| Summary: | The study was designed to investigate the fecal microbiome and resistome of broiler chickens infected with multidrug-resistant (MDR) <i>Escherichia coli (E. coli)</i>. Fecal samples (<i>n</i> = 410) from broiler chickens were collected from thirteen randomly selected sites of Khyber Pakhtunkhwa and screened for the presence of MDR <i>E. coli</i>. Upon initial screening, thirteen (13) MDR <i>E. coli </i>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 <i>iro</i>N, <i>iut</i>A,<i> iss</i>, and <i>iuc</i>A. 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 <i>E. coli</i>, <i>Salmonella enterica</i>,<i> Klebsiella pneumoniae</i>, and <i>Shigella flexneri</i> 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 <i>E. coli</i> of Khyber Pakhtunkhwa for the first time using NGS.
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