Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n = 4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.