Multilocus Sequence Typing of Mycoplasma pneumoniae, Japan, 2002–2016

In Japan, Mycoplasma pneumoniae resistance to macrolides is high. To compare sequence types (STs) of susceptible and resistant isolates, we performed multilocus sequence typing for 417 isolates obtained in Japan during 2002–2016. The most prevalent ST overall was ST3, for macrolide-resistant was ST19, and for macrolide-susceptible were ST14 and ST7.

In Japan, Mycoplasma pneumoniae resistance to macrolides is high. To compare sequence types (STs) of susceptible and resistant isolates, we performed multilocus sequence typing for 417 isolates obtained in Japan during 2002-2016. The most prevalent ST overall was ST3, for macrolide-resistant was ST19, and for macrolide-susceptible were ST14 and ST7.
M acrolide-resistant Mycoplasma pneumoniae was first isolated in Japan in 2000 (1). Macrolide-resistant M. pneumoniae is highly prevalent in Asia and has been reported from several parts of the world (2)(3)(4)(5)(6)(7). During a 2011-2012 outbreak in Japan, the resistance rate was as high as 90% (8). Molecular typing methods have been developed for M. pneumoniae and include multilocus variable-number tandem-repeat analysis (MLVA), P1 typing, and others (9). In recent years, multilocus sequence typing (MLST) involving molecular analysis of 7-8 housekeeping genes has been applied to various bacterial pathogens (10,11). In 2014, MLST for M. pneumoniae was devised by Brown et al. (12). Using this MLST method for M. pneumoniae, we compared sequence types (STs) of macrolide-susceptible and macrolide-resistant M. pneumoniae isolates from Japan and examined their evolutionary relationships. As is commonly performed for this pathogen, we also typed the P1 adhesin gene.

The Study
Using the random function of Excel 2010 (Microsoft, Redmond, CA, USA), we randomly selected 417 M. pneumoniae isolates (372 from children, 45 from adults) from 1,084 isolates obtained from patients throughout Japan who had had pneumonia or bronchitis during 2002-2016. Samples were not collected in 2014. The strains were isolated from clinical samples sent from 31 medical institutions in Japan to the Department of Infectious Diseases at Keio University School of Medicine in Tokyo. Of these strains, 232 (55.6%) were macrolide-susceptible M. pneumoniae and 185 (44.4%) were macrolide-resistant M. pneumoniae that had 23S rRNA point mutations. According to M. pneumoniae numbering, these mutations were A2063G (n = 163), A2063T (n = 10), A2064G (n = 10), A2063C (n = 1), or C2617A (n = 1). We used microdilution methods with pleuropneumonia-like organism broth (Difco, Detroit, MI, USA) to determine MICs for 6 antimicrobial agents against these strains of macrolide-susceptible and macrolide-resistant M. pneumoniae (Table 1).
Typing of the P1 adhesin gene in M. pneumoniae was performed as previously described (13). After PCR, the purified DNA products were treated with a restriction enzyme (HaeIII) for 1 hour. The standard stains M129 (ATCC 29342) and FH (ATCC 15531) were used as controls.
When we compared the relationship between year of strain isolation and ST (Table 2), ST3 accounted for most macrolide-susceptible M. pneumoniae, but ST14 also was present in every year; in other words, 2 STs were consistently common among macrolide-susceptible M. pneumoniae. In 2016, infection with macrolidesusceptible M. pneumoniae, especially ST7 and ST14, was epidemic. Among macrolide-resistant isolates, most were ST3. Although ST3 was identified more frequently in macrolide-susceptible isolates during 2002-2006, its identification in macrolide-resistant isolates has increased rapidly since then, and macrolide-resistant ST3 became more common in 2008. ST19 has been identified since the M. pneumoniae epidemic in 2006.
Data showing the relationship between CC and STs according to eBURST (Figure 2) include the 417 strains in this study and 62 strains registered to the MLST database from other countries. Of the 2 CC clusters, the center of CC1 was ST3, from which new STs including ST19 and ST17 arose with >1 alleles mutated. CC1 appears more frequently in macrolide-resistant M. pneumoniae (p<0.001). The center of CC2 was ST2, and almost all strains in Japan belonged to ST7 and ST14. ST22 did not belong to either CC1 or CC2, but 5 of 8 of its alleles were identical to those of CC1. In certain areas of Japan, ST22 has emerged in macrolide-resistant and macrolidesusceptible M. pneumoniae.
P1 typing analysis indicated that M. pneumoniae STs belonging to CC1 and ST22 were type 1, and STs belong to CC2 were type 2. ST14 and ST15 were type 2a, which is a subtype of P1 type 2.