Campylobacter jejuni and Campylobacter coli are the predominant thermophilic species responsible for foodborne gastroenteritis worldwide. Elevated resistance to certain antibiotics was observed due to antimicrobial therapy in farm animals and humans, while reduced antimicrobial usage partially reduced antibiotic resistance. Monitoring the antimicrobial resistance demonstrated a substantial fraction of multi-resistant isolates, indicating the necessity of reliable tools for their detection. In this study, resistance determinants in 129 German and 21 Vietnamese isolates were selected to establish a novel multiplex real-time PCR (qPCR), facilitating the simultaneous detection of four resistance determinants. These comprised tet(O) gene variants associated with tetracycline resistance, point mutations GyrA_T86I and GyrA_T86V associated with ciprofloxacin resistance, and the erm(B) gene together with the point mutation A2075G in the 23S rRNA gene, associated with erythromycin resistance. Moreover, the performance of the qPCR assay was evaluated by comparing the results of qPCR to phenotypic antimicrobial resistance profiles, obtained with standardized EUCAMP3 microdilution panel, which showed 100% similarity (inclusivity and exclusivity). Variation in measurement methods, including qPCR machines and master mixes showed robustness, essential for laboratories. The assay can be used for the rapid detection of resistance determinants, and is beneficial for monitoring the spread of antibiotic resistance in C. jejuni and C. coli.

The EU commission established Regulation (2017/1495) in 2017 to reduce Campylobacter on chicken skin and to decrease the number of human cases of campylobacteriosis attributable to the consumption of poultry meat. A Process Hygiene Criterion based on colony-forming unit data was set to a maximum of 1000 CFU Campylobacter spp. per gram chicken neck skin at slaughterhouses. Confronted with stressors, including cold, oxidative stress or antibiotic treatment, live cells may enter into a viable but non-cultivable state (VBNC) and lose the ability to grow, in reference to the plate count ISO 10272-2:2017 method, but still possess the potential to recover and cause infections under favorable conditions. In this study, a droplet digital PCR combined with the intercalating dye propidium monoazide (PMA) was established for quantification of C. coli and C. jejuni in chicken meat rinses. The PMA was used to inactivate DNA from dead cells in this technique. This method was successfully validated against the reference method according to ISO 16140-2:2016 for accuracy and relative trueness. Additionally, it presented a 100% selectivity for Campylobacter jejuni and C. coli. Moreover, the technical measurement uncertainty was determined according to ISO 19036:2019, and the applicability of ddPCR for quantifying C. coli and C. jejuni in chicken meat rinses was investigated on naturally contaminated samples from slaughterhouses and supermarkets. Results obtained from this study demonstrated a strong correlation to qPCR as well as the classical microbiological reference method.