Comprehensive analysis of chromosomal and plasmid-borne AMR determinants in Campylobacter coli Italian isolates from broilers

Abstract Campylobacter coli is a key zoonotic pathogen and, owing to its close phylogenetic relationship and shared ecological niches with Campylobacter jejuni , an important reservoir of antimicrobial-resistance (AMR) determinants that can be horizontally transferred between these two species. Here, we provide a genomic overview of Italian C. coli isolates collected from broilers during the 2022 EU harmonised monitoring program, with a specific focus on the contribution of plasmids and other mobile genetic elements to the maintenance and spread of acquired AMR. A total of 52 isolates were analysed with Illumina short-read whole-genome shotgun sequencing and in silico genomic characterization, including cgMLST, screening for acquired AMR and virulence genes and resistance-associated mutations. Eight isolates were additionally sequenced with Oxford Nanopore long reads and reconstructed via hybrid assembly (Unicycler v0.4.9 and v0.5.1; Plassembler v1.6.2) to resolve chromosomal and plasmid structures and to obtain a complete genomic characterization. Core-genome analysis revealed a highly clonal population structure, with cgMLST enabling discrimination into closely related clusters that largely shared AMR genotypic profiles. AMR determinants were widespread, including high prevalence of tet O (92%, 48/52), bla OXA genes (71%, 37/52), and the fluoroquinolone-associated gyr A T86I substitution (88%, 46/52). Hybrid assemblies showed that, despite the canonical association of acquired AMR with plasmids, most resistance determinants were located on the chromosome, usually in contexts suggestive of transformation or transposition (e.g., IS-associated arrangements), while plasmid-borne ARGs were unequivocally identified only in a subset of isolates. Overall, our findings support the role of C. coli as a reservoir of clinically relevant AMR and virulence-related genomic determinants, both in the chromosome and plasmids, and highlight the added value of hybrid sequencing for accurately reconstructing complete genomic structures enabling comprehensive characterization of these genomes. Furthermore, a custom cgMLST scheme has been created as a highly sensitive and discriminatory tool for comparison of genomically homogeneous Campylobacter populations.