Changes in Molecular Characteristics of Mycoplasma pneumoniae in Clinical Specimens from Children in Beijing between 2003 and 2015

The molecular characteristics of 480 Mycoplasma pneumoniae polymerase chain reaction-positive specimens (331 were previously reported and 149 were newly reported) collected from pediatric patients in Beijing, China, between 2003 and 2015 were analyzed. Genotype M4-5-7-2/P1 were the most prevalent across the 13-year study period, although the isolation and mutation rates for this genotype varied between the periods 2003–2007, 2008–2013, and 2014–2015. In addition, there was a close association between the M4-5-7-2 genotype and macrolide resistance.


Introduction
Mycoplasma pneumoniae is a common bacterial pathogen that causes upper and lower respiratory tract infections in humans, particularly children and young adults [1]. Worldwide epidemics of M. pneumoniae infection occur every 3-7 years [2]., and after the last epidemic around 2012, there were significant increases in the number of M. pneumoniae-associated pneumonia cases reported in 2015 in China, Japan, England, and Wales [3,4,5]., there may be another new epidemic started in 2015. Analysis of the molecular characteristics of specimens collected over this period is therefore very important.
The most prevalent M. pneumoniae genotypes, along with drug resistance profiles, can differ between regions and countries [6,7]., and even within the same region, type-shifts in predominant P1 genotypes have been recorded in Japan at intervals of about 10 years [8]. However, there is little epidemiological information regarding trends in P1 and multiple locus variable-number tandem repeat analysis (MLVA) genotypes and macrolide resistance mutations in the 23S rRNA gene of M. pneumoniae in China. Therefore, this study aimed to elucidate changes in the molecular characteristics of M. pneumoniae specimens in China over a 13-year period. M. pneumoniae-positive specimens were identified using real-time PCR, as described previously [11].

P1 gene typing
Nested PCR-restriction fragment-length polymorphism analysis was used for P1 genotyping, and was performed as previously described [9] directly from DNA extracted from the PCRpositive specimens. The PCR products of type 2 specimens were sequenced to identify type 2 variants.

MLVA typing
Multiplex PCR amplification-linked capillary electrophoresis of four loci (Mpn13, Mpn14, Mpn15, and Mpn16) was used for the amended MLVA genotyping, and was performed according to previously described [10] and the international guidelines [12].

Detection of macrolide resistance
Detection of macrolide resistance mutations, including the common macrolide resistance point mutations at positions 2063, 2064, 2611, and 2617 (M. pneumoniae numbering), was performed as previously described [13].  Fig 1A). As 2007 and 2012 were epidemic years in China [3], the specimens were also grouped into three time periods. In the 5-year period from January 2003 until December 2007, types 2 and 2c accounted for 17.4% (4/23) of specimens, while between 2008 and 2013, after the epidemic of 2007, the prevalence of type 2 and 2c specimens significantly decreased to only

Detection of macrolide resistance mutations
This study examined changes in the molecular characteristics of M. pneumoniae in clinical specimens collected from children in Beijing over a 13-year period. The genotype M4-5-7-2/ P1 was most prevalent in children with M. pneumoniae infection, especially from 2008 to 2013. However, the prevalence of the two main MLVA types, M4-5-7-2 and M3-5-6-2, along with the main P1 gene types, 1 and 2, varied between the three epidemic periods. In particular, some genotypes were only detected in Beijing during and after the last worldwide epidemics.
Our study also provides more evidence that there is an association between MLVA type M4-5-7-2 and macrolide resistance.
However, this study also has some limitations. As the number of specimens from 2003-2007 was small, examination of a greater number of specimens will help to better understand the epidemic trends. Moreover, other typing methods, such as pulsed-field gel electrophoresis and multilocus sequence typing, could be used in conjunction with the current methods to enhance the results of further studies [19,20]. Although a large amount of sequencing is required with multilocus sequence typing, this method has better discriminatory power than MLVA and P1-restriction fragment-length polymorphism analysis. Therefore, more studies using a combination of these methods should be undertaken to investigate associations between clinical infections and specific M. pneumoniae molecular characteristics.