Antimicrobial susceptibility studies
Emerging multiple mutations and high-level fluoroquinolone resistance in methicillin-resistant Staphylococcus aureus isolated from ocular infections

https://doi.org/10.1016/j.diagmicrobio.2006.04.017Get rights and content

Abstract

We investigated the prevalence of mutations in the gyrA, gyrB, grlA, and grlB genes in 21 clinical isolates of methicillin-resistant Staphylococcus aureus recovered from ocular infections. These isolates were classified into 16 groups on the basis of pulsed-field gel electrophoresis (PFGE) patterns. Among these nonidentical strains, 9 different combinations of mutations were observed. We observed an unusually high frequency of mutations: 10 (62.5%) of the isolates had four or five mutations, 5 (31.3%) of the isolates had double mutations, and 1 isolate had a single mutation. Different mutations were detected in strains of the same PFGE type, and this is consistent with the independent acquisition of mutations. The minimun inhibitory concentrations values ranged from 0.5 to 512 μg/mL for levofloxacin and 0.125 to 128 μg/mL for gatifloxacin. Only 1 isolate was found to be susceptible to both agents. These results suggest that application of high concentration fluoroquinolone ophthalmic solutions selects for isolates presenting such mutations that may result in a high level of resistance to fluoroquinolones.

Introduction

Fluoroquinolone antibacterial agents have potent activities against both Gram-positive and Gram-negative bacteria and are currently used for various bacterial infections. In the field of ophthalmology, low toxicity, safety, good ocular surface penetration, prolonged tear-film concentration, stability at room temperature, and ready availability have made fluoroquinolone-based ophthalmic solutions a widely used, first-choice topical therapy for bacterial infections of the eye. In addition, corneal concentrations of fluoroquinolones after 1 to 2 doses have been reported to be higher than those in the serum after systemic administration (Kowalski et al., 1998).

Staphylococcus aureus is the leading cause of bacterial keratitis, conjunctivitis, and dacryoadenitis. Recent reports have documented increasing fluoroquinolone resistance in S. aureus isolated from ocular as well as non-ocular infections (Goldstein et al., 1999, Kowalski et al., 2001, Marangon et al., 2004, Morrissey et al., 2004). Moreover, the development of fluoroquinolone-resistant, methicillin-resistant S. aureus (MRSA) has been widespread (Marangon et al., 2004). The mechanisms that underlie fluoroquinolone resistance in S. aureus have been studied extensively. Although overexpression of norA, which encodes the multidrug efflux pump NorA, can reduce susceptibility to fluoroquinolones (Kaatz et al., 1993, Kaatz and Seo, 1995, Yoshida et al., 1990), resistance is believed to be related mainly to mutations in genes encoding the target enzymes. The primary target of fluoroquinolones is DNA topoisomerase IV, which is composed of the GrlA and GrlB subunits, encoded by the grlA and grlB genes, respectively (Hooper, 2000, Hooper, 2002, Ng et al., 1996, Trucksis et al., 1991, Yamagishi et al., 1996). The secondary target is DNA gyrase, which is composed of the GyrA and GyrB subunits, encoded by the gyrA and gyrB genes, respectively (Ito et al., 1994). In most cases, mutations are in the highly conserved quinolone resistance-determining regions (QRDRs) of the grlA and gyrA genes; a combination of mutations in both genes can cause high-level resistance even to the newer fluoroquinolones (Guirao et al., 2001, Schmitz et al., 1998a, Wang et al., 1998).

In response to the increasing frequency of resistant strains, new ophthalmic solutions of fluoroquinolones with excellent corneal penetration, such as gatifloxacin (GFLX), which target both DNA topoisomerase IV and DNA gyrase, have been developed (Takei et al., 2001). Recent studies have shown that GFLX is more potent than levofloxacin (LVFX), ciprofloxacin, and ofloxacin (OFLX) against S. aureus isolated from ocular infections (Kowalski et al., 2003, Mather et al., 2002). For this reason, GFLX was expected to have greater efficacy against S. aureus including MRSA. However, there has been growing concern in Japan that the frequent use of OFLX and LVFX since the 1990s may lead to increased resistance as well as cross-resistance to other fluoroquinolones. Fluoroquinolone resistance in MRSA isolated from the eye has not been well characterized. Furthermore, the impact of increasing fluoroquinolone resistance on the effectiveness of GFLX for treatment of eye infections has not been studied.

To address whether high concentrations of ophthalmic fluoroquinolone solutions and the common use of such solutions to treat eye infections lead to high-level fluoroquinolone resistance, we investigated the prevalence of mutations in the gyrA, gyrB, grlA, and grlB genes in MRSA isolated from ocular infections at a medical center and an eye clinic in Japan. We also examined the effect of the mutations on the minimun inhibitory concentration (MIC) of GFLX and LVFX.

Section snippets

Bacterial isolates

A total of 21 clinical MRSA isolates (Table 1) were obtained from 16 patients; 13 isolates were recovered from 8 patients admitted to Ehime University Hospital (Ehime Prefecture, Shikoku Island, Japan) between January 2003 and March 2004. The other 8 isolates were collected from outpatients seen at the Inoue Eye Clinic in Okayama Prefecture (southern Honshu Island, Japan) between May 2003 and August 2004. Isolates E001, E002, E017, and E047 were recovered from the same patient; likewise,

PFGE typing

Twenty-one isolates were classified into 11 PFGE types by PFGE fingerprinting patterns (Fig. 1). PFGE types A, B, and I were further subdivided into 3, 2, and 2 subtypes, respectively. All isolates recovered from the same patient showed the same PFGE type. Isolates E001, E002, E017, and E047; isolates E043 and E046; and isolates E044 and E092 were PFGE types A, B, and C, respectively.

Genetic analysis in the QRDRs of the gyrA, gyrB, grlA, and grlB genes

Mutations detected in the QRDRs of the gyrA, gyrB, grlA, and grlB genes are summarized in Table 3. Nine

Discussion

To investigate the prevalence of mutations in the QRDR, we examined the gyrA, gyrB, grlA, and grlB genes from 21 clinical MRSA isolates recovered from ocular infections. We also determined the susceptibility of these isolates to LVFX and GFLX.

We found 1 and 3 novel mutations in the grlA and grlB genes, respectively. With respect to the grlB mutations, it is not clear whether these mutations are typical of ocular isolates, and the role of these mutations in fluoroquinolone resistance remains

Acknowledgments

We acknowledge Dr. Takahashi for careful reading the manuscript.

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