A rapid increase in macrolide resistance in Streptococcus pyogenes isolated in Poland during 1996–2002

Katarzyna Szczypa1,*, Ewa Sadowy2, Radoslaw Izdebski2 and Waleria Hryniewicz1,2

1 Department of Epidemiology and Clinical Microbiology,, and 2 Department of Molecular Microbiology, National Institute of Public Health, Chelmska str 30/34, 00-725 Warsaw, Poland

Received 20 May 2004; returned 10 June 2004; revised 20 July 2004; accepted 4 August 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: The aim of this study was to investigate Polish clinical isolates of Streptococcus pyogenes collected during a 7 year period using phenotypic and genotypic techniques.

Methods: A total of 816 isolates of S. pyogenes recovered from 33 medical centres in Poland were tested for their susceptibility to various antimicrobial agents. Erythromycin-resistant isolates were analysed by PFGE, multilocus sequence typing and emm typing methods.

Results: The tetracycline resistance rate was high (43%) among all S. pyogenes strains. Ninety-eight (12%) isolates were resistant to erythromycin. A low prevalence of the M phenotype (5.1%) associated with the presence of the mef(A) gene was found. All the isolates of the iMLSB phenotype harboured the erm(TR) gene. Out of the cMLSB isolates, 71.4% and 28.6% carried erm(TR) and erm(B), respectively. All isolates with erm(B) were resistant to telithromycin. PFGE analysis discerned 13 different patterns, A–N, with two predominant PFGE profiles—A (41 isolates) and B (25 isolates)—that in multilocus sequence typing corresponded, respectively, to a novel sequence type (ST) 367 and ST63. Overall, the representatives of these clones accounted for >90% of isolates of the iMLSB phenotype.

Conclusions: A significant increase in erythromycin resistance was observed among clinical S. pyogenes collected in Poland over a 7 year period driven by the spread of two epidemic clones.

Keywords: S. pyogenes , erythromycin resistance , phenotypes , genotypes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Since the 1940s, penicillin has been the treatment of choice for Streptococcus pyogenes infections, whereas erythromycin and clindamycin are usually recommended as alternative antibiotics. The first erythromycin-resistant strain of Streptococcus was described in 1959 in the UK and since then in many countries.1 Whereas data regarding the prevalence of S. pyogenes resistance to macrolides have been reported worldwide, the problem in Poland has remained unstudied until now.

The aim of this study was to investigate the susceptibility patterns of Polish clinical isolates of S. pyogenes, with particular emphasis on macrolide resistance and the underlying genetic determinants of this resistance. The clonal structure of macrolide-resistant isolates was studied by PFGE of SmaI-restricted bacterial DNA as well as multilocus sequence typing (MLST) and emm typing of selected strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial isolates

Eight hundred and sixteen isolates of S. pyogenes were collected during 1996–2002 at 33 medical centres in Poland. The number of strains collected in the periods 1996–1997, 1998–1999 and 2000–2002 were 266, 258 and 292, respectively. The isolates were recovered from throat swabs (n=438), pus (n=331), blood (n=17), sputum (n=8) and other sources (n=22). Of these, 361 isolates were obtained from children and 455 from adult patients. S. pyogenes were identified by standard procedure using a commercially available agglutination kit (Streptex, Murex Biotech Limited, UK).

Determination of MICs and erythromycin resistance phenotypes

MICs were determined according to the NCCLS guidelines2 by the standard microdilution method. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain. The MIC breakpoints were interpreted according to the NCCLS.2 Breakpoints for spiramycin were those proposed by the French Society for Microbiology.3 For all strains, susceptibility to the following antimicrobials was tested: penicillin G, erythromycin, tetracycline (Sigma-Aldrich, Steinheim, Germany) and clindamycin (Pharmacia Upjohn, Inc., Kalamazoo, MI, USA). Erythromycin-resistant strains (MIC≥1 mg/L) were additionally tested for susceptibility to clarithromycin (Abbott Laboratories Chicago, IL, USA), roxithromycin (Roussel Uclaf, Paris, France), azithromycin (Pliva Kraków, Poland), spiramycin, (Rhone-Poulenc Rorer, Collegeville, PA, USA), telithromycin, quinupristin/dalfopristin (Aventis Pharma, Romainville, France), linezolid (Pharmacia & Upjohn, Inc., Kalamazoo, MI, USA) and moxifloxacin (Bayer AG, Wuppertal, Germany). All erythromycin-resistant isolates were assigned to their particular phenotypes, such as inducible MLSB (iMLSB), constitutive MLSB (cMLSB) and efflux-mediated resistance (M phenotype) on the basis of the double erythromycin–clindamycin disc test.4

Detection of erythromycin resistance genes

PCR detection of erm(B) and mef(A) genes was carried out as reported by Sutcliffe et al.5; the erm(TR) gene was detected with primers described by Kataja et al.4

PFGE analysis, MLST and emm typing

Chromosomal DNA of erythromycin-resistant isolates was digested with SmaI restriction enzyme (MBI Fermentas, Lithuania) and analysed by PFGE, as described elsewhere.6 PFGE patterns were analysed according to the criteria proposed by Tenover et al.7 MLST was performed on representatives of all PFGE types and subtypes, following the method established by Enright et al.8; particular allele numbers and sequence types (STs) were identified using the MLST database (www.mlst.net). emm types for the isolates characterized by MLST were determined according to the recommendations of the Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, S. pyogenes emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/doc.htm).

Statistical analysis

Comparison of frequencies between groups was performed using {chi}2 analysis. A P value of <0.05 was considered statistically significant.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
All 816 clinical S. pyogenes isolates examined in our study were highly susceptible to penicillin G. The highest rate of resistance was found for tetracycline (43%) and the rate was higher among isolates from adults (62%). These results are similar to those observed by Jasir et al.9 in Iran and once again indicate that tetracycline is not an appropriate choice for empirical therapy of S. pyogenes infections.

Several European countries reported an increase in erythromycin resistance in S. pyogenes at the beginning of the 1990s.1 In our study, altogether 98 isolates (12.0%) were resistant to erythromycin. The proportion of these isolates in adults was higher (64%) than in children. The frequency of erythromycin-resistant strains showed a rising trend over the years, from 1.8% in 1996–1997, to 12% in 1998–1999, to 25.1% in 2000–2002. The P values between 1.8% and 12%, 12% and 25.1% and 1.8% and 25.1% were 0.001, 0.01 and 0.001, respectively, showing a statistical significance. All erythromycin-susceptible isolates were also susceptible to clindamycin (MICs, 0.003–0.25 mg/L). Among the 98 erythromycin-resistant isolates, 65 (66.6%) exhibited the iMLSB and 28 (28.2%) the cMLSB phenotype (Table 1). All the iMLSB phenotypes harboured the erm(TR) gene. In the more diverse group of cMLSB isolates, 20 (71.4%) and eight (28.6%) contained erm(TR) and erm(B), respectively. Overall, 85 isolates (86.7%) showed the presence of erm(TR). Such a high prevalence of the erm(TR) isolate among macrolide-resistant S. pyogenes in Poland is unusual compared with other countries.4,1012 The M phenotype occurred in only five isolates (5.1%) and all possessed the mef(A) gene. Similarly, a low prevalence of the M phenotype (10%) was found in a study conducted in Berlin.10 However, other investigators have demonstrated that the M phenotype accounted for >80%.1 In our study, all iMLSB and M-phenotype isolates were susceptible to clindamycin and telithromycin. In contrast, all cMLSB isolates were resistant to clindamycin. In the case of ketolides, we found eight (8.1%) erm(B)-positive cMLSB isolates that were resistant to telithromycin (Table 1). The data presented here, which confirmed that the isolates with the erm(B) gene were resistant to telithromycin, are in accordance with studies from Spain11 and from other European countries.12 In the present study, we determined the susceptibilities of macrolide-resistant S. pyogenes to antibiotics belonging to various groups. All tested isolates were fully susceptibility to linezolid, moxifloxacin and quinupristin/dalfopristin. These agents demonstrated good in vitro activity independent of the macrolide resistance phenotype present (Table 1). Hence these antibiotics could be used as alternatives for the treatment of S. pyogenes infections in selected cases.


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Table 1. Susceptibility patterns of 98 isolates of erythromycin-resistant S. pyogenes according to macrolide-resistance phenotypes

 
Until now, no data have been available on the genetic diversity of Polish S. pyogenes isolates resistant to macrolides. PFGE analysis was performed on 95 of the 98 macrolide-resistant strains (three M-phenotype isolates were not typeable). Altogether, 13 different PFGE patterns, designated A–N, were discerned among the isolates (Table 2), with two predominant PFGE profiles A (n=41) and B (n=25). Among the type A and B isolates, five (A1–A5) and four (B1–B4) PFGE subtypes were found, respectively. Whereas all the 41 isolates of the PFGE subtypes A1–A51 showed the iMLSB phenotype, six isolates of the PFGE type B1 had the cMLSB phenotype and 19 (B1-B4) were iMLSB. All the isolates belonging to the A and B clones harboured the erm(TR) gene (Table 2) and were resistant to tetracycline. Thirty-three (80%) isolates of the A type were recovered from wounds, whereas the majority of the B type (76%) were from throat samples. Overall, the isolates belonging to clones A and B accounted for almost 90% of isolates of the iMLSB phenotype. It can be concluded, therefore, that S. pyogenes of the iMLSB phenotype in Poland are highly clonal. In contrast, the cMLSB isolates showed a polyclonal nature (six different PFGE types).


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Table 2. Profile PFGE, emm types and MLST of erythromycin-resistant S. pyogenes isolates

 
The representatives of all PFGE types and subtypes were further subjected to MLST analysis and emm typing, resulting in 10 different STs and eight emm types (Table 2). The majority of STs found in this study, in particular: ST63 (characteristic of all the subtype B1–B4 isolates as well as isolates of types F, G, H); ST28 (subtypes C1–C3); ST36 (subtypes D1–D3 and three non-typeable isolates); ST46 (type K) and ST52 (type N) have been previously found among erythromycin-resistant S. pyogenes in Germany.13 In contrast, the main clone of PFGE subtypes A1–5 represented a novel ST367 (emm type 44/61). This clone, in which resistance developed presumably locally, constitutes a single local variant of ST25, isolated in the 1950s and 1970 in Northern and Central America.8 Erythromycin and tetracycline resistance may, in this case, represent a strong selective advantage, which drives successful spread of the new clone under antibiotic pressure.

In summary, the present study demonstrates the alarming increase in macrolide resistance in S. pyogenes in Poland over the few past years. A significant part of this increase can be attributed to the successful dissemination of two epidemic S. pyogenes clones, each involved in a different type of infection. Unfortunately, these clones, and thus the majority of macrolide-resistant strains possess the MLSB phenotype conferring resistance to all macrolides, lincosamides and streptogramin B. Moreover, these clones are resistant to tetracycline. Resistance to a novel ketolide, telithromycin, albeit presently low, may grow under increased selective pressure as strains with this characteristic circulate within the population. Although several therapeutic options still remain for the treatment of S. pyogenes infections, judicious use of all available antimicrobials and continued monitoring of susceptibility of this pathogen are critical for the future.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank T. Kaminska and B. Chmylak for their excellent technical assistance and S. Murchan for critical review of the manuscript. We acknowledge the use of the MLST database located in Imperial College in London and founded by the Wellcome Trust.


    Footnotes
 
* Corresponding author. Tel: +48-22-851-46-70; Fax: +48-22-841-29-49; Email: kazbunda{at}cls.edu.pl


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