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Antimicrob Agents Chemother. May 2006; 50(5): 1896–1899.
PMCID: PMC1472204

Macrolide-Resistant Streptococcus pyogenes in Norway: Population Structure and Resistance Determinants

P. Littauer,1,* D. A. Caugant,2,3 M. Sangvik,1 E. A. Høiby,2 A. Sundsfjord,1,2 G. S. Simonsen,1,2 and the Norwegian Macrolide Study Group

Abstract

A 2.7% prevalence of macrolide resistance in 1,657 Norwegian clinical Streptococcus pyogenes isolates was primarily due to erm(TR) (59%) and mef(A) (20%). Four clonal complexes comprised 75% of the strains. Macrolide resistance in S. pyogenes in Norway is imported as resistant strains or locally selected in internationally disseminated susceptible clones.

The emergence of macrolide resistance in one of the most challenging human bacterial pathogens, Streptococcus pyogenes (group A streptococci [GAS]), is an increasing problem worldwide (11, 14, 27, 36). Although penicillin is the drug of choice, macrolides are used for patients who are allergic to beta-lactams and in combination therapy for severe infections. The principal mechanisms of macrolide resistance in GAS include target site modification encoded by erm genes and active drug efflux due to mef genes (34, 37, 39). Two classes of methylase genes, erm(B) and erm(A) subclass erm(TR) [hereafter designated erm(TR)], (29) have been described in GAS and encode cross-resistance to macrolides, lincosamides, and streptogramin B (MLSB). They are expressed either constitutively (cMLSB phenotype) or inducibly (iMLSB phenotype). Efflux-encoding mef genes have been assigned to a single class of mef(A) genes (29), but recent work distinguishes between mef(A) (first identified in GAS) and mef(E) (originally described in S. pneumoniae) because of important genetic differences (6). Two new subclasses of mef genes have recently been reported: mef(I) in pneumococci (4) and mef(O) in GAS (30). Giovanetti et al. have described a putative novel efflux system, which contributes to high-level macrolide resistance in some erm(TR)-positive GAS strains (12), and msr(D) has also proved necessary for drug efflux (5). The aim of the present study was to examine the prevalence of macrolide-resistant GAS in Norway and perform a population structure analysis of resistant strains and their resistance determinants.

A total of 1,657 GAS from clinical infections were consecutively collected within three separate periods from 1993 to 2002 in Norway (Table (Table1).1). Forty-four strains (2.7%) showed reduced susceptibility to erythromycin (MIC ≥ 1 mg/liter) and were further investigated. Antimicrobial susceptibility testing and determination of macrolide resistance phenotype and genotype and tetracycline resistance genotype was performed as previously described (9, 16, 25, 32, 33, 37, 38). Subtyping of mef was achieved by BamHI digestion of the 346-bp mef amplicon (22). mef genes were DNA sequenced and compared using BLAST as previously described (30). T-typing, opacity factor analysis, emm typing, and multilocus sequence typing (MLST) were performed as previously described (18) and according to published protocols (http://www.cdc.gov/ncidod/biotech/strep/protocols.htm and http://spyogenes.mlst.net).

TABLE 1.
Epidemiological and clinical origin of 44 Norwegian macrolide-resistant S. pyogenes isolates included in the study

Of 1,657 S. pyogenes isolates, 44 (2.7%) were macrolide resistant (Table (Table1).1). The prevalence of resistance was significantly higher in isolates from wounds (5.8%) than in isolates from the respiratory tract (2.3%) (P = 0.002). iMLSB dominated among the 33 of 44 (75%) strains displaying an MLSB phenotype encoded by erm(TR) (n = 26), erm(B) (n = 6), or erm(TR) and mef(E) (n = 1). M phenotype strains (n = 11) harbored either mef(A) (n = 9) or a recently described mef gene (n = 2) (30), which is hereafter designated mef(O). Tetracycline resistance (MIC ≥ 8 mg/liter) was found in 31 of 44 (70%) strains, predominantly MLSB strains (29 of 33 [88%]), and was encoded by either tet(M) (n = 23) or tet(O) (n = 8). erm(B) and mef(O) were exclusively associated with tet(M), whereas erm(TR) was also found in strains possessing tet(O) (Table (Table2).2). The iMLSB, erm(TR)-carrying strains harboring tet(M) had lower erythromycin MICs compared to strains carrying tet(O) (data not shown).

TABLE 2.
Macrolide resistance genotypes and phenotypes, tetracycline resistance genotypes, T-types, emm types, and MLST of 44 S. pyogenes isolates

The emm types were all present in the Centers for Disease Control and Prevention database, and 31 of 44 strains (70%) had sequence types (STs) previously defined in the MLST database (Table (Table2)2) (10, 20, 28, 36). A total of 13 strains revealed new allele combinations and were assigned to new STs (Table (Table33).

TABLE 3.
Allelic profiles of new STs in macrolide-resistant S. pyogenes from Norway

The prevalence of resistance was low (2.7%), which is comparable to rates in Denmark (3%) and Canada (4.7%), but in contrast to rates in Finland (7.4%), Poland (12%), Germany (14%), Spain (29.7%), and Greece (38%) (2, 3, 23, 27, 35, 36, 40). erm(TR) dominated in our material as in Poland, Bulgaria, and Canada (8, 36, 40) but in contrast to reports from Spain, Belgium, Greece, Argentina, and Finland where M type resistance encoded by mef(A/E) predominates (2, 7, 14, 17, 35).

A significant association between macrolide usage and macrolide resistance in S. pyogenes has been documented in Finland (31). Albrich et al. found a direct association between the antibiotic selection pressure and the prevalence of macrolide resistant S. pyogenes on a national level in 16 industrialized countries from 1994 to 2000 (1). Total macrolide usage in Norway is comparable to Finland, but the prevalence of macrolide resistance in S. pyogenes has remained lower in our country. Goossens et al. reported that azithromycin usage is widespread in Finland, whereas erythromycin is more commonly used in Norway (13). Whether this observation can explain the differences in macrolide resistance rates warrants further investigation. Coresistance was primarily associated with tetracycline, and tet(M) was the most common determinant. Since coresistance to tetracycline predominated among MLSB strains in our study, it is likely that tetracycline resistance determinants were genetically linked to erm genes rather than to mef(A)-containing elements.

Population structure analysis revealed four clonal complexes (CCs) defined by emm typing, T typing, and MLST (33 of 44 [75%]). Three of the CCs displayed the erm(TR)-encoded MLSB phenotype and were coresistant to tetracycline. ST46 in CC1 has been associated with erm(B), mef(A/E), or macrolide susceptibility in other countries (20, 28, 36), whereas ST63 in CC2 contained erm(TR) in Poland and Germany in accordance with our findings (28, 36). The remaining six strains in CC2 belonged to ST369, which is a single locus variant (SLV) of ST63 not previously registered in the MLST database. CC3 comprised erm(TR)-positive ST340, which is a novel SLV of the Australian ST205 but carrying a mef gene (21). CC4 was monoresistant to macrolides due to mef(A). This clone has previously been observed in Finland, Germany, Spain, and England (14, 20, 26, 28).

McGeer et al. have recently described local selection and clonal spread as two fundamentally different evolutionary mechanisms for the emergence and dissemination of antimicrobial resistance (19). Our findings elucidate the relationship between these two mechanisms in Norway. Most isolates (43 of 44 [98%]) were indistinguishable or closely related to internationally reported clones, suggesting import and clonal spread. However, several STs (ST340, ST369, and ST46) displayed differences in their content of resistance genes. This may indicate that they have acquired their determinants in response to local antibiotic selection, but the paucity of macrolide susceptible strains in our study precludes any definite conclusion on this issue. The regional dissemination of the M type ST39 clone in both high- and low-consumption countries is intriguing, and the selective advantage of this clone warrants further studies. Furthermore, mef(A) was recently shown to predominate among macrolide resistant S. pneumoniae in Norway (16). The evolutionary mechanism(s) for the successful spread of this efflux mechanism between streptococci remain(s) to be elucidated.

Acknowledgments

We thank the Northern Norway Regional Health Authority for financial support.

We thank Torill Alvestad, Bjørg Haldorsen, Aase-Mari Kaspersen, and Jan Oknes for technical assistance. This publication made use of the Multi Locus Sequence Typing Web site (http://www.mlst.net), which was developed by Man-Suen Chan and is maintained by the Wellcome Trust Centre for the Epidemiology of Infectious Disease, University of Oxford, Oxford, United Kingdom.

Members of the Norwegian Macrolide Study Group include Signe H. Ringertz, Bitten Rasmussen (Aker University Hospital), Martin Steinbakk, Siri Haug (Akershus University Hospital), Fredrik Müller, Miriam Sundberg (Bærum Hospital), Hjørdis Iveland, Ann Elise Johansen (Central Hospital of Buskerud), Liisa Mortensen, Karstein Korsvik (Central Hospital of Nordland), Arne Mehl, Eldbjørg Berg (Central Hospital of Nord-Trøndelag, Levanger), Gerd Skjervold, Lise Haaland (Central Hospital of Nord-Trøndelag, Namsos), Ingunn Haavemoen, Kari Ødegaard (Central Hospital of Oppland, Lillehammer), Linda Schildman, Lene Bjøntegård (Central Hospital of Hedmark, Elverum), Eivind Ragnhildstveit, Eva Madsen (Central Hospital of Østfold), Elisebet Haarr, Tone Roa (Central Hospital of Rogaland), Reidar Hjetland, Berit Ose (Central Hospital of Sogn og Fjordane), Sølvi Noraas, Torill S. Larsen (Central Hospital of Vest-Agder), Rolf Schøyen, Astrid Lia (Central Hospital of Vestfold), Liv J. Sønsteby, Pirrko-L. Kellokumpu (Central Hospital of Hordaland, Haugesund), Einar Vik, Margreet B. Sandhaug (County Hospital of Møre og Romsdal, Molde), Reidar Hide, Fillip Angeles (County Hospital of Møre og Romsdal, Ålesund), Asbjørn Digranes, Hilde Bekkeheien (Haukeland Hospital), Mette Walberg, Magli Bøvre (National Hospital, University of Oslo), Yngvar Tveten, Inger Johanne Lunde (Telelab A/S, Skien), Gaute Syversen, Thea Bergheim (Ullevål University Hospital), Gunnar S. Simonsen, Siv-H. Barkhald (University Hospital of North Norway), Trond Jacobsen, Mariann Hulsund (University Hospital of Trondheim), Wibeke Aasnæs, and Anne K. Andersen (Laboratory of Clinical Medicine, Oslo).

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