Study On The Susceptible Breakpoint And Molecular Resistance Mechanism For Mequindox And Florfenicol Against Animal-derived E. Coli

The interpretive criteria or breakpoint for antimicrobial susceptibility testing was the key technical criteria to conduct resistance surveillance and to analyze the resistance rate and resistance level. The authoritative organizations to set the breakpoint included the CLSI in the US and EUCAST in the Europe. For the lack of interpretive criteria, the breakpoint abroad was employed in China. However, there was no interpretive criteria for the new antimicrobials or formulations firstly devoloped in China such as mequindox and florfenicol powder. These two antimicrobials were extensively used in China and has played an important role in the control of infections caused by Escherichia coli. However, the lack of breakpoint hindered the implement of resistance surveillance for mequindox and florfenicol. Thus, this study aimed to investigate the susceptibility phenotype and resistance genotype as well as to set the susceptible breakpoint for these two drugs against E. coli.In vitro efficacy of these two drugs were studied including minimum inhibitory concentration (MIC^minimum bactericidal concentration (MBC)、mutant prevention concentration (MPC) and post-antimicrobial effect (PAE) It showed that mequindox presented a concentration-dependent bactericidal activity against enteropathogenic E. coli (MBC=1-2MIC), while florfenicol showed concentration-dependent bacteriostatic activity against enteropathogenic E. coli (MBC=4-16MIC). Both drugs have an in vitro PAE although their mutation selective window (MSW) was wide. A total of1123E. coli isolates were collected from domestic animals in China from the1970s to2013, and mequindox and florfenicol susceptibility was tested by broth microdilution. The percentages of E. coli isolates with increased mequindox MICs (≥64μg/ml) and florfenicol MICs (≥32μg/ml) showed a rising trend each year throughout the study period. The E. coli from chicken was more sensitive to mequindox than that from pigs while strains of both origins have the similar susceptibility pattern towards florfenicol. By plotting the histogram of MIC distribution for mequindox and florfenicol and also using the statisticial analysis, the wide-type cut-coff (COWT) was set for these two drugs against E. coli.PCR was performed to detect the olaquindox resistance gene oqxAB and all the florfenicol resistance genes in these strains. It revealed that the oqxAB was the dominant resistance gene (94.4%) mediating increased mequindox MICs (MIC≥64μg/ml), while floR gene was the most prevalent gene mediating increased florfenicol MICs (MIC≥32μg/ml) and percentages of these genes showed a rising trend in E. coli during the70s to2013. The cfr gene was detected for the first time in the plasmid of one porcine E. coli isolate and the cfr-positive strain showed florfenicol MICs of64μg/ml. The genotype tested was in agreement with the COWT.By sequencing, we found that both oqxAB and floR genes showed high identities (99.2%and99.6%, respectively) in strains of different animal origins. The genetic environment of oqxAB and cfr was investigated and both genes were found to be bracketed by the IS26and the element was not stable and was readily to be looped out via IS26mediated recombination in the form of a circular intermediate. The PK-PD relationship for mequindox injection against E. coli was studied in neutropenic murine thigh infection model. The most relevant PK-PD parameter is the AUC/MIC and the PK-PD targets were0.85h for bacteriostatic and4.3h for bactericidal effect, respectively. The PK-PD relationship for florfenicol powder against E. coli was investigated in porcine fistula and the serum ex vivo model. However, the bactericidal test was impracticable ex vivo for the complicated environment in intestinal tract. The concentration-time process of florfenicol in the intestinal tract was investigated and it revealed that the florfenicol was present in the ileum for a short time and could not be detected after3h. The PK-PD relationship in porcine serum ex vivo was simulated using Hill model and it showed that the bacteriostatic and bactericidal effect was achieved when the AUC24h/MIC attained15.79h and49.49h, respectively. Considering the MIC distributions for wild-type E. coli strains (MIC≤16μg/mL), the regime of administration twice one day may not be practical to cure the system infection caused by some of the wild-type strains.In conclusion, the percentage of E. coli isolates with decreased mequindox and florfenicol susceptibility showed a rising trend during the70s and2013. The oqxAB and floR gene was the dominant resistance gene mediating low-level susceptibility of mequindox and florfenicol, respectively. The COwTwas set for mequindox and florfenicol against E. coli, respectively. The PK-PD target for mequindox against E. coli infection in mice is AUC/MIC≥4.3h. PK-PD relationship in porcine intestinal tract as well as serum ex vivo was explored and administration regime was recommended. The findings in this study provided theory basis and data support for the final breakpoint and may promote the implement of resistance surveillance as well as the resistance risk assessment of these two drugs against E. coli.