This study was conducted to investigate the effects of in-feed encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination on antimicrobial resistance (AMR) phenotypes and genotypes of Escherichia coli isolates recovered from feces of 6-, 16-, 23-, and 27-day-old broiler chickens. The five dietary treatments including the basal diet (negative control [NC]) and the basal diet supplemented with 55 ppm of bacitracin (BAC), 100 ppm of encapsulated CIN, 100 ppm of encapsulated CIT, or 100 ppm each of encapsulated CIN and encapsulated CIT (CIN+CIT). Antimicrobial susceptibility testing of 240 E. coli isolates revealed that the most common resistance was to β-lactams, aminoglycosides, sulfonamides, and tetracycline; however, the prevalence of AMR decreased (P < 0.05) as birds aged. The prevalence of resistance to amoxicillin–clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, and sulfonamide was lower (P < 0.05) in isolates from the CIN or CIN+CIT groups than in isolates from the NC or BAC groups. Whole genome sequencing of 227 of the 240 isolates revealed 26 AMR genes and 19 plasmids, but the prevalence of some AMR genes and the number of plasmids were lower (P < 0.05) in E. coli isolated from CIN or CIN+CIT birds than in isolates from NC or BAC birds. The most prevalent resistance genes were tet(A) (108 isolates), aac(3)-VIa (91 isolates), aadA1 (86 isolates), blaCMY-2 (78 isolates), sul1 (77 isolates), aph(3)-Ib (58 isolates), aph(6)-Id (58 isolates), and sul2 (24 isolates). The numbers of most virulence genes carried by isolates increased (P < 0.05) in chickens from 6 to 27 days of age. The prevalence of E. coli O21:H16 isolates was lower (P < 0.05) in CIN and CIN+CIT, and the colibacillosis-associated multilocus sequence type (ST117) was most prevalent in isolates from 23-day-old chickens. A phylogenetic tree of whole genome sequences revealed a close relationship between 25 of the 227 isolates and human or broiler extraintestinal pathogenic E. coli strains. These findings indicate that AMR and virulence genotypes of E. coli could be modulated by providing encapsulated CIN or CIN+CIT feed supplements, but further investigation is needed to determine the mechanisms of the effects of these supplements.

  • AMR genes were less prevalent in E. coli isolates from birds fed CIN or CIN+CIT.

  • AMR gene prevalence and plasmids numbers were low in E. coli from CIN and CIN+CIT birds.

  • AMR of E. coli isolates decreased as birds aged.

  • The distribution of E. coli virulence genes was affected by CIN, CIN+CIT, and bird age.

  • Some E. coli isolates had high potential for virulence in humans.

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