Antimicrobial Resistance Phenotypes and Genotypes of Cecal Digesta Escherichia coli of Pullets Fed Omega-3 Fatty Acids or Yeast Bioactives (Glycobolites)

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Antimicrobial resistance (AMR) is a significant threat to poultry production and food safety. In laying hens, commensal Escherichia coli can serve as a reservoir of AMR and virulence genes. Although omega-3 fatty acids (N-3 FA), yeast bioactives (YB), and spacing allowance (SA) influence gut health and immunity, their combined effects on AMR profiles of gut bacteria remain unclear. A total of 2,832 chicks were raised in enriched cages under high (HSA, 348 cm 2 /bird) or low (LSA, 284 cm 2 /bird) SA and fed a control diet (C), C+3% N-3 FA, or C+0.05% YB. At 4, 16, and 35 weeks of age (woa), cecal contents were cultured on ChromoCult agar to isolate E. coli. Susceptibilities of isolates to 14 antibiotics were determined. Of the 428 isolates, 35.7% were resistant to at least one antimicrobial, and overall AMR prevalence decreased with age ( P < 0.05 ). At each of 4 and 16 woa, N-3 FA-fed birds showed the lowest ampicillin resistance ( P < 0.05 ). Streptomycin resistance was higher in N-3 FA than in YB-fed birds at 16 woa ( P = 0.02 ) but lower than in control-fed birds. Whole-genome sequencing and analysis of 237 selected isolates identified 19 antimicrobial resistance genes (ARGs) and 29 plasmids, with 15 (78.9%) ARGs and 19 (65.5%) plasmid replicons affected by either diet, SA, or age ( P < 0.05 ). Several virulence genes were identified in sequenced E. coli isolates, with those encoding fimbriae, pili, protectin, and toxins being higher in younger pullets ( P < 0.05 ). Overall, isolates from phylogroups A and B1 were predominant; however, at 4 woa, phylogroup D isolates were most prevalent, depending on diet and SA ( P < 0.05 ). Isolates of serotype O23:H16, ST2 were the most prevalent. Of the 237 sequenced isolates, 13 were related to human ExPEC strains. Overall, these findings suggest that N-3 FA YB and SA modulate AMR and virulence genotypes in laying hens, highlighting their potential in mitigating AMR in the poultry production system.