Campylobacter spp. (C. spp.) are the leading cause of human gastroenteritis worldwide. RE-CmeABC is a newly identified resistance-enhancing multidrug efflux pump of C. spp. that confers high-level resistance to fluoroquinolones, phenicols, macrolides, and tetracyclines, all of which are critical drugs in both human and veterinary medicine. In this study, we analyzed the presence and antimicrobial susceptibility of RE-cmeABC-positive Campylobacter isolates of food-animal origin from three representative regions (Shandong, Shanghai, and Guangdong) in China over three successive years, from 2014 to 2016. A total of 1088 Campylobacter isolates (931 C. coli and 157 C. jejuni) were recovered from the RE-cmeABC screening. We detected 122 (11.2%) RE-cmeABC-positive isolates of chicken origin, including 111 (70.7%) C. jejuni and 11 (1.2%) C. coli. This multidrug efflux pump is more prevalent among C. jejuni than C. coli. The level of resistance was significantly different in 111 RE-cmeABC-positive C. jejuni versus 46 RE-cmeABC-negative C. jejuni for florfenicol, clindamycin, and erythromycin (P < 0.05), but not for ciprofloxacin (CIP), tetracycline (TET), and gentamicin. However, the isolates harboring RE-cmeABC could shift the minimum inhibitory concentration distribution to the higher range for CIP and TET. Pulsed-field gel electrophoresis (PFGE) analysis suggested that horizontal transmission might be involved in the dissemination of RE-cmeABC in Shanghai and Guangdong, while clonal expansion was predominant in Shandong. Three isolates shared the indiscriminate PFGE types of RE-cmeABC-positive C. jejuni isolates in Shanghai and Guangdong, and
four isolates in Shanghai and Shandong. Our study suggests the possibility of a wide dissemination of RE-cmeABC in Campylobacter of food-animal origin, which would pose a significant threat to public health.