Periodic Genotype Shifts in Clinically Prevalent Mycoplasma pneumoniae Strains in Japan

Abstract

Nationwide increases in Mycoplasma pneumoniae pneumonia cases in Japan were reported in 2011, 2012, 2015, and 2016. In this study, we isolated 554 M. pneumoniae strains in 4 areas in Japan (Kanagawa, Okayama, Osaka, and Saitama) between 2006 and 2019, and performed genotyping analysis. More than 80% of the strains isolated in 2011 and 2012 harbored type 1 p1 adhesin gene; however, strains harboring type 2 or its variant p1 gene increased in 2015 and 2016 and dominated after 2017. These findings suggested that a shift in the prevalent genotype of M. pneumoniae clinical strains occurred recently in Japan. More than 90% of the type 1 strains isolated after 2010 harbored macrolide-resistance mutations in their 23S rRNA gene, whereas most type 2 lineage strains had no such mutations. Consequently, the increase in type 2 lineage strains in Japan has reduced the macrolide resistance rate of clinical M. pneumoniae strains. During this analysis, we also identified M. pneumoniae strains carrying a novel variant type 1 p1 gene, and we classified it as type 1b. We then sequenced the genomes of 81 selected M. pneumoniae strains that we collected between 1976 and 2017 in Japan, and compared them with 156 M. pneumoniae genomes deposited in public databases to provide insights into the interpretation of M. pneumoniae genotyping methods, including p1 typing, multiple-locus variable-number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST), and typing by 8 single-nucleotide polymorphism markers (SNP-8). As expected, p1 typing, MLST, and SNP-8 results exhibited good correlation with whole-genome SNP analysis results in terms of phylogenetic relationships; however, MLVA typing results were less comparable to those of the other methods. MLVA may be useful for the discrimination of strains derived from a single outbreak within a limited area; however, is not reliable for classification of strains collected from distantly separated areas at different time points. This study showed the usefulness of genome-based comparison of M. pneumoniae for molecular epidemiology. Genome sequencing of more strains will improve our understanding of global propagation routes of this pathogen and evolutionary aspects of M. pneumoniae strains.

Keywords: MLST; MLVA; Mycoplasma pneumoniae; genotyping; infectious diseases surveillance; macrolide resistance; p1 gene; whole-genome SNP.

Figure 1

Results of p1 genotyping and macrolide resistance (MR) detection of the 554 M. pneumoniae isolates collected in this study. (A) Annual numbers of total p1 type isolates. Merged data of (B–E). (B–E) Annual numbers of p1 type isolates in the 4 study areas (Kanagawa, Okayama, Osaka, and Saitama) are shown. Numbers of isolates carrying MR mutations are shown in parentheses. (F) LAMP and culture test results for the 221 swab specimens collected in Saitama. (G) Annual numbers of isolates shown by type 1 and 2 lineages. Fraction and rate of MR of two lineages are shown. (H) Numbers and types of MR mutations detected in this study. (I) Occurrence of type 1 lineage isolates. Bars indicate annual isolation number of type 1 lineage isolates, the dotted line indicates the MR rate of type1 lineage isolates. (J) Occurrence of type 2 lineage isolates. Bars indicate annual isolation number of type 2 lineage isolates; the dotted line indicates the MR rate of type 2 lineage isolates. (K) Annual isolation rates of type 1 (blue) and 2 (red) lineages. The dotted line indicates the total MR rate of isolates.

Figure 2

PCR-RFLP analysis of the new type p1 gene. (A) RFLP patterns of the RepMP4 region obtained using the ADH1-2 primer set. (B) RFLP patterns of the RepMP2/3 region obtained using ADH3-4 primer set. PCR products were digested with HaeIII and analyzed by 2% agarose-gel electrophoresis. Lane M: 100-bp ladder marker, lane 1: M129 (type 1), lane 2: KP2440 (type 1b), lane 3: FH (type 2), lane 4: 309 (type 2a), lane 5: KCH-402 (type 2b), and lane 6: KCH-405 (type 2c). (C) Dendrogram of 13 p1 gene sequences reported to date. The tree was generated by alignment of p1 gene sequences using ClustalW v.2.1. The names of representative strains of each type (black) and GenBank accession nos. of the sequences (green) are shown. Type 2b is also reported as type 2V (Diaz et al., 2017). Type 2b2 is also reported as type 2e or type 2bv (Alishlash et al., ; Gullsby et al., 2019). Type 2e of strain Mp100 was originally reported as type 2d (Xiao et al., 2014). In this study, the p1 types were classified as shown in this figure for systematic classification.

Figure 3

Integrated data of genotyping of M. pneumoniae isolates and surveillance of M. pneumoniae pneumonia in Japan. (A) Integrated data of p1 gene typing of M. pneumoniae isolates over the past 40 years in Japan. Blue and red bars show annual detection rates of type 1 and type 2 lineage strains. The dotted line shows the MR rate of isolates after 2006. Detailed integrated data are shown in Supplementary Figure 4. (B) Surveillance data of M. pneumoniae pneumonia in Japan by the National Epidemiological Surveillance of Infectious Diseases (NESID) (https://www.niid.go.jp/niid/ja/10/2096-weeklygraph/1659-18myco.html). Surveillance data of primary atypical pneumonia (red line) for April 1981–March 1999, before the start of M. pneumoniae pneumonia surveillance (blue line) in April 1999 (Yamazaki and Kenri, 2016) are shown. The years of introduction of clarithromycin and azithromycin for the treatment for M. pneumoniae pneumonia in Japan are indicated.

Figure 4

Phylogenetic tree of 237 M. pneumoniae genomes based on whole-genome SNP analysis. p1, orf6, MLVA, MLST, and SNP-8 types, MR, origin, year of isolation, and GenBank accession nos. of the strains are shown at the right side of the trees. Bootstrap values are indicated at the branches. (A) Phylogenetic tree of 136 genome data of p1 type 1 lineage strains. (B) Phylogenetic tree of 101 genomes of p1 type 2 lineage strains. Magnifications of the trees are shown in Supplementary Figure 5.

Figure 5

Alignment-based comparison of the Mpn13 VNTR marker region in strains M129, FH, and KPI-200. VNTR repeat units are marked by blue color. The Mpn13 region of strain KPI-200 exhibits irregular repeat number (2.3 units).