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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Introduction |
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Materials and methods |
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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 MuellerHinton 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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Discussion |
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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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Notes |
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References |
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2 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility TestingNinth Informational Supplement M100-S9. NCCLS, Villanova, PA.
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Andrews, J. M., Ashby, J. P., Jevons, G. M. & Wise, R. (1999). Tentative minimum inhibitory concentration and zone diameter breakpoints for moxifloxacin using BSAC criteria. Journal of Antimicrobial Chemotherapy 44, 81922.
4 . 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, 27604.[Abstract]
5 . Pan, X. S., Ambler, J., Mehtar, S. & Fisher, L. M. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 23216.[Abstract]
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Jones, M. E., Sahm, D. F., Martin, N., Scheuring, S., Heisig, P., Thornsberry, C. et al. (2000). Prevalence of gyrA, gyrB, parC, and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from worldwide survillance studies during the 19971998 respiratory season. Antimicrobial Agents and Chemotherapy 44, 4626.
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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, 959.
8 . Perichon, B., Tankovic, J. & Courvalin, P. (1997). Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 11667.[Abstract]
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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, 798801.
Received 7 September 2000; returned 2 January 2001; revised 7 February 2001; accepted 20 February 2001