Molecular characterisation of macrolide resistance mechanisms of Streptococcus pneumoniae and Streptococcus pyogenes isolated in Germany, 2002–2003

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Abstract

In the present study, a real-time PCR protocol was developed for the detection of macrolide resistance determinants and was validated in a nationwide study in Germany covering a total of 236 Streptococcus pyogenes and 241 Streptococcus pneumoniae strains collected from children ≤16 years of age with community-acquired infections. Macrolide resistance was observed in 19.9% of pneumococcal strains and 14% of S. pyogenes isolates. Of the erythromycin A-resistant S. pyogenes strains, 93.9% showed the efflux type mef(A); 62.5% of the S. pneumoniae strains were mef(A)- and 37.5% erm(B)-positive. The correlation of the results of real-time PCR assay genotyping in the present study compared with those of conventional PCR genotyping and resistance phenotyping was 100%. Macrolide resistance is of growing concern in Germany. This highly sensitive and specific PCR assay to detect macrolide resistance has the potential to provide sufficiently rapid results to improve antibiotic treatment of streptococcal infections.

Introduction

Streptococcus pyogenes and Streptococcus pneumoniae continue to be significant causes of morbidity and mortality in humans and are responsible for both respiratory tract infections and invasive diseases [1], [2].

Macrolides are widely used for treatment of respiratory tract infections and offer treatment alternatives to penicillin; this is valuable especially in patients allergic to β-lactams, but resistance to this class of antibiotics is being increasingly reported for both S. pneumoniae and S. pyogenes in many countries [3], [4], [5]. In addition, cases of breakthrough pneumococcal bacteraemia caused by macrolide-resistant pneumococci in patients being treated with macrolides have been observed [6], [7]. Despite the limitations of available clinical data, concern is being expressed in clinical practice about the possibility of treatment failure with macrolides threatening the important role of these agents in the treatment of respiratory tract infections [8].

Target site modification is mostly based on dimethylation of an adenine residue (A2058) of the 23S rRNA domain V through the action of a family of enzymes encoded by erm class genes. Two classes of methylase genes, the erm(B) (S. pneumoniae and S. pyogenes) and the erm(A) (S. pyogenes), subclass erm(TR), have been described. These strains show cross-resistance to macrolides, lincosamides and the streptogramin B component (MLSB resistance). MLSB resistance can be expressed either constitutively (cMLSB phenotype) or inducibly (iMLSB phenotype) [9]. iMLSB-B and iMLSB-C strains generally possess the erm(A) methylase gene which is usually the predominant erm gene in S. pyogenes, while the iMLSB-A and the constitutively resistant (cMLS) isolates have the erm(B) methylase gene. Some of the constitutively resistant erm(B) positive S. pyogenes strains have been found to be telithromycin-resistant [10].

Quantitative PCR assay with the LightCycler (Roche Diagnostics, Mannheim, Germany) amplification and detection system is being increasingly used to detect both the pathogen [11] and resistance determinants [12]. This technology combines rapid thermocycling using glass capillaries with online fluorescence detection of the PCR amplicon.

The aim of the present study was (i) to evaluate the prevalence of macrolide resistance in S. pyogenes and S. pneumoniae isolates in Germany, and (ii) to develop a method for the rapid detection of macrolide resistance determinants in these streptococcal species that is amenable to high-throughput sample processing.

Section snippets

Material and methods

Between November 2002 and March 2003 consecutive clinical strains were collected from children ≤16 years of age with community-acquired respiratory tract infections.

Strains were shipped to a central laboratory (National Reference Centre for Streptococci, Aachen, Germany), where compliance of the isolates with the criteria for inclusion in the study was checked. Confirmation of the identification of isolates was provided by positive bile solubility and inhibition by optochin for S. pneumoniae.

Results

In total, 477 isolates comprising 236 S. pyogenes and 241 S. pneumoniae isolates were collected by 10 centres. Pneumococcal strains were isolated from the following sources: nasopharynx (n=107 (44.4%)), ear swabs (n=75 (31.1%)), eye swabs (n=37 (15.4%)), paracentesis (n=11 (4.6%)) and other sources (n=11 (4.6%)). Streptococcus pyogenes isolates were mainly isolated from the throat (n=163 (69.1%)), 15 (6.4%) strains from other respiratory sources, 24 (10.2%) strains from ear swabs, 9 (3.8%) from

Discussion

Macrolide resistance is spreading among streptococci [24], [25], [26]. Furthermore, there is increasing evidence that macrolide resistance may result in clinical failure [6], [7], [27] so that, as resistance to macrolides increases and clinical failures in children become more common with this class of antimicrobials, judicious use of macrolides in children is needed [28], [29]. On the other hand, this class of antibiotics is widely used in children because of the favourable adverse event

Acknowledgments

S. pyogenes reference strains were kindly provided by Dr. Helena Seppälä, Turku, Finland (strain A200, erm(A)/erm(TR)-positive), Dr. Aftab Jasir, Lund, Sweden (strains 544 and 517R, erm(B), and Dr. Joyce Sutcliffe, Groton, CT, USA (strain O2C1064, mef(A)-positive). We thank Nelli Neuberger and Claudia Cremer for excellent technical assistance. We thank Susanne Reinert (SR Medical Communications GmbH, Germany) for organising and monitoring the study. We thank André Bryskier for providing the

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