No abstract

No abstract

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.

A novel bacterial strain, designated HSG9, was isolated from aisolated from a rotten tropical mangrove root. Cells of strain HSG9 were aerobic, Gram-stain-negative, yellow, oxidase-negative and catalase-positive. Growth was observed in 0.5-9 % sea salt (optimum 3 %, w/v), at 10-42 °C (optimum 25-35 °C) and at pH 6.0-8.0 (optimum 7.0-8.0). Gelatin, esterase and Tweens 20, 40, 60 and 80 were hydrolysed, but starch, protein, cellulose and casein were not. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain HSG9 formed an independent lineage related to the family Flavobacteriaceae. The dominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 1 G. The respiratory quinone was identified as MK-6 and the polar lipids were phosphatidylethanolamine, three unidentified phospholipids and an unidentified lipid. The DNA G+C content was 37.1 mol%. The combined genotypic and phenotypic data indicated that strain HSG9 represents a novel species of a new genus within the family Flavobacteriaceae, for which the name Croceivirgaradicis gen. nov., sp. nov. is proposed. The type strain is HSG9 (=MCCC 1A06690=KCTC 52589).

No abstract