Phylogenetic Association and Genetic Factors in Cold Stress Tolerance in Campylobacter jejuni

Campylobacter jejuni is a major foodborne pathogen transmitted to humans primarily via contaminated poultry meat. Since poultry meat is generally processed, distributed, and stored in the cold chain, the survival of C. jejuni at refrigeration temperatures crucially affects human exposure to C. jejuni. Here, we investigated genetic factors associated with cold stress tolerance in C. jejuni. Seventy-nine C. jejuni strains isolated from retail raw chicken exhibited different survival levels at 4°C for 21 days. Multilocus sequence typing (MLST) clonal complex 21 (CC-21) and CC-443 were dominant among cold stress-tolerant strains, whereas CC-45 was common among cold stress-sensitive strains. Genome-wide average nucleotide identity (ANI) analysis identified a phylogenetic cluster associated with cold stress tolerance. Moreover, a pangenome analysis revealed 58 genes distinctively present in the cold stress-tolerant phylogenetic cluster. Among these 58 genes, cfrA, encoding the ferric enterobactin receptor involved in ion transport and metabolism, was selected for further analysis. Remarkably, the viability of a ∆cfrA mutant at 4°C was significantly decreased, while the levels of total reactive oxygen species and intracellular iron exceeded those of the wild type. Additionally, a knockout mutation of cfrA also significantly decreased the viability of three cold stress-tolerant isolates at 4°C, confirming the role of cfrA in cold stress tolerance. The results of this study demonstrate that unique phylogenetic clusters of C. jejuni associated with cold stress tolerance exist and that cfrA is a genetic factor contributing to cold stress tolerance in C. jejuni.