Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt

Abstract

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.

Keywords: Campylobacter species; FQ resistant; PCR-RFLP; PDR; gyrA.

Figure 1

Prevalence of Campylobacter jejuni and Campylobacter coli resistance against 24 antimicrobial agents. ns: non-significant, * p < 0.05, ** p < 0.01.

Figure 2

Prevalence of Campylobacter species resistance among human and chicken sources. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Antimicrobial resistance patterns of human and chicken Campylobacter isolates (A) and C. jejuni and C. coli (B). n: number, ns: non-significant, * p < 0.05, ** p < 0.01.

Figure 4

Agarose gel electrophoresis showing typical amplification products of 23S rRNA gene for confirmation of genus Campylobacter. Lane L: 100 bp DNA ladder “Marker”, lane 1: negative control (PCR grade water), lane 2: positive control (C. jejuni NCTC11322), lanes 3–15: positive Campylobacter isolates from human stool swabs and lanes 16–40: positive Campylobacter isolates from chicken samples.

Figure 5

PCR amplification products of mapA gene specific for C. jejuni (A) and ceuE gene specific for C. coli (B). Lanes 1–11: C. jejuni from human origin, lanes 12–29: C. jejuni from chicken samples, lanes 30–31: C. coli from human samples, lane 32–38: C. coli from chicken origin, lane L: 100 bp DNA ladder “Marker”, lane P: positive controls (C. jejuni NCTC11322 and C. coli NCTC11366), lane N: negative control (PCR-grade water).

Figure 6

Prevalence of molecularly identified Campylobacter species in different samples. H: human stool swabs, Cl: chicken liver, Cbm: chicken breast meat, Ccs: chicken cloacal swabs, ns: non-significant, * p< 0.05, ** p < 0.01.

Figure 7

PCR-restriction fragment length polymorphism patterns obtained after digestion of gyrA PCR products with FastDigest RsaI enzyme. Lane L: 100 bp DNA ladder and lanes 1–38: PCR-RFLP fragments obtained for Campylobacter species isolated from human and chicken samples.