Preliminary Study On Emergence Of Florfenicol Resistance In E.Coli O157:H7

E. coli O157:H7is an important zoonosis pathogen. Florfenicol is an effective antimicrobial using for prevent and treatment colibacillosis, however, howere, E. coli O157:H7has emergency serious resistance against florfenicol in recent years. This study aimed to investigate the drug resistance emergency mechanism of E. coli O157:H7against florfenicol, for providing a theoretical basis to slow down bacterial resistance to florfenicol and to lay a foundation for providing more effective antibacterial drugs in clinical.This study determined the in vitro minimum inhibitory concentration (MIC), mutant prevention concentration (MPC), resistant mutant selection window (MSW) and selection index (SI) of two stains of E. coli O157:H7against florfenicol. E. coli O157:H7stains which high sensitive to florfenicol was induced to be high level florfenicol resistant strains by sub-inhibitory concentrations drug-induced method, and established a sensitive determination based of enterobacteriaceae against florfenicol. E. coli O157:H7florfenicol sensitive strains and resistance strains which induced in vito were used as investigate strains, florfenicol resistance gene (floR) in plasmids of investigated strains was detected by the methods of PCR and loop-mediated isothermal amplification (LAMP). The florfenicol resistance of the plasmid was confirmed by plasmid transformation, or conjugation and elimination experiments. Otherwise, the efflux pumps phenotypic of E. coli O157:H7was detected.The results showed that, the MIC, MPC, MSW, SI of E. coli O157:H7RS2and FC1was4μg/mL,7.6μg/mL,4μg/mL-7.6μg/mL,SI1.9, respectively.The MIC of RS2and FC1strains of E.coli O157:H7against florfenicol was increased from4μg/mL to256μg/mL by in vitro florfenicol-resistant induced artificially32generations and36generations, respectively.Established a sensitive determination based of enterobacteriaceae against florfenicol:MIC≤8μg/mL, determined as sensitive, MIC=16μg/mL, determined as intermediary, MIC≥32μg/mL, determined as resistant.The results of plasmid detection by agarose gel electrophoresis showed that, the florfenicol-resistant induced stains contain with two plasmids with1,200bp and3,500bp. The LAMP detection and PCR detection results showed that, florfenicol resistance gene floR locate in the plasmids of E. coli O157:H7florfenicol sensitive stains and florfenicol-resistant induced strains.The plasmid of E. coli O157:H7florfenicol resistant stains was transformed or conjugated to the same genus or different genus bacteria which sensitive to florfenicol, the results showed that the MIC of receptor bacteria against florfenicol increased16-64times. The PCR detection results showed that gene floR were not detected from receptor bacteria before transformed or conjugated, while floR were detected from positive receptor bacteria which resistance to florfenicol after plasmid ransformed or conjugated.E. coli O157:H7florfenicol resistant stains were cultured200generations under a condition without florfenicol,the MIC of subculture strains against florfenicol were decreased from256μg/mL to8μg/mL, the florfenicol resistant stains were recover sensitivity to florfenicol. After subcultured, the plasmids of subculture strains were not lost, but the copy number of floR was decreased from0.768ng/μL to0.166fg/(μLThe MIC of E. coli O157:H7florfenicol-resistant stains against florfenicol were decreased from256μg/mL to64μg/mL, by SDS-temperature alternately, but the plasmids were not lost. The results showed that SDS-temperature alternately has little effect on eliminated the plasmids of this stains.With the action of efflux pump inhibitors of carbonyl cyanide chlorophenyl hydrazone (CCCP), omeprazole sodium and reserpine, the MIC of E. coli O157:H7florfenicol sensitive stains were decreased2-8times, the MIC of florfenicol resistant stains were decreased2-32times. The results showed that, E. coli O157:H7contain efflux pumps which depend on ATP hydrolysis or proton driven.The results of this study indicated that, the plasmid that carrying gene floR is a reason for E. coli O157:H7emergency florfenicol resistance.