Mutations in the quinolone resistance determining region in Staphylococcus epidermidis recovered from conjunctiva and their association with susceptibility to various fluoroquinolones

Abstract

Background:Staphylococcus epidermidis is one of the prominent pathogens in ocular infection. The prevalence of mutations in the quinolone resistance determining region (QRDR) area in S epidermidis isolated from the ocular surface and its association with fluoroquinolone resistance has not been fully elucidated.Methods:Mutations in the QRDR of gyrA, gyrB, parC, and parE genes of 138 isolates of S epidermidis recovered from the human conjunctival flora were analysed. The minimal inhibitory concentrations (MICs) of four fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin) against these isolates were also determined using agar dilution methods.Results:The MIC90 values of levofloxacin, gatifloxacin, moxifloxacin and tosufloxacin were 3.13, 1.56, 0.78 and 3.13 μg/ml, respectively. The MIC values of all fluoroquinolones showed a bimodal distribution (susceptible strain and less susceptible strain). Mutations with amino acid substitution in the QRDR were present in 70 (50.7%) isolates. 19 different combinations of mutations were detected: 3 isolates (2.2%) had four mutations, 8 (5.8%) had three mutations, 43 (31.2%) had double mutations and 16 (11.6%) had single mutations. Isolates with mutations in the QRDR of both gyrA and parC (n = 53) were less susceptible to fluoroquinolones.Conclusions:The present findings show that approximately half the S epidermidis isolates from the normal human conjunctiva have mutation(s) in the QRDR. The presence of mutations in both gyrA and parC is strongly associated with reduced susceptibility to fluoroquinolones.

Emerging 8-Methoxyfluoroquinolone Resistance among Methicillin-Susceptible Staphylococcus epidermidis Isolates Recovered from Patients with Endophthalmitis

“…Low-level resistance isolates demonstrated only a single point mutation (Ser84Phe) in gyrA, whereas isolates carrying a second point mutation (Ser84Phe and Glu88Lys) showed high-level resistance. Previous work has also shown that the additional substitution at codon 88 from a glutamic acid to lysine in the gyrA of S. epidermidis ocular isolates is related to a higher MIC of 8-methoxyfluoroquinolones (18,19). Although overexpression of the efflux pump might play a role in the high-level fluoroquinolone resistance, our present and previous data suggest that the level of resistance for 8-methoxyfluoroquinolones increases as additional mutations are included in the QRDR of gyrA (28,29).…”

“…Fluoroquinolone-resistant CoNS isolates from the normal conjunctiva microbiota have been recovered from patients undergoing intraocular surgery and intravitreal injection (4,5,(22)(23)(24). In addition, two studies found mutations within the QRDR in around 50% to 58% of S. epidermidis isolates from in the conjunctival sacs before preoperative instillation of antibiotic from patients undergoing intraocular surgeries (18,19). Although a double point mutation was found for a few isolates in both studies, 38% to 41.6% of S. epidermidis isolates from conjunctival sacs preoperatively harbored at least one point mutation in the gyrA gene.…”

“…Amplification of QRDRs of the gyrA and parC genes was performed as previously described (19) using the following primer pairs: gyrA forward, 5=-ATGCGTGAATCAT TCTTAGACTATGC-3=, and reverse, 5=-GAGCCAAAGTTACCTTGAC C-3=, designed to generate an amplification product of 284 bp, and parC forward, 5=-TCGCAATGTATTCAAGTGGG-3=, and reverse, 5=-ATCGT TATCGATACTACCATT-3=, designed to amplify a region of 197 bp in length. PCR was set up using 10 l HotStarTaq master mix (Qiagen; final concentrations of 1.25 U HotStarTaq DNA polymerase, 1.5 mM MgCl 2 , and 200 M each deoxynucleoside triphosphate), 0.5 M each primer, and 1 l of DNA template.…”

“…Some studies have reported increasing resistance rates to fluoroquinolones as well as lower in vitro poten-cies against MSSA and methicillin-susceptible CoNS ocular isolates (13)(14)(15)(16). Several studies have confirmed the development of multiple mutation profiles in the quinolone resistancedetermining region (QRDR) of the topoisomerase subunits GyrA (DNA gyrase) and ParC (topoisomerase IV) and also its association with the level of fluoroquinolone resistance in S. epidermidis recovered from the normal conjunctival microbiota as well as from infected eyes (17)(18)(19). In this study, we report the emergence of low-and high-level resistance to 8-methoxyfluoroquinolones associated with multiple mutations in the QRDRs of the gyrA and parC genes among methicillinsusceptible S. epidermidis (MSSE) isolates recovered from endophthalmitis patients.…”

In Vitro Activity of Ozenoxacin against Quinolone-Susceptible and Quinolone-Resistant Gram-Positive Bacteria

“…If one of the strains was found to be resistant to LVX and MXF, it was allocated to the QR group, although they were susceptible to the remaining fluoroquinolones. The quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes were amplified by PCR using a set of previously described primers (10)(11)(12)(13)(14).…”