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Antimicrobial Agents and Chemotherapy, February 2005, p. 846-848, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.846-848.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Stability of Fluoroquinolone Resistance in Streptococcus pneumoniae Clinical Isolates and Laboratory-Derived Mutants

Heather J. Smith-Adam,^1* Kimberly A. Nichol,² Daryl J. Hoban,^1,2 and George G. Zhanel^1,2,3

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba,¹ Departments of Medicine,³ Clinical Microbiology, Health Sciences Centre, Winnipeg, Manitoba, Canada²

Received 26 September 2004/ Accepted 21 October 2004

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The stability of fluoroquinolone resistance in Streptococcus pneumoniae was evaluated in laboratory-derived mutants and clinical isolates. Isolates with various genotypes and phenotypes were subcultured for 20 days on antibiotic-free media and were monitored by E-tests to identify any alterations in resistance. Fluoroquinolone resistance mechanisms, whether efflux or chromosomally mediated, remained stable in both clinical and laboratory-derived mutants.

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Streptococcus pneumoniae is a primary cause of numerous respiratory tract infections, resulting in significant morbidity and mortality worldwide. Respiratory fluoroquinolones, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, are increasingly used in the empirical therapy of community-acquired respiratory infections likely caused by S. pneumoniae as a result of the rise in resistance to ß-lactams and macrolides. The respiratory fluoroquinolones have demonstrated excellent bacteriological and clinical cure rates, including against penicillin-resistant, macrolide-resistant, and multiple-antibiotic-resistant S. pneumoniae (10).

Fluoroquinolones impede DNA synthesis by inhibiting two essential enzymes, DNA gyrase and topoisomerase IV. DNA gyrase and topoisomerase IV are heterotetramers comprised of two A, gyrA and parC, and two B, gyrB and parE, subunits, respectively (3, 10). Fluoroquinolone resistance primarily results from spontaneous chromosomal mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC (3, 10). Active efflux has also been implicated in fluoroquinolone resistance (8, 10).

Many publications have investigated both clinical and laboratory-derived fluoroquinolone-resistant isolates; however, the stability of the resistance mechanisms has not been specifically evaluated. The purpose of this study was to assess the stability of fluoroquinolone resistance, both efflux and chromosomal, in the absence of antibiotic selective pressure.

The S. pneumoniae clinical isolates investigated in this study were collected as part of an ongoing national respiratory organism surveillance program (11, 12). The isolates were obtained from 25 medical centers in 9 of the 10 Canadian provinces between 1997 and 2003 (11). Isolates were identified using conventional methodology and were deemed to be significant respiratory pathogens by each laboratory's existing protocol. MICs were determined using the NCCLS broth microdilution technique (6) after the isolates were subcultured twice from frozen stock, grown on blood agar, and incubated at 37°C in 5% CO₂ for 24 h (11). Antibiotics tested included ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin. The susceptibility interpretive criteria for gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin were the same as those described in the NCCLS M100-S12 document (6), and ciprofloxacin nonsusceptibility was defined as a MIC of 4 µg/ml. American Type Culture Collection strains S. pneumoniae 49619 and S. aureus 29213 were used as controls for all MIC determinations. All MICs were determined a minimum of three times on separate days to ensure precision. Three additional isolates studied were received from G. Doern: 984, 1146, and 1292 (2). The isolates were selected such that various phenotypes and genotypes, including fluoroquinolone-susceptible, efflux-positive, ParC mutations, efflux-positive ParC mutations, GyrA mutations, and mutations in both ParC and GyrA, were evaluated. In total, 17 clinical isolates were studied.

Twenty-six previously described laboratory-derived mutants were selected for this study (9). Briefly, fluoroquinolone-resistant laboratory-generated mutants were obtained by plating an approximately 10¹⁰-CFU/ml concentration on Mueller-Hinton agar plates containing 5% lysed horse blood at 1x, 2x, 4x, 8x, or 16x the MIC of each fluoroquinolone (ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, or moxifloxacin). Mutants were created from isolates with various genotypes and phenotypes, including fluoroquinolone-susceptible, efflux-positive, ParC mutations, GyrA mutations, and mutations in both ParC and GyrA. The most resistant mutant generated with each fluoroquinolone from every originating genotype was selected. Additionally, mutants with a onefold lower level of resistance to levofloxacin and moxifloxacin were studied when possible. For the mutants created from the originating isolate containing a GyrA mutation, mutants with a onefold lower level of resistance to ciprofloxacin and gemifloxacin were selected. The genotypes and phenotypes of all isolates are presented in Table 1.

View this table: [in this window] [in a new window]

TABLE 1. Resistance mechanisms, QRDR mutations in GyrA or ParC, and efflux present in the clinical and laboratory-derived mutants evaluated for stability and observed MIC decreases

Prior to the stability study, the fluoroquinolone resistance mechanisms of each isolate was molecularly characterized. Primers previously described by Morrissey and George were used to generate PCR products of the QRDRs of gyrA and parC followed by sequencing of the QRDRs in the forward and reverse directions (5). An ABI PRISM Big Dye Terminator kit and ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Mississauga, Ontario, Canada) were used to conduct the sequencing (12). The presence of efflux was tested by ciprofloxacin agar dilution MICs on Mueller-Hinton agar plates plus 5% sheep blood in the presence or absence of reserpine (10 µg/ml). Isolates for which the antibiotic MIC was reduced fourfold or greater in the presence of reserpine were considered positive for reserpine-sensitive efflux (1).

The 43 clinical and laboratory isolates were subcultured every day for 20 days on antibiotic-free trypticase soy agar plates containing 5% lysed sheep blood. The MICs of ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin were evaluated by E-test every day for the first 10 days and again at day 20. Gemifloxacin MICs could not be evaluated, as gemifloxacin E-test strips were not commercially available. As described in the E-test insertional document, a 0.5 McFarland was prepared for each organism, streaked onto Mueller Hinton plates containing 5% lysed sheep blood, and allowed to dry prior to the application of the E-test strips (4). Isolates were stocked on days 5, 10, and 20 for further investigation. The study was repeated in order to ensure consistency.

Each isolate was confirmed to be genotypically identical prior to and following the 20-day study by pulsed-field gel electrophoresis (PFGE). Preparation of genomic DNA for pulsed-field gel electrophoresis was performed as described previously by Nichol et al. (7). SmaI restriction fragments were resolved in a contour-clamped homogenous electric field apparatus (CHEF DRIII; Bio-Rad Laboratories, Hercules, Calif.) for 18 h at 6 V/cm with switch times of 2 to 30 s and an included angle of 120°. DNA banding patterns were digitized for analysis using Molecular Analyst (Fingerprinting Plus, version 1.12) software, and a dendrogram was calculated by the unweighted pair group method with arithmetic averages.

Nearly all fluoroquinolone resistance mechanisms remained stable in clinical and laboratory-derived mutants in the absence of antibiotic selective pressure. MIC changes of greater than twofold were considered significant alterations. The MIC decreases are presented in Table 1. The MIC for one laboratory-derived mutant showed slight decreases over the course of the study. The isolate, 2663M4, was an efflux-positive isolate that had been selected on moxifloxacin medium. During the course of study, the ciprofloxacin and moxifloxacin MICs decreased by 3.5- and 3-fold, respectively. Active efflux was still present at the termination of the study, regardless of the observed MIC decreases. Each isolate had the same pulsed-field pattern prior to and following the stability study.

Fluoroquinolone resistance mechanisms, efflux and chromosomally mediated, remain stable in both clinical and laboratory-derived mutants. These findings demonstrate that laboratory-derived mutants are a valuable tool in the analysis of fluoroquinolone resistance development. Additionally, this study implies that fluoroquinolone-induced alterations in S. pneumoniae are not likely to wane, even without the selective pressure of the antibiotics. Fluoroquinolone-resistant S. pneumoniae do not appear to spontaneously return to wild type in the absence of fluoroquinolone selective pressure.

 
H. Smith-Adam is supported by the CIHR-ICID training program. This research was funded in part by the University of Manitoba and Abbott Laboratories, Ltd., AstraZeneca Canada, Inc., Aventis Pharma, Bayer, Inc., Bristol-Myers Squibb Pharmaceutical Group, GlaxoSmithKline, Janssen-Ortho, Inc., Merck Frosst Canada & Co., Pfizer/Pharmacia Canada, Inc., and Wyeth-Ayerst Canada, Inc.

 
* Corresponding author. Mailing address: Clinical Microbiology, Health Sciences Centre, MS673-820 Sherbrook St., Winnipeg, Manitoba, R3A 1R9 Canada. Phone: (204) 787-4684. Fax: (204) 787-4699. E-mail: smithhj14{at}hotmail.com.

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Antimicrobial Agents and Chemotherapy, February 2005, p. 846-848, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.846-848.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Smith-Adam, H. J. Articles by Zhanel, G. G. Search for Related Content PubMed PubMed Citation Articles by Smith-Adam, H. J. Articles by Zhanel, G. G.