Multilocus variable-number tandem-repeat analysis of Mycoplasma pneumoniae clinical isolates from 1962 to the present: a retrospective study

Abstract

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.

Fig 1

MLVA typing capillary electrophoresis electropherogram of reference strains FH (1996) and FH (2009). (A) FH (1996) electropherogram displaying a single Mpn1 VNTR allele along with Mpn14 and Mpn16 VNTR loci. (B) FH (2009) electropherogram displaying two Mpn1 VNTR alleles along with Mpn14 and Mpn16 VNTR loci. (C) FH (1996) electropherogram displaying Mpn13 and Mpn15 VNTR loci. (D) FH (2009) electropherogram displaying Mpn13 and Mpn15 VNTR loci. The number of tandem repeats for each locus is indicated by parentheses. Relative fluorescence units are indicated by the y axis, and fragment size in base pairs is indicated by the top x axis.