Multiplex amplicon sequencing for the comprehensive genotyping of Mycoplasma pneumoniae

Abstract

Major genotyping methods used to characterize Mycoplasma pneumoniae strains are based on various experimental approaches that need to be implemented in parallel for each strain. In this study, we developed a comprehensive workflow based on amplicon sequencing using next-generation sequencing. This workflow comprised PCR amplification with seven tubes, collection into single tubes, shotgun sequencing, de novo assembly separating each target into individual contigs, and in silico genotyping. The results for p1, orf6, multilocus sequence types, 23S ribosomal RNA gene mutations conferring macrolide resistance, and single-nucleotide polymorphisms identifying the p1 type 1 lineage were obtained simultaneously. The genotyping accuracy was confirmed by comparing the sequences with the whole-genome sequences of 40 M. pneumoniae isolates collected in Tokyo, Japan. The workflow described not only enables high-throughput comprehensive data collection but also enables the detection of novel genotypes with single-nucleotide resolution.

Importance: Genotyping plays a central role in the molecular epidemiology of pathogenic bacteria, and many methods have been developed to identify prevalent lineages, infection routes, and antimicrobial resistance. Whole-genome sequencing generally provides most of the genetic information targeted by classic PCR-based schemes and has contributed to the construction of a simplified workflow for many bacterial species. However, several issues concerning the Mycoplasma pneumoniae genome, such as the presence of repetitive elements of the p1 gene, prevent the collection of genotyping results from a single run of short-read shotgun sequencing. Herein, we describe a simplified workflow using amplicon sequencing that covers most of the major genotyping schemes for M. pneumoniae, including p1 genotyping. The workflow effectively characterized M. pneumoniae clinical isolates. This workflow could help advance research on the molecular epidemiology of M. pneumoniae and the detection of novel genotypes.

Keywords: Mycoplasma pneumoniae; antibiotic resistance; genome analysis; genotypic identification; molecular epidemiology; phylogenetic analysis.

Fig 1

Schematic representation of the genotyping workflow.

Fig 2

Phylogenetic tree showing the phylogenetic relationship between the different clades. The SNPs-based non-rooted tree for p1 types 1 (A) and 2 (B) are shown with the clades specified by combination of genotypes which can be acquired by the comprehensive workflow. The p1 and MLST genotypes were represented using colored plots and branches, respectively. Interpretations for each clade are summarized in boxes, and the clade names for type 1 tree are shown. Red arrowheads represent the positions of M. pneumoniae strains from Tokyo in this study. Scale bars = number of nucleotide substitutions. Full details of these trees are shown in Fig. S2 and S3.