Although the presence of live microbes in utero remains under debate, newborn gastrointestinal bacteria are undoubtedly important to infant health. Measuring bacteria in meconium is an ideal strategy to understand this issue; however, the low efficiency of bacterial DNA extraction from meconium has limited its utilization. This study aims to improve the efficiency of bacterial DNA extraction from meconium, which generally has low levels of microflora but high levels of PCR inhibitors in the viscous matrix. The research was approved by the ethical committee of the Xiamen Maternity and Child Health Care Hospital, Xiamen, China. All the mothers delivered naturally, and their newborns were healthy. Meconium samples passed by the newborns within 24 h were collected. Each sample was scraped off of a sterile diaper, transferred to a 5-ml sterile tube, and stored at −80°C. For the assay, a freeze-thawing sample preparation protocol was designed, in which a meconium-InhibitEX buffer mixture was intentionally frozen 1–3 times at −20°C, −80°C, and (or) in liquid nitrogen. Then, DNA was extracted using a commercial kit and sequenced by 16S rDNA to verify the enhanced bacterial DNA extraction efficiency. Ultimately, we observed the following: (1) About 30 mg lyophilized meconium was the optimal amount for DNA extraction. (2) Freezing treatment for 6 h improved DNA extraction at −20°C. (3) DNA extraction efficiency was significantly higher with the immediate thaw strategy than with gradient thawing at −20°C, −80°C, and in liquid nitrogen. (4) Among the conditions of −20°C, −80°C, and liquid nitrogen, −20°C was the best freezing condition for both improving DNA extraction efficiency and preserving microbial species diversity in meconium, while liquid nitrogen was the worst condition. (5) Three freeze-thaw cycles could markedly enhance DNA extraction efficiency and preserve the species diversity of meconium microflora. We developed a feasible freeze-thaw pretreatment protocol to improve the extraction of microbial DNA from meconium, which may be beneficial for newborn bacterial colonization studies.

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BackgroundCaesarean section (CS) is associated with newborns’ health risks due to the blocking of microbiome transfer. The gut microbiota of CS-born babies was different from those born vaginally, which may be attributed to reduced exposure to maternal vaginal microbes during labour. To understand the microbial transfer and reduce CS disadvantages, the effect of vaginal microbiota exposure on infant gut microbiota composition was evaluated using 16s rDNA sequencing-based techniques.ResultsPregnant women were recruited in the Women and Children’s Hospital, School of Medicine, Xiamen University from June 1st to August 15th, 2017. Maternal faeces (n = 26), maternal vaginal fluids (n = 26), and neonatal transitional stools (n = 26) were collected, while the participants underwent natural delivery (ND) (n = 6), CS (n = 4) and CS with the intervention of vaginal seedings (I) (n = 16). 26 mothers with the median age 26.50 (25.00-27.25) years showed no substantial clinical differences. The newborns’ gut microbiota altered among ND, CS and I, and clustered into two groups (PERMANOVA P = 0.001). Microbial composition of ND babies shared more features with maternal vaginal samples (PERMANOVA P = 0.065), while the microbiota structure of ND babies was obviously different from that of sample of maternal faeces. The genus Bacteroides in CS-born babies with intervention approached to vaginal-born neonates, compared with CS-born neonates without intervention.ConclusionsNeonatal gut microbiota was dependent on the delivery mode. And the gut microbiota CS newborns with vaginal seeding shared more features with those of ND babies, which hinted the aberrant gut microbiota composition initiated by CS might be partly mitigated by maternal vaginal microbiota exposure.