National Reference Laboratory of Veterinary Drug Residues(HZAU)/MOA Key Laboratory of Food Safety Evaluation, Huazhong Agricultural University, Wuhan 430070, P. R. China.
Macrolide antibiotics are important for clinical treatment of infections caused by Campylobacter jejuni. Development of resistance to this class of antibiotics in Campylobacter is a complex process and the dynamic molecular changes involved in this process remain poorly defined. Multiple lineages of macrolide-resistant mutants were selected by stepwise exposure of C. jejuni to escalating doses of erythromycin or tylosin. Mutations in target genes were determined by DNA sequencing and the dynamic changes in the expression of antibiotic efflux transporters and the transcriptome of C. jejuni were examined by real-time RT-PCR, immunoblotting, and DNA microarray. Multiple types of mutations in ribosomal proteins L4 and L22 occurred in the early stepwise selection. On the contrary, the mutations in 23S rRNA gene, mediating highly resistant to macrolides, were only observed in the late-stage mutants. Upregulation of antibiotic efflux genes was observed in the intermediate-level resistant mutants, and the magnitude of upregulation declined as the occurrence of mutations in the 23S rRNA. DNA microarray analysis revealed differential expression of 265 genes, most of which occurred in the intermediate mutant, including upregulation of genes encoding ribosomal proteins and downregulation of genes involved in energy metabolism and motility. These results indicate 1) that mutations in L4 and L22 along with temporal overexpression of antibiotic efflux genes precede and may facilitate the development of high-level macrolide resistance and 2) that the development of macrolide resistance affects the pathways important for physiology and metabolism in C. jejuni, providing an explanation for the reduced fitness of macrolide-resistant Campylobacter.