Simultaneous Detection of Fluoroquinolone- and Macrolide-Resistant <i>Campylobacter jejuni/Campylobacter coli</i> in Retail Chicken Meat Using Multiplex-PCR

<i>Campylobacter</i> contamination in chicken meat, particularly with fluoroquinolone- and macrolide-resistant strains, is a significant public health concern. Therefore, developing an easy and rapid detection method for fluoroquinolone- and macrolide-resistant <i>Campylobacter</i> will improve food safety. In this study, we designed and evaluated a multiplex-Polymerase Chain Reaction (PCR) method for the simultaneous detection of fluoroquinolone- and macrolide-resistant <i>Campylobacter jejuni</i> and <i>Campylobacter coli</i> in chicken meat. Our multiplex-PCR-targeted mutations in the <i>gyrA</i> and <i>rrl</i> genes of <i>Campylobacter</i> species confer resistance to fluoroquinolones and macrolides, respectively. Overall, this method could detect <i>Campylobacter</i> contamination and identify <i>C. jejuni</i> and <i>C. coli</i> as well as their susceptibility to fluoroquinolone and macrolide. Spiking of chicken meat samples revealed that multiplex-PCR can detect at least 0.72 Colony-Forming Unit (CFU)/g <i>C. jejuni</i>/<i>coli</i>. Among 277 retail chicken meat samples, <i>C. jejuni</i>/<i>coli</i> were detected in 54 samples using multiplex-PCR, with isolation from 52 samples using microbial culture. The susceptibility of the isolates to fluoroquinolones and macrolides was determined using both the PCR and culture methods, and a comparison of these methods showed that the accuracy, specificity, and sensitivity were all above 95%. The culture method required approximately 8 days for <i>Campylobacter</i> isolation and antimicrobial susceptibility testing. Multiplex-PCR could determine the contamination with <i>C. jejuni</i>/<i>coli</i> and susceptibility to fluoroquinolones and macrolides within 2-3 days from the enrichment culture to final determination. These results indicate that the proposed multiplex-PCR enables rapid and simultaneous detection of fluoroquinolone- and macrolide-resistant <i>C. jejuni</i>/<i>coli</i> in chicken meat and its utility for monitoring their contamination in food.