Detection of Drug Resistance Genes and Effect of Efflux Pump Inhibitors on Biofilm of Escherichia coli Isolated from Hequ Horses
[Objective]The experiment was conducted to understand the resistance situation of Escherichia coli(E.coli)isolated from Hequ horses and provide theoretical guidance for rational use of antibacterial agents in veterinary medicine.[Method]Bacterial isolation and identification were carried out using bacterial morphology,Gram staining microscopy and 16S rDNA sequence analysis.Broth micro-broth dilution method and PCR method were conducted for antibacterial sensitivity tests against antibiotics and detection of antibacterial resistance genes,integrase genes,and efflux pump genes in the isolated E.coli.Meanwhile,a modified semi-quantitative crystal violet staining method was employed to assess the biofilm-forming ability.The biofilm removal ability of the combination of 13 efflux pump inhibitors with doxycycline was determined using a checkerboard method.[Result]The isolates were purple-black colonies with green metallic luster on eosin-mercian blue agar medium,and blue colonies on E.coli/coliform chromogenic medium,and pink short rod-shaped bacteria were seen on Gram staining microscopy.64 strains were highly similar to E.coli by 16S rDNA sequencing.Among 64 strains of E.coli isolates,relatively high resistance rates were observed against quinolones,sulfonamides,and tetracyclines,with multiple drug-resistant strains emerging.Among them,20.31％(13/64)showed resistance to five classes of antibiotics.Twenty-one resistance genes,one integrase gene and six efflux pump genes were positively detected.The detection rate of integrase gene intl1 was 89.06％.The number of E.coli strains with strong,medium,weak and no biofilm forming ability were 8(12.50％),34(53.13％),20(31.25％),and 2(3.12％),respectively.The combination of efflux pump inhibitors with doxycycline enhanced the biofilm removal ability.[Conclusion]The resistance of E.coli from Hequ horses was severe and it was necessary to further strengthen the supervision of the rational use of antibacterial agents in the racing horses in Henan Mongolian Autonomous county of Qinghai province to reduce and avoid the emergence and widespread transmission of multidrug-resistant strains.
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