In this study, we evaluated a recently developed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method for the molecular typing of Mycoplasma pneumoniae. The method is based on GeneScan analysis of five VNTR loci throughout the genome which define a specific genotype based on the number of tandem repeats within each locus. A retrospective analysis of 154 M. pneumoniae clinical isolates collected over the last 50 years and a limited (n ؍ 4) number of M. pneumoniae-positive primary specimens acquired by the CDC was performed using MLVA. Eighteen distinct VNTR types were identified, including two previously unidentified VNTR types. Isolates from several M. pneumoniae community outbreaks within the United States were also analyzed to examine clonality of a specific MLVA type. Observed in vitro variability of the Mpn1 VNTR locus prompted further analysis, which showed multiple insertions or deletions of tandem repeats within this locus for a number of specimens and isolates. To our knowledge, this is the first report showing variation within the Mpn1 locus, thus affecting precise and reliable classification using the current MLVA typing system. The superior discriminatory capability of MLVA provides a powerful tool for greater resolution of M. pneumoniae strains and could be useful during outbreaks and epidemiological investigations.
Mycoplasma pneumoniae is one of the most common bacterial etiologies of community-acquired pneumonia (CAP), representing 15 to 20% of cases in some studies (28). It is also a significant cause of community-wide outbreaks which are reported to occur in 3-to 7-year intervals with various incidence rates (10,12,28). Upper and lower respiratory tract symptoms are often mild and self-limiting; however, occasional extrapulmonary complications may develop, sometimes resulting in death (5,7,23).Strain subtyping is an important epidemiological tool for surveillance and outbreak investigations. Until recently, molecular typing of M. pneumoniae had been hindered by its minimal and highly uniform genome. Molecular typing was often restricted to a single gene encoding the P1 protein. Methods such as PCR-restriction fragment length polymorphism (RFLP) analysis of the P1 gene (4, 18) and real-time PCR followed by high-resolution melt analysis (HRM) targeting the region of M. pneumoniae repetitive elements 2 and 3 (RepMp2/3) of the P1 gene (19,20) were used to genotype M. pneumoniae into two subtypes (PCR-RFLP) and variants of subtypes 1 and 2 (PCR-HRM). Both sequencing (9) and pyrosequencing (6, 21, 22) techniques have also been developed to differentiate subtypes of M. pneumoniae. Recently, Degrange et al. (6) developed a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) method for M. pneumoniae based on whole-genome analysis that was able to differentiate 26 distinct VNTR types. This procedure takes advantage of the variation in the copy numbers of tandemly repeated sequences within five different loci within the genome and has proven to be highly discriminatory (...