Comparative pangenomics of the Corynebacterium kroppenstedtii complex reveals species structure and accessory genome diversity.

BACKGROUND: The Corynebacterium kroppenstedtii complex (CKC) is increasingly recovered from mastitis, but its species boundaries and genomic diversity remain poorly resolved. METHODS: Following quality screening, we analysed 56 publicly available genomes of C. kroppenstedtii (Ck), C. parakroppenstedtii (Cpak), and C. pseudokroppenstedtii (Cpse). Taxonomic assignments were supported by average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), average amino acid identity (AAI)-derived protein similarity, and a core-genome-based phylogeny. We then examined pangenome structure, mobile elements, resistance genes, functional enrichment, and conserved gene neighbourhoods. RESULTS: ANI and core-gene phylogeny resolved Ck, Cpak, and Cpse as three species. The 4,706-cluster pangenome was open (fitted exponent = 0.232) and comprised 1,751 core and 2,955 accessory clusters. Accessory-gene profiles further resolved three Cpak lineages, Cpak-P1 to Cpak-P3, concordant with the core-genome phylogeny. Insertion-sequence counts correlated with accessory cluster counts (Spearman's ρ = 0.640, p = 1.12 × 10-7), and accessory clusters more frequently overlapped predicted mobile genetic elements (MGEs) than core clusters (402/2,955 versus 98/1,751; odds ratio = 2.66, p = 3.31 × 10-19), supporting an MGE-accessory association without implying causality. Seven ARG types were detected, most commonly ermX, cmx, APH(6)-Id, and tet(W). AC410-like sequences occurred in 35/56 genomes and were strongly associated with antimicrobial-resistance gene (ARG) carriage (34/35 versus 4/21; odds ratio = 120.5, p = 9.93 × 10-10). In five of eight complete genomes, AC410-like sequences were located on the chromosome rather than on independent plasmid contigs. Functional enrichment and conserved gene neighbourhoods identified a candidate lipid-iron locus shared by Cpak-P1 and Cpak-P2. CONCLUSION: These findings provide a lineage-resolved genomic framework for the CKC, demonstrating an open pangenome with substantial species- and lineage-associated accessory diversity. The results further identify AC410-like resistance-associated genomic backgrounds and a candidate lipid-iron locus for future functional investigation.