Prospective study on the analysis of intestinal microflora changes and prediction on metabolic function in severe burn patients at early stage by 16S ribosomal RNA high-throughput sequencing

Abstract

Objective: To analyze the changes of intestinal microflora and to predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA (rRNA) high-throughput sequencing. Methods: In this prospective observational study, 48 patients with severe burns who met the inclusion criteria were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included in burn group, and 40 healthy volunteers who met the inclusion criteria and underwent physical examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Fecal samples were collected from patients in burn group in about 1 week after admission and from volunteers in healthy group on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the feces of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software to analyze the dominant bacteria. The OTU number, Chao1 index, Ace index, and Shannon index of fecal microflora were analyzed by QIIME1.9.0 software. The principal component analysis for relative abundance of fecal microflora was performed by Canoco Software 5.0. The metabolic function of fecal microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample t test, and Mann-Whitney U test, and Bonferroni correction. Results: The relative abundance of Bacteroides, Enterococcus, Acinetobacter, Macrococcus, and Staphylococcus in feces of patients in burn group was significantly higher than that of volunteers in healthy group (Z=-5.20, -2.37, -5.17, -4.41, -6.03, P<0.05 or P<0.01), and the relative abundance of unclassified-Helicobacillae, Prevotella, Cecobacteria, unclassified-Rumencocci, Pseudobutyrivibrio, Brautia, and unclassified-Digiestive Streptococcaceae (Z=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, P<0.01) and other 12 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group. The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the main dominant microflora of volunteers in healthy group were Bacteroides, unclassified-Helicobacillae, Prevotella, unclassified- Enterobacteriaceae, Brautia, Parabacteroides, Escherichia coli, etc., and the main dominant microflora of patients in burn group were Bacteroides, Prevotella, unclassified-Enterobacteriaceae, and Parabacteroides. The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group (t=10.325, 11.972, 12.224, 11.662, P<0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, P<0.01. The fecal microflora of volunteers in healthy group were more concentrated on principal component 2, the fecal microflora of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, P>0.05. The metabolic levels of alanine-aspartate-glutamate, arginine- proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group (Z=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, P<0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group (Z=-6.07, -4.51, P<0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group (P>0.05). Conclusions: There are significant differences in intestinal microflora between severe burn patients at the early stage and healthy people, and the species and diversity of microflora are decreased, and the nutrient metabolism level is decreased in burn patients by 16S rRNA high-throughput sequencing.