Development of a Multilocus Sequence Typing Scheme for Molecular Typing of Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).

FIG 1

Phylogenetic trees were constructed based on concatenated sequences of eight housekeeping loci for 12 unique STs using maximum-likelihood (A) and neighbor-joining (B) methods. Bootstrap support values of >70% are shown. STs are indicated by differential shading.

FIG 2

eBURST version 3 was used to analyze the 12 unique STs resolved for all 57 M. pneumoniae isolates. Two main clonal complexes (CC) were defined. The size of each dot is proportional to the number of isolates included in the analysis for each ST.

FIG 3

Prediction of recombination in the M. pneumoniae isolate chromosomes. Regions of variation in the genomes of the 35 clinical M. pneumoniae isolates and the type strain M129, which are predicted to have arisen by homologous recombination, are shown on the right. Red blocks indicate recombination predicted to have occurred on internal nodes, and blue indicates taxon-specific recombination. Isolates are ordered according to the phylogenetic tree displayed on the left. The track along the top of the figure displays the M129 chromosome and annotation, in which protein-coding sequences (CDS) are indicated in light blue.

FIG 4

Distribution of MLVA, P1 type, and ST for 57 M. pneumoniae isolates (each group defined by lines below).