Investigation on the mechanism of double zone drug resistance of Enterobacteriaceae to amikacin

Objective To investigate the mechanism of double zone(DZ) drug resistance of Enterobacteriaceae to amikacin in the K-B agar diffusion test.Methods Five isolates of Escherichia coli(E.coli) and 2 Klebsiella pneumoniae(Kpn) with DZ resistant to amikacin in the K-B test were collected.Meanwhile,2 strains of E.coli and 2 K.pneumoniae with completely resistant(CR) to amikacin were collected for the control.The drug sensitivity of these isolates to amikacin were detected with the Vitek 2 Compact system and the agar dilution method,respectively.The aminoglycoside modification gene,integron and 16S rRNA methylase genes were determined by specific PCR and DNA sequence analysis,and the homology of these isolates by the repetitive extragenic palindromic(REP)-PCR.Results The results for the Vitek 2 Compact system showed that three isolates were sensitive to amikacin and four intermediary in the 7 isolates with DZ resistant to amikacin,and the control strains were resistant to amikacin.However,all isolates or strains were highly resistant to amikacin(MIC512 mg/L) in the agar dilution method.All the isolates carried different aminoglycoside modification genes and the main was aac(6)-Ⅰ.All the isolates or strains were positive for intlⅠ,8 with aadA5-dfrA17 gene and 3 with aadA2-dfrA12.All the isolates with DZ resistant to amikacin were positive for armA and the control strains negative.REP-PCR revealed that there was high homology among the isolates with DZ resistant to amikacin,and there was large difference with the control strains.Conclusion The DZ drug resistance of Enterobacteriaceae to amikacin was correlated with the expression of 16S rRNA methylase armA.The Vitek 2 Compact system might not be suitable for the detection of susceptibility of Enterobacteriaceae with the DZ resistant to amikacin in the K-B method.