Identification of Aminoglycoside-Modifying Enzymes by Susceptibility Testing: Epidemiology of Methicillin-Resistant Staphylococcus aureus in Japan

Abstract

A multiple-primer PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 381 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The technique used three sets of primers delineating specific DNA fragments of the aph(3′)-III, ant(4′)-I , and aac(6′)-aph(2") genes, which influence the MICs of gentamicin, tobramycin, and lividomycin. Isolates with none of the three genes detected were susceptible to all three agents. Isolates with the aph(3′)-III gene showed resistance to lividomycin (MIC > 1,024 μg/ml), and those with the ant(4′)-I gene were resistant to tobramycin (MIC ≥ 8 μg/ml). Isolates with only the aac(6′)-aph(2") gene were resistant to gentamicin (MIC ≥ 8 μg/ml) and tobramycin in decreasing order; those with both the ant(4′)-I and aac(6′)-aph(2") genes also were resistant to gentamicin and tobramycin, but in increasing order. Susceptibility testing, then, could detect specific genes. In 381 Japanese MRSA isolates, the ant(4′)-I, aac(6′)-aph(2") , and aph(3′)-III genes were prevalent in 84.5, 61.7, and 8.9%, respectively. Isolates with only the ant(4′)-I gene had coagulase type II or III, but isolates with both the ant(4′)-I and aac(6′)-aph(2") genes included all coagulase types. Most isolates with coagulase type IV or VII carried the aac(6′)-aph(2") gene. Of the MRSA isolates with ant(4′)-I and/or aac(6′)-aph(2") genes, 97% were resistant to aminoglycosides in clinical use, but a new aminoglycoside, arbekacin, had excellent activity against these isolates.