Identification of Novel Gene-Specific Markers for Differentiating Various Pathogenic Campylobacter Species Using a Pangenome Analysis Approach

<i>Campylobacter</i> spp. are the causative agents of campylobacteriosis, a major foodborne illness globally, with millions of cases reported annually. These pathogens pose significant risks to both human and animal health. Conventional culture-based diagnostic methods are labor-intensive and time-consuming, underscoring the need for more efficient molecular detection strategies. This study employed a pangenomic analysis to identify novel gene-specific markers for pathogenic <i>Campylobacter</i> species and subspecies, laying the groundwork for their application in diverse diagnostic assays. A curated dataset of 105 high-quality genomes, representing 33 species and 9 subspecies, was analyzed using the Roary ILP Bacterial Annotation Pipeline. The results revealed substantial genomic diversity within the genus, with core gene counts varying across different nucleotide identity thresholds. Ribosomal genes such as <i>rpsL</i>, <i>rpsJ</i>, <i>rpsS</i>, <i>rpmA</i>, <i>rpsK</i>, <i>rpsU</i>, <i>rpsG</i>, <i>rpmH</i>, and <i>rpsZ</i> were consistently identified in the core genome, whereas accessory genes exhibited marked variability. This study uncovered novel and highly specific genetic markers for various <i>Campylobacter</i> species, including <i>petB</i>, <i>clpX</i>, and <i>carB</i> for <i>C. coli</i>; hypothetical proteins for <i>C. jejuni</i> and <i>C. fetus</i>; <i>porA2</i> for <i>C. lari</i>; and <i>mdtJ</i> for <i>C. upsaliensis</i>. These markers demonstrated a specificity of at least 90% with minimal cross-reactivity with non-target organisms. The findings underscore the genomic heterogeneity within <i>Campylobacter</i> and provide essential genetic targets for the enhanced molecular detection of its pathogenic species, subspecies, and biovars.