Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter coli Isolated from the Cecal Contents of Cattle in the United States

Genomic analyses were performed on florfenicol-resistant (FFN(r)) Campylobacter coli isolates recovered from cattle, and the cfr(C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFN(r) C. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr(C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr(C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFN(r) isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3′)-III, hph, ΔaadE (truncated), bla(OXA-61), cfr(C), and tet(O) genes plus a mutation of GyrA (T86I). The cfr(C), aph (3′)-III, hph, ΔaadE, and tet(O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50 kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2, virB4, virB5, VirB6, virB7, virB8, virb9, virB10, virB11, and virD4. The cfr(C) gene conferred resistance to florfenicol (8 to 32 μg/ml), clindamycin (512 to 1,024 μg/ml), linezolid (128 to 512 μg/ml), and tiamulin (1,024 μg/ml). Phylogenetic analysis showed SNP differences ranging from 11 to 2,248 SNPs among the 16 isolates. The results showed that the cfr(C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies. IMPORTANCE Campylobacter is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFN(r)) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [cfr(C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr(C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.