1Institute of Medical Microbiology, National Reference Center for Streptococci, University of Aachen, Pauwelsstrasse 30, D-52074 Aachen; 2Institute of Medical Microbiology and Virology, University Hospital Düsseldorf, Germany
Received 13 July 2001; returned 29 November 2001; revised 6 February 2002; accepted 19 February 2002.
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Abstract |
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Introduction |
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The aim of the present study was to characterize mutations in the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE of pneumococcal strains isolated in Germany, with ciprofloxacin MICs of 4 mg/L. In addition, the study was aimed at determining the activity of a panel of quinolone agents against these strains and the detection of multiply resistant isolates.
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Materials and methods |
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The MIC of ciprofloxacin was determined in the presence and absence of reserpine (20 mg/L; Sigma, Frankfurt, Germany), an inhibitor of efflux pumps, in order to determine the presence of an efflux mechanism, as described previously.5 MIC determinations in the presence and absence of reserpine were conducted three times. Efflux activity was assumed if a difference of at least two doubling dilutions in ciprofloxacin MICs with and without reserpine was observed. As controls, isolates with no efflux activity or three doubling dilutions difference derived from selection experiments described by Boos et al.6 were used. Prepared chromosomal DNA was used as a template for PCR amplification of target QRDRs. The primers and PCR conditions were those previously defined.79
The PCR products were purified using a PCR purification kit (Qiagen, Ratingen, Germany) and sequenced using the dye terminator method in both forward and reverse directions. The products were then resolved and analysed automatically using an ABI PRISM 310 DNA sequencer. In all isolates with reduced susceptibility to fluoroquinolones and with decreased sensitivity to erythromycin, clindamycin or tetracycline, the underlying resistance mechanisms were characterized. For amplification of tetM the following primers were used: 5'-TGG AAT TGA TTT ATC AAC GG-3' (position 24962515) and 5'-TTC CAA CCA TAC AAT CCT TG-3' (position 35753556). For the detection of erm and mef the following primers were chosen: erm, 5'-CGA GTG AAA AAG TAC TCA ACC-3' (position 362382), 5'-GGC GTG TTT CAT TGC TTG ATG-3' (position 978958); mef, 5'-AGT ATC ATT AAT CAC TAG TGC-3' (position 5777), 5'-GTA ATA GAT GCA ATC ACA GC-3' (position 551532).3
Pneumococcal strains were serotyped by Neufelds Quellung reaction using type and factor sera provided by the Statens Serum Institut, Copenhagen, Denmark.
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Results and discussion |
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The significance of parE and gyrB mutations in fluoroquinolone resistance remains unclear. Previous authors have assigned no role to alterations of ParE and GyrB in isolates with reduced susceptibility to fluoroquinolones.10 In our pneumococcal strains, no alterations were found in gyrB, whereas seven of 14 strains displayed the frequently identified Ile-460Val change, which seemed to have no clear-cut effect on MICs. Eleven of the 14 S. pneumoniae strains displayed ciprofloxacin MICs two to five doubling dilutions lower in the presence of reserpine, indicating the important contribution of efflux to decreased susceptibility to quinolones. The greatest efflux activity was observed in a serotype 16F isolate (PS 2378), which exhibited no alterations in GyrA or ParC.
The serotype distribution of strains with reduced susceptibility to fluoroquinolones differs widely from those found among penicillin non-susceptible (predominant serotypes 23F and 6B) and macrolide-resistant isolates (predominant serotypes 14 and 6B). Multiple resistance among strains with reduced susceptibility to fluoroquinolones is rare in Germany. Only four of 14 strains showed resistance equal to or more than three classes of antibiotic. Of the pneumococci showing decreased susceptibility to fluoroquinolones, one also showed reduced sensitivity to penicillin G, three strains were erythromycin resistant (one strain mef positive, one strain erm positive, one strain neither erm positive nor mef positive), and two strains were also tetracycline resistant (all tetM positive). Cross-resistance is more likely to occur among strains with resistance to ß-lactams, macrolides or tetracycline compared with those with reduced susceptibility to fluoroquinolones.
In conclusion, the rate of strains showing decreased sensitivity to fluoroquinolones is very low in Germany, as documented by the present work and other studies from Germany. Nevertheless, the broad usage of new fluoroquinolones may lead to increases in the prevalence of fluoroquinolone resistance among pneumococci.
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Footnotes |
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References |
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2
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Reinert, R. R., Al-Lahham, A., Lemperle, M., Tenholte, C., Briefs, C., Haupts, S. et al. (2002). Emergence of macrolide and penicillin resistance among invasive pneumococcal isolates in Germany. Journal of Antimicrobial Chemotherapy 49, 618.
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Reinert, R. R., Simic, S., Al-Lahham, A., Reinert, S., Lemperle, M. & Lütticken, R. (2001). Antimicrobial resistance of Streptococcus pneumoniae recovered from outpatients with respiratory tract infections in Germany from 1998 to 1999: results of a national surveillance study. Journal of Clinical Microbiology 39, 11879.
4 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA.
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Boos, M., Mayer, S., Fischer, A., Kohrer, K., Scheuring, S., Heisig, P. et al. (2001). In vitro development of resistance to six quinolones in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 45, 93842.
7 . 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]
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Schmitz, F. J., Jones, M. E., Hofmann, B., Hansen, B., Scheuring, S., Luckefahr, M. et al. (1998). Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC. Antimicrobial Agents and Chemotherapy 42, 124952.
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10 . Munoz, R. & De La Campa, A. G. (1996). ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype. Antimicrobial Agents and Chemotherapy 40, 22527.[Abstract]