Journal of Antimicrobial Chemotherapy (2001) 47, 655-658
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Target site modifications and efflux phenotype in clinical isolates of Streptococcus pneumoniae from Hong Kong with reduced susceptibility to fluoroquinolones
a Department of Microbiology, f School of Professional and Continuing Education, The University of Hong Kong; b Department of Microbiology, Queen Mary Hospital; c Department of Clinical Pathology, Tuen Mun Hospital; d Department of Clinical Pathology, Queen Elizabeth Hospital; e Department of Clinical Pathology, Princess Margaret Hospital, Hong Kong SAR, China
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Abstract |
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Ciprofloxacin-susceptible (n = 7) and -resistant (MIC
4 mg/L) (n = 15) clinical isolates of Streptococcus pneumoniae from diverse sources in Hong Kong were studied for target site modifications and efflux phenotype. Reserpine-inhibited efflux of ciprofloxacin and/or levofloxacin was common in both susceptible and non-susceptible isolates. The ParC substitutions K137N and/or S79F or Y were associated with increased ciprofloxacin MICs. The GyrA substitution S81F was only found in isolates with full resistance to ciprofloxacin (MIC 16 mg/L) and levofloxacin (MIC 8 mg/L). Among clinical isolates of S. pneumoniae, accumulation of target site mutations in strains with an efflux mechanism was associated with increasing MICs of fluoroquinolones. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The primary targets of fluoroquinolones are DNA gyrase and topoisomerase IV. Both enzymes are tetramers with two different subunits. In pneumococci, fluoroquinolone resistance generally occurs as a result of amino acid substitutions in the quinolone resistance-determining region (QRDR) of these enzymes. In addition, active efflux was found to be a mechanism of resistance to some fluoroquinolones in Streptococcus pneumoniae. Although resistance to the fluoroquinolones remains rare in most countries, the percentage of S. pneumoniae non-susceptible to fluoroquinolones increased from <0.5% for ofloxacin to 5.5% for levofloxacin (MIC > 2 mg/L) between 1995 and 1998 in Hong Kong. In this study, fluoroquinolone resistance mechanisms in clinical isolates of S. pneumoniae with increased MICs of ciprofloxacin were characterized.1 The mutations in gyrA, gyrB, parC and parE associated with fluoroquinolone resistance were determined. The presence of a quinolone efflux mechanism was determined by comparing MICs in the presence and absence of reserpine (a known efflux pump inhibitor).
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Materials and methods |
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Bacterial strains
One-hundred and eighty-one non-duplicate isolates of S. pneumoniae were isolated from consecutive clinical specimens submitted to four hospital laboratories during the second half of 1998.1 These four laboratories together serve about half of the population in Hong Kong. Of the 181 isolates, 15 ciprofloxacin-resistant and seven ciprofloxacin-susceptible isolates were examined in the present study. The number of isolates from each laboratory was as follows: A (nine isolates), B (two isolates), C (three isolates) and D (eight isolates). All except one (from a patient with bacteraemia) were isolated from sputa of patients with lower respiratory tract infections. Organisms were stored on porous beads at –70°C before testing. S. pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were used as quality control strains.
Antimicrobial agents and MIC determinations
Ciprofloxacin and levofloxacin powders with known potencies were kindly provided by Bayer China Company, Ltd (Hong Kong, China) and the R. W. Johnson Pharmaceutical Research Institute (Raritan, NJ, USA), respectively. Etest strips containing penicillin, trovafloxacin and moxifloxacin were purchased from AB Biodisk (Solna, Sweden). MICs were determined by the Etest and NCCLS broth microdilution (BMD) methods.2 All MICs were determined at least twice. Discrepant results were repeated. MIC results were interpreted according to NCCLS criteria and, for moxifloxacin, the criteria of the British Society for Antimicrobial Chemotherapy.2,3 For ciprofloxacin, MIC 4 mg/L was used to define resistance.
Active efflux of fluoroquinolones
MICs of ciprofloxacin and levofloxacin were determined by the BMD method with and without reserpine (10 mg/L) in cation-adjusted Mueller–Hinton broth supplemented with 2.5% lysed horse blood. MIC determinations were performed at least twice. Discrepant results were repeated. A reproducible reduction of MIC (by two-fold or more) in the presence of reserpine was considered to indicate active efflux.
PCR and DNA sequencing
The QRDRs of gyrA, gyrB, parC and parE were amplified using primers described previously.4,5 Nucleotide sequencing was performed by Bigdye dideoxynucleotide chain termination method using the ABI PRISM 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA, USA). For all the isolates, the sequences of both strands of the amplicons were determined.
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Results |
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The Table
shows the MICs of four quinolone antibiotics, together with the efflux phenotype and mutations in the gyrA, gyrB, parC and parE genes for 22 isolates of S. pneumoniae. All MICs were reproducible on repeated testing. Efflux of ciprofloxacin was found in 6/7 of the ciprofloxacin-susceptible and 14/15 ciprofloxacin-resistant isolates. Efflux of levofloxacin was found in 3/12 levofloxacin- susceptible and 4/10 levofloxacin-non-susceptible isolates. In GyrA, eight isolates had mutations that resulted in Ser-81 being substituted by phenylalanine. No amino acid change was detected in GyrB. Sixteen isolates had at least one amino acid substitution in ParC and seven of these had two substitutions, being a S79F or Y plus K137N pair. In ParE, 20/22 isolates had at least one substitution, and three of these had two and one had three substitutions.
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Discussion |
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This study revealed target site modifications in all the isolates with reduced susceptibility to fluoroquinolones. Similar to mutants selected in the laboratory, most of the amino acid substitutions in the QRDR were present in ParE and ParC, while few changes occurred in GyrA. Resistance that occurs as a result of substitutions in GyrA alone is not commonly encountered in clinical isolates. Of 69 isolates examined by Jones et al.,6 only five had a single amino acid substitution in GyrA alone. In the present study, all GyrA substitutions were found in the levofloxacin-resistant (MIC
8 mg/L) isolates, and they occurred together with various substitutions in ParC and ParE. In comparison, group 2 isolates with changes in ParC and ParE alone remained susceptible to levofloxacin. These findings thus suggested that first-step mutants (ParC and ParE) might be an initial stage in the development of high-level fluoroquinolone resistance, which has arisen in vivo by an additional GyrA substitution. Gene sequencing of gyrA, parC and parE in this study shows that substitutions with a possible association with quinolone resistance in clinical strains of pneumococci are S81F in GyrA, S79F, S79Y and K137N in ParC, and D435N, P454S, I460V and E474K in ParE. Similar to previous reports,6 the two classical substitutions, S81F in GyrA and S79F or Y in ParC, were clearly associated with resistance among our strains. This study shows that ParE substitutions, including I460V, E474K and D435N, are not obviously associated with significant resistance to any of the fluoroquinolones. Absence of effect on susceptibility for I460V and D435N in ParE has been reported by some,6,7 while others8 found that they might produce low-level resistance to some fluoroquinolones. Although K137N in ParC was suggested to be unimportant,6 our finding indicated that the S81F (GyrA) + K137N (ParC) pair can bring about resistance to levofloxacin, trovafloxacin and moxifloxacin. Nonetheless, the effect of double mutations in ParC (S79F or Y and K137N) with and without S81F in GyrA did not seem to be additive. This study identified reserpine-inhibited efflux of ciprofloxacin in almost all the ciprofloxacin-susceptible and -resistant isolates, similar to that reported earlier.7 Efflux of levofloxacin was also common, although at a lower frequency in both levofloxacin-susceptible and levofloxacin-non-susceptible isolates. This study and those of others7 indicate that antimicrobial drug efflux on its own is not a common cause of high-level resistance. It might, however, facilitate mutational resistance by permitting short-term bacterial survival.9 Patient information was available for nine of the 15 patients with ciprofloxacin-resistant pneumococci. Seven had chronic obstructive pulmonary disease. Six patients had been treated with one or more fluoroquinolones [ofloxacin (2/6), ciprofloxacin (2/6) and levofloxacin (5/6)] before isolation of these organisms. In conclusion, increasing MICs to a panel of fluoroquinolones were associated with accumulation of target site modifications in those strains with a reserpine-inhibited efflux mechanism. To prevent resistance development, the use of efflux-susceptible and the less active fluoroquinolones for pneumococcal infection should be avoided.
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Acknowledgments |
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We thank Frankie Chow and Terrence Cheung for technical assistance, and Frances Wong for secretarial services. This study was supported by a grant from the Research Committee, The University of Hong Kong. Part of this work was presented at the Thirty-ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999.
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Notes |
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* Correspondence address. Division of Infectious Diseases, Department of Microbiology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Pokfulam, Hong Kong SAR, China. Tel: +852-2855-4897; Fax: +852-2855-1241; E-mail: plho{at}hkucc.hku.hk
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References |
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1 . Ho, P. L., Que, T. L., Tsang, D. N., Ng, T. K., Chow, K. H. & Seto, W. H. (1999). Emergence of fluoroquinolone resistance among multiply resistant strains of Streptococcus pneumoniae in Hong Kong. Antimicrobial Agents and Chemotherapy 43, 1310–3.
2 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing—Ninth Informational Supplement M100-S9. NCCLS, Villanova, PA.
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Janoir, C., Zeller, V., Kitzis, M. D., Moreau, N. J. & Gutmann, L. (1996). High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrobial Agents and Chemotherapy 40, 2760–4.
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Broskey, J., Coleman, K., Gwynn, M. N., McCloskey, L., Traini, C., Voelker, L. et al. (2000). Efflux and target mutations as quinolone resistance mechanisms in clinical isolates of Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 45, Suppl. 1, 95–9.
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Beyer, R., Pestova, E., Millichap, J. J., Stosor, V., Noskin, G. A. & Peterson, L. R. (2000). A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrobial Agents and Chemotherapy 44, 798–801.
Received 7 September 2000; returned 2 January 2001; revised 7 February 2001; accepted 20 February 2001
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