[Analysis of the dynamic changes in gut microbiota in patients with extremely severe burns by 16S ribosomal RNA high-throughput sequencing technology]

Abstract

Objective: To analyze the dynamic change rule of gut microbiota in patients with extremely severe burns using 16S ribosomal RNA (rRNA) high-throughput sequencing technology. Methods: Five patients with extremely severe burns who were admitted to Hwa Mei Hospital of University of Chinese Academy of Sciences from February to June 2017 and conformed to the inclusion criteria were included in the prospective observational study. All patients were males with age of 32-48 years. Fecal samples were collected in the shock stage (within 3 days after injury), early stage of acute infection (4-14 d after injury), middle stage of acute infection (15-28 d after injury), late stage of acute infection (from 29 d after injury to 1 week before discharge) and within 1 week before discharge. The number of samples was 5 in each stage. The fecal pH value was measured using a pH meter. High-throughput sequencing technology was applied for sequencing of 16S rRNA V3 and V4 regions of fecal samples. QIIME software was used to analyze the number of operational taxonomic units (OTUs), α diversity (Chao1 index and Shannon index), and the relative abundance of gut microbiota at the phylum and family levels. Unweighted pair group method with arithmetic mean clustering method was used to analyze the β diversity of gut microbiota, and Tax4Fun was used to predict functional changes of gut microbiota. Data were statistically analyzed with one-way analysis of variance for repeated measurement, Bonferroni method, Wilcoxon rank sum test for paired samples, and Bonferroni correction. Results: (1) The pH value of feces in the early and middle stages of acute infection in patients with extremely severe burns in this group was 7.40±0.45 and 7.56±0.45 respectively, which were significantly higher than 6.68±0.36 in the shock stage (P<0.05 or P<0.01). (2) A total of 2 333 584 efficient and high-quality sequences were obtained, and the length of the sequences was about 415 bp. A total of 1 209 OTUs were obtained. The sequencing coverage of all samples was over 99.0%. The number of OTUs and Chao1 index in the early, middle, and late stages of acute infection in patients with extremely severe burns in this group were significantly lower than those in the shock stage (Z=2.023, P<0.05). The number of OTUs and Chao1 index within 1 week before discharge were significantly higher than those in the early, middle, and late stages of acute infection, and Shannon index within 1 week before discharge was significantly higher than that in the early and middle stages of acute infection (Z=2.023, P<0.05). (3) The structure of gut microbiota in the shock stage in patients with extremely severe burns in this group was highly similar to that within 1 week before discharge, and lowly similar to that in the early, middle, and late stages of acute infection. The analysis of individual sample showed that the clustering rule of most of the samples was in accordance with that of the staged samples. The weighted Unifrac distance of gut microbiota in the shock stage was significantly shorter than that in the early, middle, and late stages of acute infection (Z=3.326, 2.570, 2.690, P<0.05 or P<0.01), while the weighted Unifrac distance of gut microbiota in the other stages was similar. (4) At the phylum level, compared with that in the shock stage, the relative abundance of Firmicutes was decreased in the early, middle, and late stages of acute infection, while the relative abundance of Bacteroidetes and Proteobacteria increased. However, the relative abundance of the above three phyla within 1 week before discharge was similar to that in the shock stage. At the family level, the top five dominant bacteria in relative abundance in different stages after injury were quite different. The relative abundance of dominant five family bacteria in the shock stage was decreased in the early, middle, and late stages of acute infection. The relative abundance of non-dominant bacteria such as Enterobacteriaceae, Streptococcaceae, and Bacteroidaceae in the shock stage increased significantly in the early, middle, and late stages of acute infection, which became new dominant families in these stages. The relative abundance of some acid-producing bacteria within 1 week before discharge resumed to the similar level in the shock stage. (5) Functions such as some amino acid metabolism, glycolysis and gluconeogenesis, and pyruvate metabolism of gut microbiota were obviously weaker in the early and middle stages of acute infection than those in the shock stage. Functions such as some amino acid metabolism and carbohydrate metabolism of gut microbiota were significantly enhanced in the late stage of acute infection compared with that in the shock stage. The distributions of functional genes in gut microbiota were similar between the shock stage and within 1 week before discharge. Conclusions: The internal environment and gut microbial compositions in extremely severe burned patients change significantly in the early and middle stages of acute infection. The pH value increases, the bacterial species and diversity decrease, especially the relative abundance of acid-produced bacteria is significantly reduced, which gradually recover with the improvement of the patient's condition. The pH value and the changes of Proteobacteria and acid-producing bacteria could be considered as suitable parameters for reflecting the disorder level of gut microbiota in patients with extremely severe burns.

Keywords: 16S ribosomal RNA; Burns; Gut microbiota; High throughput sequencing; Short chain fatty acid; Structural disorder; pH value.