Natural Transformation-mediated Transfer of Erythromycin Resistance in Campylobacter coli and Campylobacter jejuni

Abstract

Campylobacter is the most common bacterial agent causing human gastroenteritis and is mainly transmitted through foods. Campylobacter is a zoonotic agent, and commonly colonizes poultry and other meat animals. Whereas erythromycin resistance in Campylobacter jejuni rarely occurs, erythromycin resistance in Campylobacter coli from meat animals is frequently encountered, and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in C. coli has been associated with a point mutation (A2075G) in the 23S rRNA gene, acting synergistically with the CmeABC efflux pump. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli used as donor. In addition, we studied the effects of environmental factors and species (C. coli vs. C. jejuni), and fitness costs of erythromycin resistance in transformants. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli from turkeys than in swine-derived strains (P<0.01) with frequency of transformation 10&#8315;&#8315;&#8308; to 10&#8315;&#8310; in turkey-derived strains, but 10&#8315;&#8311; or less in C. coli from swine. Transformants harbored the point mutation, A2075G in the 23S rRNA gene. Erythromycin resistance was stable in transformants following serial transfers, and most transformants had high MIC values (>256&#956;g/ml), as did the C. coli donor strains. In contrast to results obtained with transformation, spontaneous mutants had relatively low erythromycin MIC (32-64&#956;g/ml) and lacked the A2075G mutation. Temperature profoundly affected frequency of transformation to erythromycin resistance in C. coli and transformation frequency at 42&deg;C was significantly higher than at 25&deg;C, 32&deg;C and even 37&deg;C. However, transformation to nalidixic acid resistance was not significantly affected by temperature. No significant difference in transformation frequency was detected between microaerobic (5-10% CO&#8322;) and aerobic conditions. Starvation conditions did not affect transformation frequency to nalidixic acid resistance. Increasing incubation time from 3-4h to 15-17h significantly increased transformation frequency to erythromycin resistance (P<0.05). Transformation of C. jejuni using genomic DNA from erythromycin resistant C. coli revealed that transformation frequency of C. jejuni to erythromycin resistance was lower than C. coli, suggesting that erythromycin resistance in C. coli may not be disseminated via transformation in C. jejuni as frequently as in C. coli. Transformants derived from C. jejuni, however, had high erythromycin MIC values (>256&#956;g/ml) and harbored the A2075G transition, similarly to C. coli transformants. When grown separately at 42&deg;C, an erythromycin-resistant transformant derived from C. coli strain 961 had a similar growth rate as its erythromycin-sensitive parental strain, whereas an erythromycin-resistant transformant derived from C. jejuni strain SC49 had a significantly longer generation time compared to its parental strain. In competitive growth studies, however, the C. coli transformant was at competitive disadvantage in relation to its parental strain in stationary phase, whereas the C. jejuni transformant was at a slight fitness advantage after 14days. Furthermore, in the mixed culture the generation time of the C. jejuni transformant was not significantly different from that of the parental strain. In conclusion, natural transformation has the potential to contribute to dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals and temperature can greatly affect transformation to erythromycin resistance, but not to nalidixic acid resistance. These findings suggest that ecological attributes may play an important role and exert differential impact on the potential of the organism to acquire antimicrobial resistance determinants via natural transformation. However, further study is necessary to characterize the fitness of erythromycin resistant transformants in Campylobacter and identify possible mechanisms underlying the relatively low frequency of erythromycin resistance in C. jejuni.