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Objectives Mycoplasma pneumoniae is a frequent cause of human lower respiratory tract infections (LRTIs) for which macrolides are the treatment of choice. The aim of this study was to determine the rate of macrolide resistance and to subtype M. pneumoniae strains in Italy. Patients and methods During an outbreak of M. pneumoniae infections in southern Italy in 2010, 48 clinical specimens from 43 paediatric patients hospitalized for LRTIs were analysed for macrolide resistance. The mutations associated with resistance (A2063G and A2064G) and M. pneumoniae subtypes were detected by sequencing the targeted domain V region of the 23S rRNA gene and a region in the MPN528a gene, respectively. Results Macrolide resistance genotypes were detected in 11 (26%) of the 43 M. pneumoniae-positive children. The A2063G mutation was identified in seven patients and the A2064G mutation was identified in the remaining four. Upon admission, the isolates from three patients showed a susceptible genotype but subsequently acquired the A2063G mutation. Genotyping revealed M. pneumoniae subtype 1 in 33 of 40 sequenced strains and subtype 2 in the remaining 7. There was no association between macrolide resistance or susceptibility and the M. pneumoniae subtypes. Conclusions This is the first report of macrolide resistance among M. pneumoniae strains in Italy. Our findings indicate an unexpected high prevalence of macrolide resistance genotypes in children, and so macrolide resistance should be carefully considered in patients who do not respond appropriately to antibiotic treatment. The epidemiological monitoring of macrolide resistance has become necessary in Italy and in the rest of Europe. [ABSTRACT FROM PUBLISHER]