Antimicrobial resistance in Streptococcus pyogenes isolates in Berlin

Mardjan Arvanda,*, Marlies Hoeckb, Helmut Hahna and Jutta Wagnera

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A total of 212 clinical Streptococcus pyogenes isolates were tested for susceptibility to various antibiotics by agar dilution. The overall frequency of erythromycin resistance was 12.7%, being higher in isolates from children (18.9%) than in those from adult patients (10.7%). Similar results were found for clarithromycin, while 2.8% of the isolates were resistant to ciprofloxacin. All strains were susceptible to penicillin and cefotaxime. Of the erythromycin-resistant isolates subjected to the double-disc diffusion test for erythromycin and clindamycin, 35% expressed constitutive and 55% inducible resistance to clindamycin. Ten per cent were susceptible to clindamycin (M-phenotype). Thus, a high rate of macrolide resistance in S. pyogenes has emerged in Berlin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Increasing antimicrobial resistance of Streptococcus pyogenes has been observed during the last decade in Europe and worldwide. In 1992 Seppälä et al.1 reported a high rate of erythromycin resistance (44%) in Finland, linked to an increased rate of erythromycin consumption in outpatients. High frequencies of erythromycin resistance have also been reported from Spain (17.1%), Italy (>40%) and the USA (32%).2,3 On the other hand, resistance rates as low as 0.5% and 1.6% have been found in The Netherlands4 and in southwestern Germany,5 respectively.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacteria

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 (17–91 years) and 53 from children (8 months–16 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 103–104 cfu were applied by means of a multipoint inoculator and plates were incubated overnight at 37°C in an atmosphere of 5–7% 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.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Of the 212 S. pyogenes isolates, 27 (12.7%) were resistant to erythromycin, and one isolate (0.5%) showed intermediate susceptibility (Table IGo). The resistance rate was higher in isolates from children (18.9%) than in those from adults (10.7%). Although the difference was not statistically significant (P = 0.16, chi-squared test), these results suggest that children might be more likely to be infected with erythromycin-resistant strains. Erythromycin resistance was 10% among invasive strains (n = 10) isolated from blood cultures and CSF, and 12.9% among non-invasive strains (n = 202). Our data are in accordance with the results of Fierek et al.,9 who found an erythromycin resistance rate of 10% among a collection (number not specified) of S. pyogenes isolates from respiratory tract infections during 1995–1996 in Greifswald, north-eastern Germany. On the other hand, in 1997 Traub & Leonhard5 reported an erythromycin resistance rate of 1.6% among 63 S. pyogenes isolates in Homburg/Saar, south-western Germany. This discrepancy might be due to differences in the time point of collecting the isolates, the site of infection or the age group of the patients studied, since these parameters were not specified in the latter study. However, the frequency of erythromycin resistance in S. pyogenes might also vary considerably between different geographical regions of Germany.


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Table I. Susceptibility to erythromycin among Streptococcus pyogenes isolates in Berlin
 
All erythromycin-resistant strains were also resistant to clarithromycin and vice versa. We did not find any significant differences between clarithromycin and erythromycin with respect to MIC range, MIC50 and MIC90. All strains were susceptible to penicillin and cefotaxime (Table IIGo), confirming that penicillin resistance has not developed in S. pyogenes.


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Table II. MICs (mg/L) of antimicrobial agents tested for 212 Streptococcus pyogenes isolates in Berlin
 
The MIC50 and MIC90 of ciprofloxacin were 0.5 and 1 mg/L, respectively. Six isolates revealed an MIC value of 4 mg/L. NCCLS7 has not defined MIC breakpoints of ciprofloxacin for S. pyogenes. According to the DIN guidelines,8 the resistant and susceptible breakpoints for ciprofloxacin are >=;4 mg/L and <=;1 mg/L, respectively, although these breakpoints have not been specifically defined for S. pyogenes. Applying these DIN breakpoints, the ciprofloxacin resistance rate was 2.8% (Table IIGo).

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.


    Notes
 
*Corresponding author. Tel: +49-30-8445-3602; Fax: +49-30-8445-3830; E-mail: arvand{at}zedat.fu-berlin.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Seppälä, H., Nissinen, A., Jarvinen, H., Huovinen, S., Henriksson, T., Herva, E. et al. (1992). Resistance to erythromycin in group A streptococci. New England Journal of Medicine 326, 292–7.[Abstract]

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, 255–60.[ISI][Medline]

3 . York, M. K., Gibbs, L., Perdreau-Remington, F. & Brooks, G. F. (1999). Characterization of antimicrobial resistance in Streptococcus pyogenes isolates from the San Francisco Bay area of Northern California. Journal of Clinical Microbiology 37, 1727–31.[Abstract/Free Full Text]

4 . Van Asselt, G. J., Sloos, J. H., Mouton, R. P., Van Boven, C. P. & Van de Klundert, J. A. (1995). Susceptibility of Streptococcus pyogenes to azithromycin, clarithromycin, erythromycin and roxithromycin in vitro. Journal of Medical Microbiology 43, 386–91.[Abstract]

5 . Traub, W. H. & Leonhard, B. (1997). Comparative susceptibility of clinical group A, B, C, F, and G beta-hemolytic streptococcal isolates to 24 antimicrobial drugs. Chemotherapy 43, 10–20.[ISI][Medline]

6 . Olsson-Liljequist, B., Larsson, P., Walder, M. & Miörner, H. (1997). Antimicrobial susceptibility testing in Sweden, methodology for susceptibility testing. Scandinavian Journal of Infectious Diseases Suppl. 105, 13–23.[Medline]

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.

9 . Fierek, O., Hinniger, P. & Panzig, B. (1998). Antibiotic sensitivity of important pathogens of bacterial respiratory tract infections in Northeast Germany. Medizinische Klinik 93, 656–61.[ISI][Medline]

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, 786–9.[ISI][Medline]

Received 5 January 2000; returned 26 March 2000; revised 9 May 2000; accepted 17 June 2000