gene-targeted qPCR) (Martin et al, 2016) of total babies studied. This difference can most possibly be ascribed to differences in the detection sensitivities of the methods used, and hence could corroborate the advantage of high sensitivity of our RT-qPCR assays.…”

“…However, some recent studies have elegantly reported the presence of bacteria in placenta (Aagaard et al, 2014; Collado et al, 2016), amniotic fluid (Oh et al, 2010; Collado et al, 2016), and umbilical cord blood (Jimenez et al, 2005), thereby manifesting that the fetus is already exposed to a variety of bacteria in utero . Several studies have also detected various bacteria in the meconium of healthy babies (Gosalbes et al, 2013; Hu et al, 2013; Del Chierico et al, 2015; Hansen et al, 2015; Collado et al, 2016), further evidencing that the gut microbial colonization may already begin before birth. However, quantitative data on the types and particularly the population levels (i.e., the absolute count) of bacteria that may be present in the meconium is still sparse.…”