Phenotypic and Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance and Virulence in <i>Campylobacter</i> spp. Isolated from Human Stool in a Croatian University Hospital

<i>Campylobacter</i> spp. is a leading cause of community-acquired gastroenteritis, with rising antimicrobial resistance (AMR) posing a significant public health challenge. This study aimed to characterize the genomic basis of resistance and virulence in <i>Campylobacter</i> spp. isolated from human stool at the University Hospital of Split, Croatia. A total of 115 unduplicated isolates (100 <i>Campylobacter jejuni</i> and 15 <i>Campylobacter coli</i>) were identified and tested for antimicrobial susceptibility, from January to June 2023. A representative number of isolates with phenotypically detected resistance were analyzed with whole-genome sequencing. Multilocus sequence typing (MLST) was determined using genomic sequences as input data against the standard MLST schemes. High rates of resistance were detected to tetracycline (45% of <i>C. jejuni</i> and 26.7% of <i>C. coli</i>) and ciprofloxacin (81% of <i>C. jejuni</i> and 46.7% of <i>C. coli</i>), while combined ciprofloxacin/tetracycline resistance was observed in 43% of <i>C. jejuni</i> and 7% of <i>C. coli</i> isolates. The majority of whole-genome sequenced isolates possess <i>tet(O/32/O)</i> gene and <i>gyrA.T861</i> mutation, conferring resistance to tetracycline and fluoroquinolones. All isolates have at least one resistance gene for β-lactams (<i>bla</i>_OXA-61, <i>bla</i>_OXA-193, <i>bla</i>_OXA-450, <i>bla</i>_OXA-451, <i>bla</i>_OXA-452, <i>bla</i>_OXA-461, and <i>bla</i>_OXA-489), while 40% of isolates possess <i>ant(6)-Ia</i> for aminoglycoside resistance. In addition, a newly emerged, phenotypically identified multidrug-resistant <i>C. coli</i>, harbored <i>tet(O/32/O)</i> and <i>tet(O)</i> genes, point mutation in <i>gyrA</i> gene, and A2075G mutation in 23S rRNA. Virulence analysis highlighted <i>C. jejuni</i>'s broad pathogenic potential, including motility, adherence, invasion, exotoxin production, and immune modulation (e.g., <i>wlaN</i> gene, linked to Guillain-Barré syndrome). On the contrary, <i>C. coli</i> isolates possess genes exclusively for motility. Nine different <i>C. jejuni</i> MLST sequence types were identified, while all <i>C. coli</i> isolates belong to the same 828 clonal complex. Escalating AMR and a broad spectrum of virulence in <i>Campylobacter</i> spp. highlight the importance of continuous surveillance on the phenotypic and genotypic level, thereby allowing more efficient health care management of these infections.