Next-Generation Sequencing Based Gut Resistome Profiling of Broiler Chickens Infected with Multidrug-Resistant Escherichia coli

Abstract

The study was designed to investigate the fecal microbiome and resistome of broiler chickens infected with multidrug-resistant (MDR) Escherichia coli (E. coli). Fecal samples (n = 410) from broiler chickens were collected from thirteen randomly selected sites of Khyber Pakhtunkhwa and screened for the presence of MDR E. coli. Upon initial screening, thirteen (13) MDR E. coli isolates were then subjected to shotgun metagenome next-generation sequencing (NGS). NGS based resistome analysis identified the multidrug efflux pump system-related genes at the highest prevalence (36%) followed by aminoglycoside (26.1%), tetracycline (15.9%), macrolide-lincosamide-streptogramin (9.6%), beta-lactam (6.6%), rifampin (2%), sulphonamide (1.3%), phenicol (0.91%), vancomycin (0.62%), trimethoprim (0.34%), colistin (0.30%), and quinolone (0.33%). The most abundant virulence-associated genes (VAGs) identified were iroN, iutA, iss, and iucA. NGS based taxonomic profiling at the phylum level revealed the predominance of Proteobacteria (38.9%) followed by Firmicutes (36.4%), Bacteroidetes (15.8%), and Tenericutes (8.9%). Furthermore, pathobionts such as E. coli, Salmonella enterica, Klebsiella pneumoniae, and Shigella flexneri belonging to the family Enterobacteriaceae were predominantly found. This study revealed the widespread presence of MDR genes, diverse VAGs, and a dysbiotic gut in the broiler chickens infected with MDR E. coli of Khyber Pakhtunkhwa for the first time using NGS.

Keywords: antibiotic resistance genes; broiler chickens; dysbiosis; fecal microbiota; multidrug resistance; shotgun metagenome sequencing.

Figure 1

Antimicrobial resistance pattern of poultry fecal microbiota to various drugs. Multidrug resistance and macrolide-lincosamide-streptogramin are abbreviated as MDR and MLS, respectively.

Figure 2

Taxonomic profiling of poultry fecal microbiota at various levels using Pavian derived from next-generation shotgun metagenomics. Different colored sidebars in the Sankey diagram show the relative abundance of bacterial communities in broiler chickens at various taxonomic levels including kingdom (K), phylum (P), class (C), order (O), family (F), and genus (G).

Figure 3

Heatmap showing the fecal microbiome profiling at the species level. All the bacterial species identified in the poultry fecal microbiome are hierarchically clustered using the Bray–Curtis dissimilarity matrix. P1 to P13 represents the sequenced shotgun metagenomic poultry samples (n = 13). The relative abundance of various bacterial species is indicated in logarithmic values (base 10).

Figure 4

The relative abundance of various virulence-associated genes (VAGS).