Phenotypic and genotypic characterization of macrolide-resistant group A Streptococcus strains in the province of Quebec, Canada

K. Weissa,c,*, J. De Azavedob, C. Restieria,c, L. A. Galarneauc, M. Gourdeauc, P. Harveyc, J. F. Paradisc, K. Salimb and D. E. Lowb

a Hôpital Maisonneuve-Rosemont, University of Montreal, Montreal, Quebec; b Mount Sinai Hospital, University of Toronto, Toronto, Ontario; c Groupe de Recherche sur les Anti-Microbiens (GRAM), Hôpital Maisonneuve-Rosemont, 5415 L'Assomption, Montreal, Quebec, Canada H1T 2M4


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Resistance to macrolides among group A streptococci is an increasing problem worldwide. We examined 496 strains phenotypically and genotypically for resistance to erythromycin and clindamycin. Strains were isolated in five different geographical areas representing about 45% of the total Quebec population. The overall resistance rate was 4.6% but varied from 0% in rural areas to 9.4% in Montreal. Of the 23 strains showing resistance to erythromycin, 15 (65%) had an identical pulsed-field gel electrophoresis pattern, were of serotype M28T28 and harboured the erm(TR) gene, suggesting the spread of a single clone. Of the remaining eight strains, two strains had the erm(B) gene, five had the mef gene and one with a different serotype also had the erm(TR) gene.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Resistance to macrolides in group A streptococci (GAS) was described in the UK in the late 1950s.1 Since then, resistance rates of <=50% have been reported in some countries, such as Italy.2 In Finland, resistance has been linked to erythromycin consumption.3 As macrolides represent the cornerstone of therapy for penicillin-allergic patients with GAS pharyngitis, emerging resistance has a clinical impact.

Resistance to macrolides in GAS arises by two distinct mechanisms: (i) ribosomal modification resulting from the presence of an Erm methylase; and (ii) drug efflux conferred by a membrane protein encoded by the mefA gene. Presence of an Erm methylase confers cross-resistance to erythromycin, lincosamides and streptogramins (MLSB phenotype). MLS resistance in GAS is encoded by two types of methylase gene: the erm(AM) [erm(B)] and the recently described erm(TR).4 The latter has also been associated with inducible resistance to clindamycin when using a double disc diffusion test. Resistance resulting from efflux is mediated by a surface protein encoded by the mefA gene. This resistance is specific for 14- and 15-member macrolides (erythromycin, azithromycin and clarithromycin); 16-member macrolides (e.g. josamycin) are not affected and neither are lincosamides or streptogramin B-type compounds (M-phenotype).5

In a previous study carried out in Ontario, 2.1% of GAS strains were resistant to erythromycin and resistance was mainly due to the presence of the mefA gene.6 In the present study, we examined macrolide resistance in strains isolated in the province of Quebec in order to determine the prevalence of erm and mef genes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Strains

A total of 496 strains of GAS were isolated from patients with pharyngitis in five acute care hospitals between September and December 1998. Three semi-rural or rural areas (Chicoutimi, Victoriaville and Rivière du Loup, 80 strains each) and two urban regions (Montreal, 156 strains; Quebec City, 100 strains) were represented. Throat samples originated mostly from community clinics; some were from hospital outpatient clinics. Each hospital collected one strain per patient. Patients comprised 270 females and 226 males with an age range of 0–64 years and a median age of 10.7 years. About 45% of the total Quebec population (7.5 million) was represented in this study. Strains were identified using standard procedures: ß-haemolysis, bacitracin sensitivity and a latex agglutination test (Streptex; Murex Biotech Ltd, Dartford, UK).

Susceptibility testing

MICs were determined by a broth microdilution method using Mueller–Hinton broth supplemented with 4% lysed horse blood. Testing and quality control were carried out following NCCLS criteria.7 Quality control was assessed by testing Streptococcus pneumoniae ATCC 49616 and Enterococcus faecalis ATCC 29212.

The following antibiotics were tested: penicillin, erythromycin, azithromycin, clarithromycin and clindamycin. A double-disc diffusion method using erythromycin (15 µg) and clindamycin (2 µg) discs was used to distinguish between an inducible and constitutive MLSB phenotype.6 M and T typing was carried out by the National Centre for Streptococci, Edmonton, Alberta, Canada.

Primers specific for erm(A), erm(B) and erm(C) (640 bp) and mefA and mefE (348 bp)8 were used to distinguish genotypically between ribosomal modification and efflux phenotypes in erythromycin-resistant strains. Primers used to amplify the erm(TR) gene were as described previously.6 Preparation of template DNA and PCR conditions were as described previously.6 DNA to be used for pulsed-field gel electrophoresis (PFGE) was extracted essentially as described by Murray et al.9 with minor modifications.

Antibiotic consumption for each area included in the study was provided by Intercontinental Medical Services Canada (IMS; Pointe Claire, Quebec, Canada). Linkage between level of resistance and antibiotic usage was done by recording the first three digits of the patient's postal code. The {chi}2 test was used to evaluate the significance of differences in group proportions.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The overall resistance of GAS to macrolides was 4.6% (23/496), but there was significant variation between the prevalence of resistance in different geographical areas. Resistance rates varied from 0% in a remote rural area to 9.4% in the Montreal region (Table IGo). The prevalence of resistant strains was not related to patients' gender or age. Of the resistant strains, 16 were from patients aged <20 years and seven (30%) from patients aged 21–39 years. Of all the macrolides tested, clarithromycin had the lowest MIC90 (0.03 mg/L), followed by erythromycin (0.06 mg/L) and azithromycin (0.25 mg/L). All the strains were highly susceptible to penicillin (MIC90 0.016 mg/L). The majority [15/23 (65%)] of the resistant strains belonged to the M28T28 serotype, had the same PFGE pattern and had inducible MLSB resistance (Table IIGo). All these strains harboured the erm(TR) determinant. All of the 16 erm(TR) strains appeared to be susceptible to clindamycin as judged by the broth microdilution assay (MIC90 0.06 mg/L) but showed inducible resistance to clindamycin in the double disc diffusion assay.


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Table I. Levels of resistance in 1998 in five geographical areas and macrolidea consumption (number of macrolide prescriptions/1000 population/year) (IMS data, Pointe Claire, Quebec, Canada)
 

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Table II. Phenotype and genotype of 23 macrolide-resistant strains
 
In Quebec, macrolide prescriptions increased from 100 prescriptions/1000 population in 1994 to 160 prescriptions/1000 population in 1998 (P < 0.001). However, whereas erythromycin usage decreased from 92/1000 in 1994 to 48/1000 in 1998 (P < 0.001), the consumption of clarithromycin and azithromycin increased (8/1000 to 112/1000; P < 0.001). There was no obvious link between macrolide usage and levels of resistance in 1998 (Table IGo).

Resistance of GAS to macrolides is a worldwide phenomenon, but the type of resistance encountered may differ from one area to the other. M-phenotype resistance, specified by the mefA gene, seems to be more prevalent in certain areas, such as Spain, where in one study, 97% of erythromycin-resistant strains were mefA positive.10 erm(B)-mediated resistance is more prevalent in certain areas and this is accompanied by high-level resistance to erythromycin (MIC90 > 128 mg/L). In Italy, 47.5% of resistant strains were categorized as MLSB resistant.2 erm- mediated resistance is more problematic clinically since macrolides may not be a therapeutic option in cases of highly resistant strains.

In vitro resistance may not necessarily correlate with clinical failure: an Italian study11 has found a limited correlation between in vitro resistance and failure to eradicate GAS in patients treated with macrolides.

The fact that 65% of the strains had a distinct clonal pattern demonstrates dissemination of a single clone throughout the province. A similar experience was noted in Spain where four clones represented 78.8% of all erythromycin-resistant strains.10 Thus, GAS resistance to macrolides may not be explained by macrolide consumption alone, but rather by clonal spread within the community.

Contrary to what has been found in Finland, where erythromycin consumption was associated with an increased level of resistance,3 preliminary data in Quebec seem to indicate lack of association with macrolide consumption. Although the number of strains in this study was low, resistant strains were either absent or very rare in certain areas, despite increased consumption of macrolides from 1996 to 1998. Preliminary data in Canada also suggest that macrolide resistance in S. pneumoniae may not correlate with antibiotic consumption since we are witnessing a decrease in the level of resistance and a marked increase in consumption of macrolides.12 Other factors—such as population density, a high proportion of children in nursery school and travel—may explain the higher prevalence of resistance in large urban centres. The fact that GAS are still universally susceptible to penicillin, despite 50 years of enormous usage of this compound all over the world, supports the concept that the equation of resistance is a little more complex than just linking antibiotic consumption and emerging resistance for this microorganism. With increasing consumption of macrolides in North America, long-term monitoring of macrolide resistance among GAS should be implemented.


    Acknowledgments
 
This study was supported in part by a grant from Abbott Canada. This study was presented in part at the Fifth International Conference on the Macrolides, Azalides, Streptogramins, Ketolides and Oxazolidinones, Seville, Spain, January 2000 (abstract 7.23).


    Notes
 
* Corresponding author. Hôpital Maisonneuve-Rosemont, 5415 L'Assomption, Montreal, Quebec, Canada H1T 2M4. Tel: +1-514-252-3817; Fax: +1-514-252-3898; E-mail: weisscan{at}aol.com Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Lowbury, E. J. L. & Hurst, L. (1959). The sensitivity of staphylococci and other wound bacteria to erythromycin, oleandromycin and spiramycin. Journal of Clinical Pathology 12, 163–9.[ISI][Medline]

2 . Giovanetti, E., Montanari, M. P., Mingoia, M. & Varaldo, P. E. (1999). Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducible resistant strains. Antimicrobial Agents and Chemotherapy 43, 1935–40.[Abstract/Free Full Text]

3 . Seppala, H., Klaukka, T., Vuopio-Varkila, J., Muotiala, A., Helenius, H., Lager, K. et al. (1997). The effect of changes in the consumption of macrolide antibiotics on erythromycin resistance in group A streptococci in Finland. Finnish Study Group for antimicrobial resistance. New England Journal of Medicine 337, 441–6.[Abstract/Free Full Text]

4 . Seppala, H., Skurnik, M., Soini, H., Roberts, M. & Huovinen, P. (1998). A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrobial Agents and Chemotherapy 42, 257–62.[Abstract/Free Full Text]

5 . Clancy, J., Petitpas, F., Dib-Hajj, F., Yuan, W., Cronan, M., Kamath, A. V. et al. (1996). Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA from Streptococcus pyogenes. Molecular Microbiology 22, 867–79.[ISI][Medline]

6 . De Azavedo, J., Yeung, R. H., Bast, D. J., Borgia, S. B. & Low, D. E. (1999). Prevalence and mechanisms of macrolide resistance in clinical isolates of group A streptococci from Ontario, Canada. Antimicrobial Agents and Chemotherapy 43, 2144–7.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.

8 . Sutcliffe, J., Grebbe, T., Tait-Kamradt, A. & Wondrack, L. (1996). Detection of erythromycin-resistant determinants by PCR. Antimicrobial Agents and Chemotherapy 40, 2562–6.[Abstract]

9 . Murray, B. E., Singh, K. V., Heath, J. D., Sharma, B. R. & Weinstock, G. M. (1990). Comparison of genomic DNAs of different enterococcal isolates using restriction endonucleases with infrequent recognition sites. Journal of Clinical Microbiology 28, 2059–63.[ISI][Medline]

10 . Perez-Trallero, E., Marimon, J. M., Montes, M., Orden, B. & De Pablos, M. (1999). Clonal differences among erythromycin-resistant Streptococcus pyogenes in Spain. Emerging Infectious Diseases 5, 235–40.[ISI][Medline]

11 . Varaldo, P. E., Debbia, E. A., Nicoletti, G., Pavieso, D., Ripa, S., Schito, G. et al. (1999). Nationwide survey in Italy of treatment of Streptococcus pyogenes pharyngitis in children: influence of macrolide resistance on clinical and microbiological outcomes. Clinical Infectious Diseases 29, 869–73.[ISI][Medline]

12 . Green, K., McGeer, A., Zhanel, G., Hoban, D., Weiss, K., Davidson, R. et al. (2000). Decreasing penicillin and macrolide resistance in Streptococcus pneumoniae in Canada: who's driving whom? In Program and Abstract of the Fifth International Congress on Macrolides, Azalides, Streptogramins, Ketolides and Oxazilidinones, Seville, Spain, 26–28 January 2000. Abstract 7.20, p. 67.

Received 7 August 2000; returned 19 September 2000; revised 9 October 2000; accepted 30 September 2000