Characterization And Proteomic Analysis Of Campylobacter Jejuni With Experimentally Induced Resistance To Erythromycin

Campylobacter jejuni is one of the most important zoonotic pathogens associated with human diarrhea. In some times, infection with Campylobacter jejuni can cause Guillain-Barre syndrome. Erythromycin is the best choice to treat human Campylobacteriosis, but with the widespread use of erythromycin, the resistance of Campylobacter jejuni to erythromycin is increasing. Therefore, it’s a great significance for development of low toxicity, efficient and selective antimicrobial agents to explore different mechanisms of Campylobacter jejuni resistance to erythromycin.With the rapid development of molecular biology, the mechanisms of Campylobacter jejuni resistance to erythromycin have made some progress, but it still can’t fully explain the formation of Campylobacter jejuni resistance to erythromycin. We all known that multifactorial and mulitistep alteration are involved in acquired erythromycin resistance of Campylobacter jejuni. Therefore, comparative proteomic analysis for protein altered during the development of erythromycin resistance is useful.The purposes of our study are:(1) develop a method that experimentally induce Campylobacter jejuni resistance to erythromycin, analysis the mutations of drug-resistant genes and the characteristics of Campylobacter jejuni resistance to erythromycin.(2) analysis the difference of protein between Campylobacter jejuni resistant and sensitive to erythromycin by two-dimensional electrophoresis, in order to explore some erythromycin resistance-associated proteins.1Induction and characteristics analysis of Campylobacter jejuni resistance to erythromycin In this study,7of Campylobacter jejuni strains(RGXY122R、d-2-3R、C31-10R、SO-12-5R、 PO-13-9R、PO-19-5R、11168R) resistance to erythromycin were obtained by serially subcultureing the wild-type strains onto CCDA agar plates containing a linear gradient of erythromycin. When comparing the stability, biochemical reaction and antimicrobial susceptibility of the induction mutants of Campylobacter jejuni with those of parent strains, we found that the strains can still grow on the CCDA agar plates containing high concentration of erythromycin after subculturing on the CCDA agar plates without antibiotics for30passages, and there was no significant effect on the metabolism. For Campylobacter jejuni resistant to erythromycin, the resistance of macrolide was increasing, but the resistance of penicillins, cephalosporins, quinolones, tetracyclines and aminoglycoside was not modified. We established a reliable model of experimentally induced Campylobacter jejuni resistance to erythromycin.Sequencing the drug-resistant genes showed that23S rRNA was the only mutation gene among the RGXY122R、d-2-3R、C31-10R, while SO-12-5R、PO-13-9R、PO-19-5R、11168R harboured two other mutation genes, rplD and rplV. The erythromycin resistant Campylobacter isolated from the nature just had the23S rRNA mutation. Comparing the point mutation in the23S rRNA gene between Campylobacter jejuni resistance to erythromycin experimentally induced and isolated from the nature, we found that the position of mutation was different, the induction mutation of Campylobacter jejuni was2074, while the nature Campylobacter was2075. There was no mutation in the cmeB in all of the Campylobacter jejuni resistance to erythromycin. We also demonstrate the significant role of an efflux pump in erythromycin resistance. From above all, we know that the mechanism of Campylobacter jejuni resistance to erythromycin is diverse and complex.When combine the results of passage times, the source of drug-resistant strains, antimicrobial susceptibility, virulence, proliferation and the mutation type of drug-resistant genes, it was showed that the mutations occurred in23S rRNA gene of Campylobacter jejuni isolated from poultry, while the mutation occurred in rplD and rplV genes of Campylobacter jejuni isolated from human. The Campylobacter jeuni which had23S rRNA gene mutation was associated with high-level resistace to erythromycin, but its adhesion and invasion to Caco-2and HD-11and proliferation to HD-11were decrease. We also found that the23S rRNA gene mutation was associated with the resistance of clindamycin. On the other side, the rplD and rplV genes mutation can cause low-level resistance to erythromycin, the decrease of proliferation.2Proteomic analysis of Campylobacter jejuni with experimentally induced resistance to erythromycinThe whole cell proteins of erythromycin-resistant strain11168R and erythromycin susceptible strain11168by method of acetone-trichloroacetic acid. It was shownd that the isoelectric point of protein was mainly between pH4-7when perform two dimensional electrophoresis using7cm pH3-10strips. We obtained a set of reproducible2-DE gel maps after performing the pH4-7strips on two-dimensional electrophoresis. Then gels were analysis by PDQest software and about500proteins were screened. Of all11different expressed proteins,9kinds of proteins were up-regulated,5were down-regulated. These proteins were identified by MALDI-TOF/TOF, they involved in the function related to energy metabolism, virulence, transcription and translation, biosynthesis. The up-regulation of proteins associated with energy metabolism mean the increase in the demand for energy during the process of the formation of drug resistant. And the up-regulation of proteins associated with virulence mean the increase of virulence after the Campylobacter jejuni obtained the resistance of erythromycin. After analysis the proteins by STRING9.0, we found that the proteins of CadF, Enolase, NusG, FusA had interaction with ribosome protein, they might be erythromycin-resistant related proteins of Campylobacter jejuni. The results of two-dimensional electrophoresis were validated by RT-PCR, and showed that expression of the genes in the level of mRNA were the same as the two-dimensional electrophoresis except fumC and fba.