Molecular Epidemiology of Mycoplasma pneumoniae: Genotyping Using Single Nucleotide Polymorphisms and SNaPshot Technology

Abstract

i Molecular typing of Mycoplasma pneumoniae is an important tool for identifying grouped cases and investigating outbreaks. In the present study, we developed a new genotyping method based on single nucleotide polymorphisms (SNPs) selected from the whole-genome sequencing of eight M. pneumoniae strains, using the SNaPshot minisequencing assay. Eight SNPs, localized in housekeeping genes, predicted lipoproteins, and adhesin P1 genes were selected for genotyping. These SNPs were evaluated on 140 M. pneumoniae clinical isolates previously genotyped by multilocus variable-number tandem-repeat analysis (MLVA-5) and adhesin P1 typing. This method was also adapted for direct use with clinical samples and evaluated on 51 clinical specimens. The analysis of the clinical isolates using the SNP typing method showed nine distinct SNP types with a Hunter and Gaston diversity index (HGDI) of 0.836, which is higher than the HGDI of 0.583 retrieved for the MLVA-4 typing method, where the nonstable Mpn1 marker was removed. A strong correlation with the P1 adhesin gene typing results was observed. The congruence was poor between MLVA-5 and SNP typing, indicating distinct genotyping schemes. Combining the results increased the discriminatory power. This new typing method based on SNPs and the SNaPshot technology is a method for rapid M. pneumoniae typing directly from clinical specimens, which does not require any sequencing step. This method is based on stable markers and provides information distinct from but complementary to MLVA typing. The combined use of SNPs and MLVA typing provides powerful discrimination of strains. Mycoplasma pneumoniae is the second leading cause of community-acquired pneumonia behind Streptococcus pneumoniae. This bacterium affects both the upper and lower respiratory tracts of individuals of all age groups, and infections occur endemically and epidemically worldwide (1, 2). Many outbreaks of M. pneumoniae respiratory infections have been reported in the community and in closed or semiclosed settings, such as military bases, hospitals, religious communities, schools, and institutions for the mentally disabled and may be associated with considerable morbidity (1, 3-6). Since 2010, a substantial increased incidence of M. pneumoniae infections has been reported in several countries (7-9). Molecular typing methods for M. pneumoniae have been developed for the identification of grouped cases and investigation of outbreaks (10). Until recently, the most common typing methods for M. pneumoniae were based on the analysis of single nucleotide polymorphisms (SNPs) within the gene encoding the major immunogenic protein P1, which is involved in adhesion to host cells. Several methodologies were used, including PCR-restriction fragment length polymorphism (11), amplification and gene sequencing (12), real-time PCR with high-resolution melt analysis (13), and pyrosequencing (14). However, due to the homogeneity of the M. pneumoniae species, few polymorphisms have been identified, and P1-based typing methods allow th...

Multiple-Locus Variable-Number Tandem-Repeat Analysis of <i>Mycoplasma pneumoniae</i> Isolates between 2004 and 2014 in Yamagata, Japan: Change in Molecular Characteristics during an 11-year Period

“…Several studies have indicated that the discrimination power of four-locus MLVA is low because the unstable Mpn 1 locus is excluded (12,21,24). Therefore, to improve the discrimination power of MLVA for M. pneumoniae, a new MLVA scheme should be developed that includes additional VNTR markers such as those identified in a recent study (33).…”

“…We also reported P1 types and the prevalence of macrolide resistance-associated mutations of M. pneumoniae isolates in Yamagata, Japan between 2004 and 2013 (22). However, few reports have described the MLVA types of M. pneumoniae and examined the association between MLVA type and macrolide susceptibility in Japan (23,24). In this study, we performed MLVA typing for M. pneumoniae isolated between 2004 and 2014 in Yamagata, Japan, and evaluated the molecular epidemiological features of M. pneumoniae including P1 type and prevalence of macrolide resistance-associated mutations during an 11-year period.…”

Inter- and Intra-Strain Variability of Tandem Repeats in Mycoplasma Pneumoniae Based on Next-Generation Sequencing Data

“…To examine intrastrain copy number variation, we used archived NGS raw reads for 26 M. pneumoniae isolates, including 454 reads (average length 509 bp) for one isolate 309 reported by Kenri et al [36]; Illumina reads (251 bp each) for 15 isolates reported by Xiao et al [8]; Illumina reads (116 bp each) for two isolates reported by Li et al [37,38]; Illumina reads (95 bp each) for seven isolates reported by Lluch-Senar et al [7]; Illumina reads (36 bp each) for the strain FH reported by Touati et al [39]. We selected only seven of the 23 isolates reported by Lluch-Senar et al [7] that represented different genome size and geographic regions, instead of using all of them because their read lengths were too short (95 bp) to cover the long repeats in Mpn14-16.…”

“…These studies expand our understanding of the VNTR variations and provide potential new MLVA schemes for M. pneumoniae geno typing. Further studies are needed of the performance of these new MLVA schemes in comparison with multilocus sequence typing [34], multiplex single nucleotide polymorphisms [39] and genome-wide single nucleotide polymorphisms and indels [7] using whole genome sequence data currently available and/or from new clinical isolates with diverse clinical and epidemiological parameters. This report presents the first successful attempt of using primarily the wealth of publicly available NGS sequences to address the fundamental questions on M. pneumoniae biology.…”

“…Many complete and fragmentary genome assemblies as well as high volumes of raw sequence reads are available for M. pneumoniae isolates from diverse geographic origins, time periods and clinical background [7][8]34,[36][37][38][39]. The wealth of genome data provides an excellent opportunity to characterize VNTRs in M. pneumoniae.…”