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Enzymatic modification results in high-level resistance to aminoglycoside (HLAR), which eliminates the synergistic bactericidal effect of combined exposure to a cell wall-active agent and an aminoglycoside. So aim of the study was to determine prevalence of HLAR enterococcal isolate and to study distribution of aminoglycoside modifying enzyme genes in them. A total of 100 nonrepeat isolates of enterococci from various clinical samples were analyzed. As per Clinical and Laboratory Standards Institute guidelines enterococci were screened for HLAR by Kirby-Bauer disc diffusion method. Minimum inhibitory concentration of all isolates for gentamicin and streptomycin was determined by E-test. Multiplex polymerase chain reaction (PCR) was carried out for HLAR enterococcal isolates to identify aminoglycoside modifying enzymes genes responsible for resistance. 60% isolates were found to be high-level gentamicin resistant (HLGR) whereas 45% isolates were found to be high-level streptomycin resistant (HLSR). By multiplex PCR 80% HLGR isolates carried bifunctional aminoglycoside modifying enzyme gene  aac(6 ′ )-Ie-aph(2 ′′ )-Ia  whereas 18 out of 45 high-level streptomycin resistant, that is, 40%, isolates carried  aph(3 ′ )-IIIa.  However,  aph(2 ′′ )-Ib, aph(2 ′′ )-Ic, aph(2 ′′ )-Id,  and  ant(4 ′ )-Ia  genes which encode other aminoglycosides modifying enzymes were not detected. Bifunctional aminoglycoside modifying enzyme gene  aac(6 ′ )-Ie-aph(2 ′′ )-Ia  is the predominant gene responsible for HLAR.

Analysis of Aminoglycoside Modifying Enzyme Genes Responsible for High-Level Aminoglycoside Resistance among Enterococcal Isolates