Study On The Quinolone Resistance Mechanism Of Vibrio Harveyi Selected In Vitro

Vibrio harveyi is mainly distributed in the marine environment and marine organisms and is an opportunistic pathogens of many marine fish and invertebrates. The prevention of Vibrio disease depends on the antibiotics and chemicals primarily. Quinolones are widely used in the aquaculture industry,while the resistance to these drugs in aquatic bacteria is becoming more serious. Currently, there is lack of the research for resistance mechanisms to quinolone of Vibrio harveyi.The reference strain ATCC33843 and quinolone sensitive strains of Vibrio harveyi from clinical were used in vitro stepwise exposure to sub-inhibitory concentration of norfloxacin,then the drug resistance and biological characteristics of strains before and after induction were detected. Two high-level quinolone resistant strains( MIC of512~1024?g/ml)were successfully constructed with stable resistance and cross-resistance.Compared with the susceptible strains, the swimming ability and biofilm formation of drug-resistant strains were significantly increased and the growth curve had no obvious change.The quinolone resistant determining region(QRDR) of gyrA、gyrB、parC and parE of strain induced were analyzed by PCR and sequencing and the MIC of different drugs were determined after treating with efflux pump inhibitors(CCCP). The results showed that mutants with significantly increased MICs were generated from the selected strain gradually. The predominant mutation profile was ser83→Ile in gyr A,then ser85→Tyr/Phe in parC.But the mutation of gyrB and parE was not detected.After adding CCCP, MIC values of resistant bacteria were declined. These findings indicated that quinolone resistant mechanisms of V. harveyi may include the target gene mutations and active efflux system.The transcriptomes of sensitive strain(VR)and resistant strain(VS)of V. harveyi were sequenced by RNA-seq technology. A total of 2,082 Unigenes were obtained after De nove and assembly. 129 genes were identified as significant differentially expression between VS and VR, among which 65 were up-regulated and 64 down-regulated in VR.After functional annotation and enrichment analysis of these differentially expressed genes, the pathways related with two-component system(ko02020), ABC transport system(ko02010),flagellar assembly(ko02040) significantly enriched, and nine genes associated with quinolone-resistance were found.Transcriptome data revealed that the resistantmechanisms of V. harveyi may include active efflux system, two-component system and flagellar assembly besides the target mutations.Ten genes differentially expressed in the transcriptome data were taken for relative quantitative validation under the optimized real-time PCR reaction conditions.The results were as the same as the trend of transcriptome analysis basically, confirming the reliability.of transcriptome sequencing and data analysis.The predicted quinolone resistance gene qnr of Vibrio harveyi was cloned and its prokaryotic expression was optimized.The results showed that the ORF of qnr gene was651 bp,encoding a protein of 216 amino acids. The optimal condition of inducible expression for QNR protein was at 28℃ in 0.05 mmol/L IPTG. for 6 hours. The obtained recombinant protein would be purified and lay the foundation for researching the struction and function of QNR and bacterial resistance mechanisms.