Resistance to macrolides, clindamycin and telithromycin in Streptococcus pyogenes isolated in Spain during 2004

J. Tamayo1, E. Pérez-Trallero2, J. L. Gómez-Garcés1, J. I. Alós1,* on behalf of the Spanish Group for the Study of Infection in the Primary Health Care Setting (IAP-SEIMC){dagger}

1 Servicio de Microbiología, Hospital de Móstoles, 28935 Móstoles, Madrid; 2 Servicio de Microbiología, Hospital Donostia, San Sebastián, Spain


* Corresponding author. Tel: +34-91-6648695; Fax: +34-91-6471917; E-mail: nachoalos{at}microb.net

Received 4 May 2004; returned 2 July 2005; revised 6 July 2005; accepted 24 July 2005


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To study the antimicrobial susceptibility and prevalence of the different phenotypes and genotypes of macrolide resistance in group A streptococci isolated in Spain in 2004, and to compare the results with those obtained in 1998 and 2001 using the same methodology and centres.

Methods: A total of 530 unique isolates of Streptococcus pyogenes collected in 21 laboratories from 16 geographic areas (regions) in Spain were used. Antimicrobial susceptibility testing was performed using the agar dilution method. Discs containing erythromycin or clindamycin were used to recognize the phenotypes of macrolide–lincosamide–streptogramin (MLS) resistance. Genes encoding macrolide–lincosamide resistance were detected by PCR.

Results: Resistance to erythromycin was 21.7% [95% confidence interval (CI) 16.5–26.3]. The resistance to azithromycin was 21.5%, whereas the resistance to miocamycin and to clindamycin was 6.6% (95% CI 3.0–8.9). Thirty-one (5.8%) of the isolates were resistant to telithromycin. Of the 115 erythromycin-resistant isolates, 67.8% had the M phenotype, representing 14.7% of all the isolates tested. Thirty-five isolates (30.5% of the erythromycin-resistant strains and 6.6% of all the isolates) had the MLSB constitutive phenotype. There was a high prevalence of resistance to telithromycin (88.6%) among the 35 strains with the MLSB constitutive phenotype. When we compared these results with those from previous studies (1998 and 2001), we found a significant increase in the MLSB constitutive phenotype (P < 0.001), and a significant decrease in the M phenotype (P < 0.005) was noted.

Conclusions: The significant increase in the prevalence of resistance to clindamycin and miocamycin, and the prevalence of resistance to telithromycin reached in a short period of time from the introduction of its use, underscore the need for continuous surveillance of antimicrobial resistance in S. pyogenes in Spain.

Keywords: M phenotype , MLSB phenotype , mef genes , erm genes


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resistance of Streptococcus pyogenes to penicillin has not been observed to-date, although this is the antibiotic most commonly recommended for the treatment of streptococcal pharyngitis, and skin and soft tissue infections. Macrolides have been recommended as an alternative in patients who are allergic to penicillin, but in recent years an increasing incidence of erythromycin resistance in S. pyogenes has been reported in several parts of the world.13

In 1998 and 2001, we performed two multicentre studies in Spain to determine the prevalence of resistance to penicillin G, six oral cephalosporins (only in 2001), several macrolides (erythromycin, azithromycin and miocamycin) and clindamycin in S. pyogenes.4,5 When we compared the results obtained in the 2 years, we observed a significant increase in resistance to erythromycin, but not to clindamycin or miocamycin (increase in the M phenotype).5 These results underscore the need for continuous surveillance of antimicrobial resistance in S. pyogenes in Spain.

The aim of this work was to study the antimicrobial susceptibility and the prevalence of the different phenotypes and genotypes of macrolide resistance in group A streptococci isolated in Spain in 2004, using the same methodology and centres as in 1998 and 2001. The results obtained in the three studies performed were compared by statistical analysis. Additionally, we tested the antimicrobial susceptibility to telithromycin, a new ketolide recently introduced in the clinical setting in Spain.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

A total of 530 unique isolates of S. pyogenes collected from outpatients in 21 laboratories in Spain from February to September 2004 were used. The country was arbitrarily divided into 16 geographic areas (regions). The sample size was proportionally stratified according to the number of inhabitants of each area, with a ratio of approximately one strain/80000 inhabitants. Throat swab samples provided 421 isolates (79.4%), and the remaining 109 were from other sources. Four hundred and thirty-two (81.5%) were isolated from children and 98 (18.5%) from adults. Identification was made by standard criteria, as described previously.5 Strains were kept frozen in skimmed milk at –40°C.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by the agar dilution method according to the guidelines of the NCCLS.6 Antibiotics were obtained, as standard reference powders of known potency, from Sigma Chemical Co. (St Louis, MO, USA), or from their manufacturers. The range of interpretative categories for each antibiotic was that recommended by the NCCLS in the 2004 supplement.7 As there are no defined MIC breakpoints for miocamycin, we used those previously published5 (miocamycin breakpoints of ≤1 and >4 mg/L for susceptibility and resistance).

To identify antibiotic resistance phenotypes, discs containing erythromycin (15 µg) or clindamycin (2 µg) were used. Three different patterns (clindamycin susceptible, erythromycin resistant; clindamycin inducible, erythromycin resistant; and clindamycin resistant, erythromycin resistant) were recognized as being related to the well-recognized phenotypes of macrolide–lincosamide–streptogramin (MLS) resistance.

Determination of genes of resistance to macrolides

Detection of the resistance genes erm(B), erm(A) (subclass TR) and mef was carried out by PCR, followed in the case of the mef gene by an restriction fragment length polymorphism with the enzymes HinfI and DraI to determine whether it was a mef(A) or a mef(E) gene.8,9

Statistical analysis

The {chi}2-test was used. A two-tailed P value of ≤0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The MIC ranges, the MICs at which 50% (MIC50) and 90% (MIC90) of the isolates are inhibited, and the percentage of susceptible, intermediate and resistant strains are given in Table 1. All isolates were fully susceptible to penicillin with an MIC90 of 0.015 mg/L. All strains were 100% susceptible to cephalosporins. However, differences in MICs were noted among the cephalosporins.


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Table 1. In vitro susceptibilities of 530 recent S. pyogenes strains to penicillin G, six oral cephalosporins, three macrolides, clindamycin and telithromycin

 
Resistance to erythromycin (MIC breakpoint ≥1 mg/L) was 21.7% [95% confidence interval (CI) 16.5–26.3]. The resistance to azithromycin (15-membered macrolide) was 21.5%, whereas the resistance to miocamycin, a 16-membered macrolide, and to clindamycin, was 6.6% (95% CI 3.0–8.9). Thirty-one (5.8%) of the isolates were resistant to telithromycin (MIC breakpoint >2 mg/L) and all of these isolates exhibited high erythromycin MICs (>16 mg/L).

The different phenotypes of susceptibility to macrolides–lincosamides were as follows: 67.8% of the 115 erythromycin-resistant strains were susceptible to clindamycin and miocamycin, and induction with erythromycin did not modify the susceptibility to the latter antibiotics; these strains were designated as having the M phenotype, and represented 14.7% of all the isolates tested. Thirty-five isolates (30.5% of the erythromycin-resistant strains and 6.6% of all the isolates) were resistant to erythromycin, azithromycin, miocamycin and clindamycin, which indicates a constitutive type of resistance. Two erythromycin-resistant strains (1.7% of the erythromycin-resistant strains and 0.4% of all the isolates) were susceptible to clindamycin, but they showed an inducible type of resistance.

All the strains with the M phenotype were susceptible to telithromycin (MIC90 0.5 mg/L), although with MICs higher than that for the strains susceptible to erythromycin (MIC90 0.015 mg/L). There was a high prevalence of resistance to telithromycin (88.6%) in the 35 strains with the MLSB constitutive phenotype (MIC range 4–>16 mg/L; MIC90 16 mg/L).

The frequency of the different phenotypes of resistance in the present study and in our two previous studies are given in Table 2, to facilitate comparison of the three sets of data. A statistically significant decrease in the number of strains with the M phenotype was observed when we compared the results obtained in the present study with those from the previous ones (1998 and 2001) (P < 0.005). However, we found an increase in the resistance to clindamycin and miocamycin, and consequently, a significant increase in the MLSB constitutive phenotype (P < 0.001).


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Table 2. Frequency of the different phenotypes of macrolide resistance in the three studies performed, and statistical comparisons

 
Sixty-two erythromycin-resistant strains were selected, 25 with the M phenotype (one or two per laboratory), and all of those with the MLSB phenotype. Twenty-four of the 25 strains with the M phenotype selected showed the presence of the mef gene when assayed by PCR. Twenty-three (92%) proved to be mef(A), one mef(E), and the remaining strain was negative in the study of the different resistance genes. All 35 strains with the MLSB constitutive phenotype had the erm(B) gene, and the two strains with the MLSB inducible phenotype had erm(A) subclass TR.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The levels of resistance to erythromycin and azithromycin show considerable fluctuation if we compare the results from the three studies. In the present study they are significantly lower in comparison with the previous study (21.7% compared with 29.7%), showing levels similar to those from the first study in 1998 (23.4%). Despite this decline, the levels of resistance are high enough to discourage, empirically, starting treatment with a macrolide without first having undertaken a susceptibility study.

The novelty in this study is the fact that the levels of resistance to miocamycin and clindamycin are significantly higher than those found in the prior studies. This reflects an increase in the prevalence of the MLSB phenotype strains. Resistance to telithromycin reached 5.8%, which is still well below the levels for erythromycin and azithromycin; telithromycin remains a good alternative in the event that a ß-lactam antibiotic cannot be used. Telithromycin shows moderate activity against phenotype M strains. Although by the present criteria 100% of these strains were susceptible to this antibiotic, their MICs were well above those for strains susceptible to erythromycin. In contrast, telithromycin was not active against those strains with the MLSB phenotype; ~90% showed MICs above the susceptibility breakpoint. Several studies in recent years have pointed to a reduction in susceptibility to telithromycin among this type of strain (MLSB phenotype).10

The significant increase in the prevalence of resistance to clindamycin and miocamycin, and the prevalence of resistance to telithromycin reached in a short time from the introduction of its use, underscore the need for continuous surveillance of antimicrobial resistance in S. pyogenes in Spain.


    Footnotes
 
{dagger} The IAP-SEIMC project participants are listed in the Acknowledgements. Back


    Acknowledgements
 
Members of the Spanish Group for the Study of Infection in the Primary Health Care Setting (IAP-SEIMC) that have participated in this work: Hospital Juan Canalejo, La Coruña (R. Villanueva); Hospital Montecelo, Pontevedra (M. García-Campello); Hospital de la Santa Creu i Sant Pau, Barcelona (B. Mirelis); C. A. P. Bon Pastor, Barcelona (B. Viñado); C. A. P. Manso, Barcelona (G. Roig); Hospital Clínico Universitario, Zaragoza (M. C. Rubio and P. Macipe Costa); Hospital Miguel Servet, Zaragoza (M. J. Revillo); Hospital del Río Hortega, Valladolid (P. Pérez-Pascual); Hospital General de Segovia, Segovia (S. García); Hospital General de Guadalajara, Guadalajara (T. Pérez-Pomata); Ambulatorio de Argüelles, Madrid (B. Orden and R. Martínez); Hospital Universitario Clínico San Carlos, Madrid (C. Betriu); Hospital Severo Ochoa, Leganés, Madrid (S. Cuetara); Hospital Universitario de Getafe, Getafe, Madrid (P. García-Hierro); Hospital Dr Pesset, Valencia (J. M. Nogueira); Hospital Morales Meseguer, Murcia (G. Yagüe and A. Menasalbes); Hospital Virgen de las Nieves, Granada (C. Miranda, A. Martínez-Brocal and M. de la Rosa); Hospital Universitario de Valme, Sevilla (J. L. García López and E. Martín); and Hospital Puerta del Mar, Cádiz (P. Marín).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 Hasenbein ME, Warner JE, Lambert KG et al. Detection of multiple macrolide- and lincosamide-resistant strains of Streptococcus pyogenes from patients in the Boston area. J Clin Microbiol 2004; 42: 1559–63.[Abstract/Free Full Text]

2 Brown SD, Ryback MJ. Antimicrobial susceptibility of Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae collected from patients across the USA, in 2001–2004, as part of the PROTEKT US study. J Antimicrob Chemother 2004; 54 (Suppl 1): i7–15.[Abstract/Free Full Text]

3 Szczypa K, Sadowy E, Izdebski R et al. A rapid increase in macrolide resistance in Streptococcus pyogenes isolated in Poland during 1996–2002. J Antimicrob Chemother 2004; 54: 828–31.[Abstract/Free Full Text]

4 Alós JI, Aracil B, Oteo J et al. High prevalence of erythromycin-resistant, clindamycin, and miocamycin-susceptible (M-phenotype) Streptococcus pyogenes: results of a multicenter study performed in 1998 in Spain. J Antimicrob Chemother 2000; 45: 605–9.[Abstract/Free Full Text]

5 Alós JI, Aracil B, Oteo J et al. Significant increase in the prevalence of erythromycin-resistant, clindamycin- and miocamycin-susceptible (M phenotype) Streptococcus pyogenes in Spain. J Antimicrob Chemother 2003; 51: 333–7.[Abstract/Free Full Text]

6 National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.

7 National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Fourteenth Informational Supplement M100-S14. NCCLS, Wayne, PA, USA, 2004.

8 Klaassen CHW, Mouton JW. Molecular detection of the macrolide efflux gene: to discriminate or not to discriminate between mef(A) and mef(E). Antimicrob Agents Chemother 2005; 49: 1271–8.[Free Full Text]

9 Sutcliffe J, Grebe T, Tait-Kamradt A et al. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996; 40: 2562–6.[Abstract]

10 Champney WS, Menstens N, Zurawick K. An examination of the differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae. Curr Microbiol 2004; 49: 239–47.[CrossRef][ISI][Medline]





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