First-pass meconium samples from healthy term vaginally-delivered neonates: an analysis of the microbiota

Background Considerable 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. Methods Healthy 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. Results The 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. Conclusion This 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