Evidence for an efflux pump in multidrug-resistant Campylobacter jejuni

Abstract

Mechanisms of drug resistance in Campylobacter jejuni were investigated. Mutant strains 34PEF r , which was resistant to pefloxacin (128-fold increase in the MIC), and 34CTX r , which was resistant to cefotaxime (32-fold increase in the MIC) and which was derived from the susceptible parent 34 s , were obtained by serial passages on pefloxacin and cefotaxime gradient plates, respectively. Both mutants showed cross-resistance to erythromycin, chloramphenicol, tetracycline, ␤-lactams, and quinolones. While the quinolone resistance of strain PEF r could be explained by a mutation at codon 86 of the gyrA gene, the multidrug resistance phenotype of both strains was further investigated. Accumulation of pefloxacin, ciprofloxacin, and minocycline was measured by fluorometry and was found to be lower in the mutant strains than in the parent strain. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone, however, completely abolished this difference. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations from both mutant strains showed overexpression of two proteins of 55 and 39 kDa which were absent from the outer membranes of the wild-type strain. These results indicate that in C. jejuni 34PEF r and 34CTX r , multidrug resistance is associated with an efflux system with a broad specificity.Campylobacter jejuni is recognized as a leading cause of diarrhea in the world (3, 5). Typically, C. jejuni diarrhea is a self-limiting disease lasting up to 5 days, but it may persist longer and may develop into severe forms, especially in children and immunocompromised patients (2, 3).Fluoroquinolones are powerful antimicrobial agents against many gram-negative enteric bacteria including C. jejuni (4,35,37). Besides their potent in vitro activity against susceptible organisms (37), quinolones can also achieve high concentrations in the gastrointestinal tract (11). Nevertheless, the resistance of C. jejuni to fluoroquinolones during therapy has been reported (1,12,15,30,31).In gram-negative bacteria three mechanisms of quinolone resistance have been described, including alterations in the DNA gyrase structure (17,26,38), decreased outer membrane permeability (10), and export through an active efflux system (10,18,21).Quinolone-resistant mutants of C. jejuni selected in vitro by single-step quinolone exposure have been shown to be due to point mutations in the gyrA gene, the molecular target of these agents (16,36). However, little is known about another possible resistance mechanism, namely, elimination of antibiotics through an active efflux system. This mechanism was investigated by stepwise in vitro selection of C. jejuni mutants resistant to the quinolone pefloxacin or to the ␤-lactam cefotaxime. Study of the cross-resistance of the generated mutants provided evidence for an active antibiotic efflux system in C. jejuni. MATERIALS AND METHODSBacterial strains and selection of resistant mutants. The pefloxacin-susceptible strain 34 s was isolated from a stool specimen...

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

“…Spontaneous multiple-drug-resistant mutants of K. pneumoniae, which have increased resistance to a range of unrelated antibiotics including tetracycline, have also been described (82). Similar mutants have been found in Serratia marcescens, Enterobacter spp., and Campylobacter (39). These mutants exhibited efflux of tetracycline, similar to the E. coli Mar mutants.…”

“…A related MexA-MexB-OprM operon has been isolated from multiple-drug-resistant Burkholderia cepacia (32), while a similar system has been identified in multiple-drug resistant Campylobacter jejuni (39). An analogous system (the mtr system) has been found in N. gonorrhoeae (161).…”

Campylobacter

“…Efflux pump with a broad specificity; preventing accumulation of antibiotics [25] this cat gene and Sagara et al [140] reported a link between kanamycin and chloramphenicol resistance in Campylobacter following a number of cloning experiments involving the cat and kan resistance genes of a plasmid from a multiple-antibioticresistant C. coli isolate. Kanamycin resistance in Campylobacter is more commonly associated with C. coli than with C. jejuni [140].…”

“…Genetic alterations in this region are often associated with high-level resistance to nalidixic acid (MIC > 64-128 µg/mL) and ciprofloxacin (MIC > 16-64 µg/mL). Similarly, resistance to fluoroquinolnes may also be associated with the activation of a multi-gene efflux pump system (see below and [25]). …”

Antimicrobial-Resistant Campylobacter Species from Retail Raw Meats

“…The remaining isolates showed a spontaneous mutation at position 2268 (from T to C), which was also found in 15 Campylobacter isolates that were susceptible to erythromycin (MIC, Յ4 g/ml) ( Table 3). Other studies have demonstrated an efflux of fluoroquinolones, which also contributes to antimicrobial resistance in Campylobacter isolates (3,7). Since campylobacteriosis is transmitted primarily through food, particularly food of animal origin, the presence of antimicrobial-resistant Campylobacter in raw meat products has important public health implications.…”