Susceptibility and resistance genes to fluoroquinolones in methicillin-resistant Staphylococcus aureus isolated in 2002

Abstract

The activity of six fluoroquinolones (FQs) was determined against 100 methicillin-resistant Staphylococcus aureus (MRSA) isolated in 2002 along with mutations in the grlA and gyrA genes and in the norA promoter of these isolates. Of the isolates tested, 97% had mutations in grlA and gyrA. A single mutation in grlA and gyrA resulted in a decrease of susceptibility to old generation FQs (norfloxacin, enoxacin, ciprofloxacin, fleroxacin, sparfloxacin and levofloxacin) but not to new generation FQs (gatifloxacin and moxifloxacin). Double mutations of both grlA and gyrA resulted in high-level resistance to all FQs tested. All norA mutants (15%) contained double mutations in grlA and gyrA and showed no decrease of MIC in the presence of reserpine, which is known to inhibit the drug-efflux pump. Our results showed that double mutations in grlA and gyrA were necessary for the expression of high-level resistance to new generation FQs. As different FQ-resistant mutants occur in the same PFGE type, FQ-resistant MRSA may well develop individually.

Introduction

Fluoroquinolones (FQs) are broad-spectrum antimicrobial agents that are effective against both Gram-positive and Gram-negative bacteria [1]. Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen as it frequently causes nosocomial infection and is resistant to various antimicrobial agents. In S. aureus, the primary target of FQs is the A subunit (GrlA) of DNA topoisomerase IV and the secondary target is the A subunit (GyrA) of DNA gyrase [2], [3], [4]. Increased use of FQs has led to greater numbers of FQ-resistant MRSA [5], [6], [7]. FQ resistance in S. aureus is due to a decrease in the affinity of the drug to the target sites by mutation(s) in the quinolone-resistance determining region (QRDR) of grlA and gyrA that encode GrlA and GyrA, respectively [3], [4]. In addition, norA of S. aureus encodes the active efflux pump (NorA) that excretes specifically hydrophilic FQs such as norfloxacin and enhanced transcription of norA by mutation in the norA promoter region is associated with FQ resistance [8], [9].

Various FQs have been developed. Old generation FQs, developed mainly before 2000, produce strong inhibition of DNA topoisomerase IV [10], while newer FQs act by inhibiting both DNA topoisomerase IV and DNA gyrase [11], [12], [13]. In the treatment of MRSA infection diseases and prevention of the spread of MRSA, it is important to investigate the susceptibility of MRSA to FQs and the distribution of FQ-resistant mutants.

The aim of the present study was to understand further the mechanism of development of FQ resistance in MRSA. To this purpose, the susceptibilities of MRSA to various FQs isolated in 2002 were compared, mutations of the QRDR in grlA and gyrA and the promoter region of norA were determined and the effect of such mutations on FQ resistance was analysed.