Background
Mycoplasma pneumoniae (
M. pneumoniae, MP) is a common pathogen that can cause moderate upper respiratory tract infection, severe lower respiratory tract infection, and extrapulmonary clinical symptoms such as encephalitis, Stevens-Johnson syndrome, myocarditis and hemolytic anemia [
1]. The most common infection of
M. pneumoniae is community-acquired pneumonia (CAP). Statistically, 10–40% of pneumonic pathogens in school-aged children and adolescents consists of
M. pneumoniae, and 4–8% consists of
M. pneumoniae in adults, whereas this proportion increased to 20–70% during the epidemic period [
2].
The adhesion of respiratory epithelial cells through the attachment organelle of
M. pneumoniae is a key step for colonization and pathogenesis [
4]. P1 is the major component of the adhesin protein complex at the surface of the organelle, which is essential for cytoadherence of
M. pneumoniae [
13]. According to the sequence differences of the
p1 gene,
M. pneumoniae can be divided into two large subtypes, type 1 and type 2, but the clinical significance of different subtypes is controversial. Although in vitro analysis of immunogenicity of different subtypes showed differences [
14], the earlier reports on
M. pneumoniae P1 typing showed no correlation with susceptibility and severity of clinical symptoms [
15,
16]. However, severe pneumonia and additional extrapulmonary clinical manifestations were reported for type 1
M. pneumoniae infection manifestations [
17]. In another study, type 2
M. pneumoniae pneumonia patients were reported with more neurological and cardiovascular symptoms [
18]. Simultaneously, the dynamic change in the proportion of two subtypes of P1 may also be related to the periodic outbreak and epidemic of
M. pneumoniae [
5]. Studies on
M. pneumoniae typing and antibiotic susceptibility analysis showed that different
p1 gene types may be associated with macrolide resistance to a certain degree, and type 2 strains may be more susceptible to macrolides [
19,
20]. Hence, it is critical to monitor the molecular epidemiological features of
M. pneumoniae since the genotypes may be related to macrolide susceptibility, disease severity and the periodic outbreak and epidemic of the pathogen.
The main treatments for
M. pneumoniae infection are antibiotics. Due to the lack of a cell wall,
M. pneumoniae is naturally resistant to antibiotics acting on the cell wall, such as β-lactam drugs, glycopeptides and fosfomycin, and it is also resistant to polymixins, sulfonamides, trimethoprim, rifampicin and linezolid. Although aminoglycosides, chloramphenicol and gentamicin have activity against
M. pneumoniae, they are not recommended for clinical use [
2,
3]. Macrolides restrained bacterial growth by binding of the 23S rRNA to inhibit protein synthesis, hence macrolides, tetracycline and fluoroquinolone have better performance for the clinical treatment of
M. pneumoniae infection. Due to the possible impact on children's development, tetracycline and fluoroquinolone are not recommended for children. Hence, macrolides, such as erythromycin and azithromycin, serve as the primary choice for the clinical treatment of
M. pneumoniae pneumonia in children. However, macrolide-resistant
M. pneumoniae is gradually increasing worldwide, especially in Asia, showing a high rate of drug resistance [
4‐
11]. In China, the drug resistance rate of macrolides can be as high as 100%, whereas it is lower than 12% in North America, Europe and Australia, and declined from 90% in 2010–2011 to 11% in 2018–2019 in Japan, which may be explained by a decrease in macrolide use and a shift in the prevalent genotype of
M. pneumoniae from macrolide-resistant type 1 to the susceptible type 2 [
12]. Studies have found that the main macrolide resistance mechanism in
M. pneumoniae is the mutation in the 23S rRNA V region, in which A2063G and A2064G mutations lead to high level resistance, and mutations at A2067G and C2617G are associated with lower resistance [
8]. Thus, it is necessary to perform epidemiological monitoring of
M. pneumoniae in different regions to monitor local epidemic characteristics.
Generally, the classification of
M. pneumoniae is mainly based on the differences between two repeated regions RepMP4 and RepMP2/3 contained in the
p1 gene. Commonly used methods for
p1 genotyping include nested PCR, PCR product restriction fragment length polymorphism (PCR–RFLP), rapid cycle PCR and real-time PCR high-resolution melt (HRM) genotyping assay. [
1,
4,
21,
22]. Nested PCR, rapid cycle PCR and PCR–RFLP have high accuracy advantage, but are time-consuming and labor intensive. Compared with traditional PCR, real-time PCR has the advantages of high sensitivity and shorter time consumption by amplifying a small target. The real-time PCR HRM genotyping assay requires amplification of the 1900 bp long region of the
p1 gene and consists of a HRM collection procedure, which may require longer time to obtain genotyping result [
22]. Hence, we aimed to establish a molecular beacon based real-time PCR genotyping method targeting the
p1 gene, which can obtain genotype results rapidly and is easy for clinical application. Meanwhile, we investigated the prevalent genotypes in Henan, China using the method established and analyzed the clinical significance of genotyping by analyzing the relationship between genotypes, macrolide resistance and clinical symptoms.
In the present study, we developed a new genotyping method that uses molecular beacon based real-time PCR for M. pneumoniae p1 gene genotyping. We examined the prevalent genotypes in Henan, China using the method established and analyzed the mutation sites of drug resistance genes by PCR and sequencing. The relationship of the clinical symptoms with the subtypes and macrolide resistance of M. pneumoniae was analyzed.
Discussion
In the present study, we described the development of a molecular beacon based
M. pneumoniae genotyping method based on real-time PCR targeting
p1 gene, in which one reaction can detect two genotypes P1-1 and P1-2. First, the accuracy of this method was evaluated by comparing the results with the ones generated by nest-PCR [
4]. Further, 100
M. pneumoniae infection samples were genotyped using this molecular beacon based method, the mutations in the domain V of 23S rRNA gene were analyzed by PCR and sequencing, and the clinical significances of genotyping were analyzed.
The reasons why
M. pneumoniae infection can lead to different manifestations are not clear. Genotypes are associated with the clinical outcomes were reported [
17,
18]. Hence, we conducted the genotyping of
M. pneumoniae infections to further analyze its relationship with clinical symptoms in the present study. According to the sequence differences of repetitive elements RepMP2/3 and RepMP4 in the P1 protein gene,
M. pneumoniae can be classified into subtype 1 and subtype 2 two major genotypes [
30]. HRM analysis based real-time PCR was reported to distinguish two subtypes of P1 protein gene by amplifying the 1.9 kb fragment [
22]. Compared to the HRM analysis based real-time PCR method, our method had a shorter amplification fragment and hence needed a shorter time ( about 1.5 h vs 2.5 h) to obtain the genotyping result. Additionally, our method can quantify the sample according to the standard curve. PCR–RFLP method was also used in the P1 gene genotyping, which could detect the subtypes by PCR amplification and agarose gel electrophoresis [
4]. In this study, we developed a molecular beacon probes based real-time PCR method that can identify two subtypes by detection of different fluorescence signals in the amplification process. The genotyping results by our method were consistent with the data generated by the nest-PCR method. Hence, we used the method established to further analyze the genotype of
M. pneumoniae infection samples in this study.
Different and changing ratios of the P1-1 and P1-2 subtype
M. pneumoniae infections were reported worldwide [
4,
22], and the incidence of P1-1 infections were usually more than P1-2 (Table
4). Genotyping is crucial for molecular epidemiological studies and the development of an effective vaccine [
31]. In total, 69 (69.0%) of 100 children analyzed in this study were infected with P1-1 M
. pneumoniae whereas the rate of P1-1 in 2019 was 76.4% and 50.0% in 2021. In 2015, 92.0% type 1 strain was reported on the basis of P1 gene PCR–RFLP analysis among 71 adults in Zhejiang province [
32]. Zhao et al. performed a multicenter study analyzing molecular characteristics of
M. pneumoniae by genotyping 154 isolates from 5 cities in mainland China in 2017–2018 and found that type 1 accounted for 76.6%, 23.4% for type 2 strains, and a large variance was found ranging from 100% type 1 in Jilin to 45.5% in Jinan [
33]. Jiang et al. reported 57.1% type 1
M. pneumoniae infection by nested PCR from children with pneumonia in Qingdao, China, in 2019 [
34]. Guo et al. analyzed the molecular features of
M. pneumoniae isolates in paediatric inpatients in Weihai, China in 2019 and found that genotype 2 was identified in 42 isolates of 82 culture-positive samples [
35]. Whistler et al. reported type 1 genotype
M. pneumoniae accounted for a ratio of 61.8% (97/157) in the rural populations of Thailand from 2009 to 2012, and no macrolide resistance mutations were detected [
36]. Kenri et al. found that the genotypes changed periodically in Japan where type 1
M. pneumoniae strains reduced from 100% of the strains isolated in 2012 to 8.3% in 2018 [
37]. Hence,
p1 subtype 1 was the prevalent genotypes in several regions analyzed, and different epidemiological genotypes were distributed in different regions in China and other countries, whereas the prevalent genotypes of Japan indicated a substantial periodical change in the epidemiological features of
M. pneumoniae.
Table 4
Distribution of p1 gene subtypes of M. pneumoniae infection in different regions
Henan, China | 2019/2021 | P1 subtype 1:76.4% in 2019 and 50.0% in 2021 | This study |
Zhejiang, China | 2015 | P1 subtype 1: 92.0% | |
Multicenter, China | 2017–2018 | Overall P1 subtype 1: 76.6%; P1 subtype 1: 100.0% in Jilin and 45.5% in Jinan | |
Qingdao, China | 2019 | P1 subtype 1: 57.1% | |
Weihai, China | 2019 | P1 subtype 2: 51.2% | |
Thailand | 2009–2012 | P1 subtype 1: 61.8% | |
Japan | 2012–2018 | P1 subtype 1: 100.0% in 2012 and 8.3% in 2018 | |
As an important causative pathogen in CAP,
M. pneumoniae increased in macrolide resistance. The high resistance rate was reported in East Asia, which was 81.6% in Japan, 87.2% in Korea, and 90% to 100% in China [
10]. In our study, the macrolide resistance rate was 100.0% and all had the high resistance related A2063G mutation, which was consistent with a study in Qingdao, China in 2021 [
34]. An earlier study in Zhejiang, China also reported A2063G mutation in all of the 71
M. pneumoniae strains isolated from adults with CAP in 2015 [
32]. It is noteworthy that high resistance rate was also found in the type 2 strains in our study, while type 2 strains were related with the lower macrolide resistance rate in other studies reported [
19,
20,
37]. Meanwhile, new mutations were found in the samples, which were C2622T, C2150A, C2202G and C2443A. Among four new mutations found, three was in P1-1 group and one was P1-2 group. The strains were needed to analyze the concrete impact of the new mutations on the macrolide resistance levels. Therefore, the high resistance rate requires special attention and macrolide antibiotic use in the clinical practice should be adjusted accordingly to reduce selection stress for the pathogen. Acute reduction in macrolide-resistant
M. pneumoniae infections among Japanese children was reported, which may indicate the importance of changing antibiotic usage and the impact of
p1 genotype distribution [
20].
Despite advances in genotyping methods to characterize different
M. pneumoniae strains, the relationship between different genotypes and specific clinical outcomes it is still unclear. Hence, we analyzed the relationship between P1-1 genotypes and clinical characteristics. Although the majority clinical outcomes of infections caused by P1-1 and P1-2 subtype
M. pneumoniae isolates are not significantly different, patients infected with P1-1 isolates had a higher lymphocyte count (2.83 × 10
9 cells/L). Fan et al. analyzed 304 cases of type 1
M. pneumoniae and 30 cases of type 2
M. pneumoniae infection (type 1 91.0%) in children with pneumonia, and found that children infected with type 1
M. pneumoniae strain had a higher risk of developing severe pneumonia and with more extrapulmonary clinical manifestations [
17]. Berlot et al. analyzed 356 cases of type 1
M. pneumoniae and 126 cases of type 2
M. pneumoniae pneumonia in children, which found that different types of
M. pneumoniae infections in patients showed different clinical features. Type 2
M. pneumoniae pneumonia patients were with more neurological and cardiovascular symptoms, but patients infected with type 1
M. pneumoniae had other clinical manifestations, which indicated that different types of
M. pneumoniae may have different pathogenicity [
18].
Our study has limitations. First, the samples collected were geographically confined to Zhengzhou, Henan, China. Second, the number of analyzed M. pneumoniae strains were still relatively small, especially the samples in 2021, and it is not enough to analyze the impact of COVID-19 to epidemiological features of M. pneumoniae. Third, although the current design does not affect the results of this study, but variant 2d may be missed by our method, which should be noted in future uses.
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