Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for Molecular Typing of Mycoplasma pneumoniae

In this study we report on the development of a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The genomic content of M. pneumoniae M129 was analyzed for VNTRs, and 5 of the 17 VNTRs identified were selected for use in an MLVA assay. The method was based on a GeneScan analysis of VNTR loci labeled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This approach was applied to a collection of 265 isolates from various European countries, Japan, and Tunisia; and 26 distinct VNTR types were found. The VNTR assay was compared to the P1 adhesin PCR-restriction fragment length polymorphism (RFLP) typing method and showed a far better resolution than the P1 PCR-RFLP method. The discriminatory power of MLVA (Hunter-Gaston diversity index [HGDI], 0.915) for the 265 isolates was significantly higher than that of the P1 PCR-RFLP method (HGDI, 0.511). However, there was a correlation between the typing results obtained by MLVA and the P1 gene PCR-RFLP method. The potential value of MLVA of M. pneumoniae as an epidemiological tool is discussed, and the use of the VNTR markers in further investigations of the potential use of MLVA in outbreaks of M. pneumoniae infections is proposed.