Benchmark taxonomic classification of chicken gut bacteria based on 16S rRNA gene profiling in correlation with various feeding strategies

Abstract

Taxonomic characterization of bacterial 16S rRNA gene has become an accepted norm for inference of complex microbial communities and ecological shifts in gut. Recently, 16S rRNA gene sequences has emerged as a taxonomic marker to investigate crosstalk between gut bacteria and host via variations in feed. Here, broiler chickens (n = 240) were subjected to dietary modulation approach to substitute antibiotics with natural plant-derived products. Five experimental groups were control, antibiotic growth promoter, organic acids, phytogenic feed additives and combination (organic acids + phytogenic feed additives). Bacterial DNA was extracted and 16S rRNA gene was amplified through polymerase chain reaction using universal primers followed by observation through gel electrophoresis and then, confirmation by direct DNA sequencing. The sequence data was analyzed for homology search, multiple sequence alignment, and construction of phylogenetic clades using software BLASTn, CLUSTAL W and MEGA 7.0. respectively. Based on the phenetic relationship, bacterial phylotypes from organic acid and phytogenic feed additive groups belongs to Enterococcus faecium and Enterococcus faecalis in both cecum and ileum. The bacterial isolate obtained from antibiotic feeding group characterized as Escherichia coli. Furthermore, sequences of probiotic bacteria (Enterococcus faecalis) were retrieved from online repositories (GenBank and RDP) and aligned with indigenously isolated strains of this study. Comparative phylogenetic analysis revealed high sequence similarities (99%) of Enterococcus faecalis KIBGE AB-6 and Enterococcus faecalis KIBGE AB5 with Enterococcus faecalis ATCC 19433, and Enterococcus faecalis DD28 respectively. Hence, under the appropriate conditions of current investigation, these bacterial strains might serve as potential candidates for probiotics in animals. To the best of our knowledge, this is the first study in Pakistan depicting taxonomic classification of chicken gut bacterial diversity from cecal and ileal samples linked with dietary modulation.