Study subjects
Neonates who hospitalized in the NICU of Hunan Children's Hospital between August 2018 and October 2019 were recruited for the present study. Eligible participants were those who: 1) The admission age was greater than 7 days, and the NBNA assessment was completed when the basic diseases had been recovered and discharge criteria had been met; 2) Vaginal delivery, birth without asphyxia, intrauterine distress, and amniotic fluid pollution; 3) Breast-fed after birth; no vomiting, hematochezia, abdominal distension and other gastrointestinal symptoms within 1 week of admission; 4) Maternal age between 20 and 35; no negative habits such as alcohol and tobacco, no special dietary habits; no pregnancy complications; and no infection within 1 month of delivery.
Nbna Assessment
The NBNA score was evaluated by trained NICU physicians, for full-term infants within 1 month after birth, and for preterm infants within 1 week after 40 weeks of corrected gestational age. The NBNA assessment was made up of five aspects, including behavioral ability, passive muscle tension, active muscle tension, primitive reflex, and general assessment, with a total score of 40 points. The adverse neurodevelopmental outcome was defined as a score of less than 35 points, indicating abnormal neurobehavioral development [8]. The peripheral environment was kept quiet and the temperature was maintained at 22–27°C. All items were conducted in the middle of the second feeding and completed within 10 minutes.
Samples And Information Collection
Fecal samples collected from neonates’ first defecation after hospitalization were immediately frozen in ice boxes and then shipped to the laboratory within 2 hours. Samples were stored in freezers at -80℃until processing. According to the unified questionnaire, maternal pregnancy status and newborn status were collected. The hospitalization data of neonates were extracted from the medical record system.
Dna Extraction And 16s Rdna Gene Sequencing
Bacterial DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit. Isolated bacterial DNA was then used as a template for the amplification of the V3-V4 region of 16S rRNA gene using the 341F/806R (341F:5’-GTGCCAGCMGCCGCGG-3’/ 806R: 5’-GGACTACVVGGGTATCTAATC-3’) primer for polymerasechain reaction (PCR). DNA was then sequenced on the Illumina MiSeq platform.
Bioinformatics Analysis
Raw reads were further filtered according to the following rules to improve the accuracy of later analysis: First, reads containing bases with a terminal quality of less than 20 and sequences with a length of less than 100 bp were removed using Trim Galore software, and the Merge sequences were splined by FLASH2 software. Then, mothur software was used to remove primers, and the usearch was used to remove sequences with a base mismatch ratio greater than 2% and a length less than 100 bp to obtain optimized sequences with high quality and reliability. The filtered sequences were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity.
α diversity was calculated using Shannon index, Chao1 index, and ACE index. Wilcoxon rank-sum test was used to compare the statistical significance of α diversity between two groups. Principal Component Analysis (PCA) based on OTUs abundance table was used to assess microbial composition and structure. The linear discriminant analysis effect size (LEfSe), an algorithm for high-dimensional biomarker discovery, was used to identify bacterial taxa that have significant differences between groups in preterm infants. The Kruskal-Wallis test (for more than two groups) or Wilcoxon rank-sum test (for two groups) was used for comparisons. Taxa that showed an LDA score > 2 and a P < 0.05 were selected. Metastats analysis was used to compare the samples among groups, and the species with significant differences between the two groups at each classification level were obtained, P < 0.05 was used as the difference significance screening threshold.
Statistical Analyses
The continuous variables (gestational age, birth weight, newborn age at the time the specimen was collected) were expressed as mean (standard deviation), and the categorical variables were expressed as counts and percentages. Differences in continuous data were evaluated by Student’s t-test (normally distributed data), while differences in categorical variables were assessed by the χ2 test. All statistical analyses were performed using SPSS version 22.0 (IBM, New York, USA) and R statistical software. The level of significance was set as a 2-sided and P value less than 0.05.