Identification of P1 Variants of Mycoplasma pneumoniae by Use of High-Resolution Melt Analysis

Abstract

Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia. Although two genetically distinct types of M. pneumoniae are known, variants of each also exist. We used a real-time PCR high-resolution melt genotyping assay to identify clinical variants which may provide greater insight into the genetic distribution of M. pneumoniae strains.Mycoplasma pneumoniae is a pervasive pathogen that accounts for up to 20% of all community-acquired pneumonias (14,15). Although its genome is highly homogeneous between strains, the P1 gene, which encodes an immunogenic adhesion protein, serves as a target to categorize isolates into either a type 1 or a type 2 genetic group (3). Numerous reports of genetic variants of each type have also been described (2-4, 6, 8). Spuesens et al. recently showed that intragenomic homologous DNA recombination occurs within the RepMP2/3 and RepMP4 regions of the P1 gene (MPN141) of M. pneumoniae and strain differences can be attributed to variation within RepMP elements (13). Dumke et al. also reported that M. pneumoniae variants differ only within the RepMP2/3 element (5). The clinical relevance of these findings is unclear, but epidemiological evidence of temporally based type-specific immunity in the population suggests these variations impart an advantage to the organism (6, 11). Moreover, the well-documented 3-to 7-year cyclic patterns of population-based, typespecific outbreaks seem to support this hypothesis (7,9,11).Historically, typing schemes were based upon restriction fragment length polymorphism (RFLP) analysis of digested PCR products of the P1 gene; however, Degrange et al. recently described a multiple-locus variable-number tandem-repeat analysis assay that was able to sort 265 strains into 26 distinct groups (1). This assay requires amplification of five loci followed by capillary electrophoresis and analysis but provides a greater level of genomic resolution. We recently reported the development of a real-time PCR assay that is able to rapidly distinguish M. pneumoniae isolates into type 1 or type 2 categories (12). We used this technique to classify 102 isolates using real-time PCR followed by high-resolution melt (HRM) analysis of a variable region partially spanning the RepMP2/3 element of the P1 gene (10, 12). The current study reports the use of this assay to identify variants of each group based upon intratype sequence deviations. Through the analysis of distinct HRM profiles from clinical isolates obtained by the CDC Respiratory Diseases Branch, we have been able to identify variants of each type. In this report, we describe the identification, sequence analysis, and unique HRM profiles of three diverse P1 variants (isolates 3, 684, and 549) and demonstrate the utility of this assay for detecting M. pneumoniae variants along with prototypical type 1 and 2 strains.M. pneumoniae culture and isolation were performed as previously described (16). M129 (ATCC 29342) and FH (ATCC 15531) were used as the reference strains for type 1 and type 2, respectively. Nuclei...

Epidemiology and Molecular Characteristics of Mycoplasma pneumoniae During an Outbreak of M. pneumoniae-associated Stevens-Johnson Syndrome

“…22,23 Typing of the major adhesion molecule P1 was performed on all isolates using a high-resolution melt assay for determination of types 1 and 2 and identification of genetic variants. 24 Classification as types 1, 2 or variant was based upon comparison of melting profiles to reference strains M129 (type 1) and FH (type 2) included in each run.…”

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…HRM analysis has been used in numerous studies since the mid-2000s on a wide range of human pathogens, including those within the genera Campylobacter, Brucella, Leishmania, Bordetella, Clostridium, Mycobacterium, Mycoplasma, Chlamydophila, Cryptosporidium, and Staphylococcus, among others (445)(446)(447)(448)(449)(450)(451)(452). Recent studies have shown HRM analysis to be a powerful technique for characterizing antibiotic resistance and typing respiratory disease agents, including M. pneumoniae, C. pneumoniae, and Chlamydophila psittaci (453)(454)(455)(456)(457)(458)(459). To our knowledge, HRM analysis for intraspecies or interspecies Legionella discrimination has been included in only a few studies (460)(461)(462)(463); however, this approach seems promising for future Legionella diagnostics or typing.…”

Multiple-Locus Variable-Number Tandem-Repeat Analysis of Mycoplasma pneumoniae Clinical Specimens and Proposal for Amendment of MLVA Nomenclature

“…Two major types of P1 genes (types 1 and 2) [11], [12], [13], three subtypes, and three variants of these subtypes have been described [14], [15], [16], [17], [18]. Recently, a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for molecular typing of M. pneumoniae was developed [19].…”