Erasmus MC-Sophia Children's hospital, Laboratory of Pediatrics, Pediatric Infectious Diseases and Immunity, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
J Microbiol Methods. 2010 Sep;82(3):214-22. doi: 10.1016/j.mimet.2010.06.004. Epub 2010 Jun 11.
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.
治疗肺炎支原体感染的首选抗生素是大环内酯类。然而，最近的几项研究表明，肺炎支原体分离株中细菌 23S rRNA 突变导致的大环内酯（ML）耐药性的流行率正在上升。因此，快速检测肺炎支原体中的 ML 耐药性至关重要，以便对患者进行适当和及时的治疗。因此，我们着手确定焦磷酸测序作为一种方便的技术来评估 ML 耐药性的效用。此外，我们研究了焦磷酸测序是否可用于肺炎支原体分离株的分子分型。为此，总共开发了四个单独的焦磷酸测序测定法。这些测定法的设计目的是从四个不同的位点确定一个短的基因组序列，即 23S rRNA 基因内的两个位置、MPN141（或 P1）基因内的一个位置和 MPN528a 基因内的一个位置。虽然 23S rRNA 区域用于确定 ML 耐药性，但后两个区域用于分子分型。焦磷酸测序测定法在 108 株肺炎支原体分离株的集合上进行。通过焦磷酸测序，容易识别集合中的 ML 耐药分离株（n=4）。此外，通过 MPN141 和 MPN528a 焦磷酸测序试验，每种菌株都正确地分型为 1 型或 2 型亚株。有趣的是，我们集合中的两个最近的分离株通过焦磷酸测序试验被鉴定为 2 型亚株，但它们携带的 MPN141 基因的新型变体，该基因中的两个重复元件（RepMP4 和 RepMP2/3）内均发生重排。总之，焦磷酸测序是一种方便的技术，可用于 ML 耐药性测定以及肺炎支原体分离株的分子分型。
