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JAC Advance Access originally published online on May 9, 2009
Journal of Antimicrobial Chemotherapy 2009 64(1):52-58; doi:10.1093/jac/dkp160
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis

O. Peuchant1, A. Ménard2, H. Renaudin1, M. Morozumi3, K. Ubukata3, C. M. Bébéar1 and S. Pereyre1,*

1 Laboratoire de Bactériologie EA 3671, Université Victor Segalen Bordeaux 2 and CHU de Bordeaux, 33076 Bordeaux cedex, France 2 INSERM U853, Université Victor Segalen Bordeaux 2, Laboratoire de Bactériologie, 33076 Bordeaux cedex, France 3 Laboratory of Molecular Epidemiology for Infectious Agents, Kitasato Institute for Life Sciences, Kitasato University, Tokyo 108-8641, Japan

Received 13 February 2009; returned 10 March 2009; revised 1 April 2009; accepted 9 April 2009

* Corresponding author. Tel: +33-5-57-57-16-25; Fax: +33-5-56-93-29-40; E-mail: sabine.pereyre{at}u-bordeaux2.fr

Objectives: Mycoplasma pneumoniae is a common aetiological agent of community-acquired respiratory tract infections for which macrolides are the treatment of choice. In France, only two macrolide-resistant isolates were reported in 1999. In contrast, several recent data reported that macrolide-resistant M. pneumoniae isolates have been spreading since 2000 in Japan. Mutations A2058G (Escherichia coli numbering), A2058C, A2059G, A2062G, C2611A and C2611G in domain V of the 23S rRNA gene were associated in vivo or in vitro with this resistance. The aim of this study was to determine whether macrolide resistance of M. pneumoniae is emerging in France.

Patients and methods: We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples.

Results: Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France.

Conclusions: The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Keywords: target gene mutation , 23S rRNA , laboratory methods , antimicrobial resistance epidemiology , low respiratory tract infections, LRTI


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