a Institut für Infektionsmedizin, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 27, 12203 Berlin; b DRK-Klinik Mark-Brandenburg, Drontheimer Strasse 39, 13359 Berlin, Germany
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Abstract |
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Introduction |
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This study was performed to determine the frequency of erythromycin resistance among S. pyogenes isolates in Berlin and to survey resistance to other antimicrobial agents. Previous studies have shown that erythromycin resistance might correlate with the patient's age; therefore, isolates from children and adult patients were examined in this study. Erythromycin-resistant strains were further analysed by a double-disc diffusion test with clindamycin and erythromycin to elucidate the underlying resistance mechanism.
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Materials and methods |
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A total of 212 consecutive isolates of S. pyogenes were collected in Berlin from November 1996 to 1998. Of these, 159 isolates were obtained from adult patients (1791 years) and 53 from children (8 months16 years); 153 isolates were from pus samples and 59 from throat swabs. Identification was performed by routine laboratory techniques including latex agglutination (Streptex, Murex Biotech Ltd, Dartford, UK).
Determination of MICs
MICs of erythromycin (Sigma, Deisenhofen, Germany), clarithromycin (Abbot, Queensborough, UK), ciprofloxacin (Bayer, Leverkusen, Germany), penicillin (Grünenthal, Aachen, Germany) and cefotaxime (Hoechst Marion Roussel, Frankfurt, Germany) were determined by an agar dilution method on Iso-Sensitest agar (Oxoid, Wesel, Germany) with 5% human blood. Inocula of 103104 cfu were applied by means of a multipoint inoculator and plates were incubated overnight at 37°C in an atmosphere of 57% CO2. The following organisms were used as controls: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212 and two S. pyogenes strains recommended by the Swedish Reference Group for Antibiotics,6 S. pyogenes Culture Collection, University of Göteborg, Sweden (CCUG) 25570 (erythromycin MIC 16 mg/L), and S. pyogenes CCUG 25571 (erythromycin MIC 0.032 mg/L). All control strains gave MICs within two dilution steps of target values. NCCLS breakpoints7 were used for all antimicrobial agents except ciprofloxacin, for which the German Institute of Antimicrobial Susceptibility Testing (DIN) breakpoint8 was used.
Erythromycin and clindamycin double-disc diffusion test
This test was performed to classify the erythromycin resistance phenotype.3 A disc containing 15 µg erythromycin was placed 20 mm from the centre of a disc containing 10 µg clindamycin on blood agar. Blunting of the clindamycin inhibition zone proximal to the erythromycin disc was taken to indicate an inducible resistance phenotype. Resistance to both erythromycin and clindamycin indicated constitutive resistance, and susceptibility to clindamycin with no blunting indicated the M-phenotype.
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Results and discussion |
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Clindamycin resistance was not detected among the erythromycin-susceptible strains by agar diffusion. However, of the 20 erythromycin-resistant strains subjected to the double-disc diffusion test for erythromycin and clindamycin, seven (35%) expressed constitutive resistance to clindamycin. Eleven strains (55%) showed resistance to clindamycin only when exposed to the double-disc diffusion test, suggesting an inducible resistance phenotype. Thus, 90% of the erythromycin-resistant isolates also expressed resistance to clindamycin, indicating a macrolide, lincosamide and streptogramin B (MLSB) resistance phenotype caused by conformational changes in the prokaryotic ribosome.10 Two (10%) strains were susceptible to clindamycin even when exposed to the double-disc diffusion test, indicating the M-phenotype, which is probably caused by a drug efflux mechanism.10 In Finland, the proportion of M-phenotype among erythromycin-resistant strains has increased from 40% in 1990 to 80% in 1994, indicating a clonal spread of this phenotype.10 Our data indicate that in Berlin the prevalence of this phenotype is rather low.
In conclusion, this study demonstrates a high rate of erythromycin resistance in S. pyogenes in the Berlin area of Germany and emphasizes the need to screen for antimicrobial resistance among S. pyogenes.
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Notes |
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References |
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2 . Betriu, C., Casado, M. C., Gomez, M., Sanchez, A. Palau, M. L., Picazo, J. J. et al. (1999). Incidence of erythromycin resistance in Streptococcus pyogenes: a 10-year study. Diagnostic Microbiology and Infectious Diseases 33, 25560.[ISI][Medline]
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7 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing; Ninth Informational Supplement M100-S9. NCCLS, Wayne, PA.
8 . Normenausschuss Medizin im DIN Deutsches Institut für Normung e.V. (1999). Susceptibility Testing of Pathogens to Antimicrobial Agents, Part 4, Suppl. 1, DIN 58940-4. DIN, Berlin, Germany.
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10 . Kataja, J., Huovinen, P., Muotiala, A., Vuopio-Varkila, J., Efstratiou, A., Hallas, G. et al. (1998). Clonal spread of group A streptococcus with the new type of erythromycin resistance. Finnish Study Group for Antimicrobial Resistance. Journal of Infectious Diseases 177, 7869.[ISI][Medline]
Received 5 January 2000; returned 26 March 2000; revised 9 May 2000; accepted 17 June 2000