Mycoplasma pneumoniae is the most prevalent cause of community-acquired pneumonia (CAP) in children, especially among hospitalized patients (Morozumi et al., 2008; Jain et al., 2015; Meyer Sauteur et al., 2020b; Meyer Sauteur et al., 2020a). M. pneumoniae regularly causes epidemics every 3–7 years that last for 1–2 years (Dumke et al., 2015). The P1 adhesin, located at the tip of the M. pneumoniae attachment organelle (Kenri et al., 2019), is an essential determinant for adherence to respiratory tract epithelial cells and for sliding movement (Krause and Baseman, 1982). Together with the P40/90 polypeptide, it forms a cross-membrane complex known as “NAP” (Scheffer et al., 2017). Additionally, P1 adhesin is recognized as one of the immunodominant proteins and performs important functions in the pathogenesis of M. pneumoniae infection (Peng et al., 2023) and immune response of infected patients (Vizarraga et al., 2020). M. pneumoniae can be divided into P1 types 1 (P1-1) and 2 (P1-2) based on differences of two repetitive DNA regions (RepMP2/3 and RepMP4) in the P1 protein-coding gene (Dumke et al., 2004; Dumke et al., 2006). Studies on the P1 genotype and its variation have been conducted worldwide (Sasaki et al., 1996; Kenri et al., 2008; Jacobs et al., 2015; Jiang et al., 2021; Guo et al., 2022; Wang et al., 2022), and changes in P1 genotype are believed to be associated with the prevalence of M. pneumoniae infections (Atkinson et al., 2008). However, findings regarding the relation between the P1 genotype and disease severity have been inconsistent. The culture-independent P1 genotype method for M. pneumoniae has been widely used due to its efficiency, cost-effectiveness, and ability to directly classify clinical samples without relying on mycoplasma culture (Dumke et al., 2006). An accurate understanding of the P1 genotype and its variation in China, as well as the correlation between genotype and disease severity, is essential for disease management and improving early warning systems.

Thus, this study investigated the changes of the P1 genotype in patients infected with M. pneumoniae in China from 2017 to 2019 and the relationship of the genotype with disease severity.

M. pneumoniae is the most common cause of respiratory tract infection, accounting for 28% -40% of CAP cases in children (Waites et al., 2017; Shah, 2019; Meyer Sauteur et al., 2020b), and it is characterized by typical periodic epidemics. The relationship between the genotype of P1 adhesin and its variation with the periodic prevalence and severity of M. pneumoniae infection is attracting increasing attention. Understanding the P1 genotype of M. pneumoniae and its migration trend of subtypes may be useful for disease surveillance and control. Our study found that M. pneumoniae-infected patients in Beijing, Shenyang, and Baotou mainly carried the P1-1 genotype from 2017 to 2019, although the prevalence of the P1-2 genotype increased over time.

We detected M. pneumoniae, performed culture-independent P1 typing, and cultivated the microbe using 1,907 clinical samples collected from 2017 to 2019. The M. pneumoniae positivity rate was 43.00%, and the positivity rate was lower in adults than in children, highlighting the importance of M. pneumoniae as a cause of CAP in children, either inpatients or outpatients. For adults, we included only inpatient CAP patients, a study on outpatient CAM patients would strengthen our findings.

At present, five main methods are utilized to study the epidemiology and drug resistance mechanism of M. pneumoniae: multi-site variable number tandem repeat analysis (MLVA), multi-site sequence typing (MLST), single nucleotide polymorphism (SNP), variable number tandem repeat (VNTR), and P1 typing based on the repeat number of P1 protein-coding gene. Among these, VNTR and MLVA are commonly used in the genotyping of infectious diseases due to their greater discriminatory power (Yan et al., 2014).

In 2009, Degrange et al. (2009) screened VNTR in the gene sequence of M. pneumoniae using software and selected 5 species-specific VNTRs, named Mpn1, Mpn13, Mpn14, Mpn15, Mpn16. A total of 26 MLVA genotypes A-Z were identified according to the number of these five VNTR loci. However, Benitez et al. (2012) found that the Mpn-1 locus in the MLVA genotyping method was unstable. After 10 generations, 75% (6/8) of strains showed changes in the number of Mpn-1 repeats. Subsequently, researchers from various countries agreed that unstable Mpn1 locus should be excluded (Chalker et al., 2015), retaining only Mpn13, Mpn14, Mpn15, and Mpn16 to improve scoring stability. However, this change caused a significant reduction in the ability of MLVA typing to identify M. pneumoniae strains. Studies (Copete et al., 2018) showed that 4-5-7-2 of the improved MLVA genotypes were equivalent to the traditional P1 types, while 3-5-6-2 and 3-6-6-2 were equivalent to P1s. MLST is another widely used method for bacterial genotyping including for M. pneumoniae. Brown et al. (2015) developed an MLST scheme for M. pneumoniae, identifying 12 different sequence types (ST). SNP Genotyping is performed by analyzing DNA sequence polymorphisms caused by single nucleotide variations. However, MLST and SNP typing are mainly used in some studies due to cost and efficiency problems.

The PCR restriction fragment length polymorphism (PCR-RFLP) method requires the isolation and purification of M. pneumoniae strains from clinical samples. Because culturing M. pneumoniae is time consuming and not routinely performed in bacteriological practice, a culture-independent P1 typing method was established (Dumke et al., 2006). This method is more economical and efficient, as it directly identifies M. pneumoniae from clinical samples, making it widely used. Moreover, studies examining the relationship between macrolide resistance in M. pneumoniae and genotyping mainly use P1 genotyping and MLVA (Spuesens et al., 2010; Guo et al., 2019).

Adhesion is a key component of the pathogenic process of M. pneumoniae, and P1 adhesin is the most important adhesion protein of M. pneumoniae (Vizarraga et al., 2020), as it forms the tip structure in combination with P40/P90 (Vizarraga et al., 2020). P1 adhesin is involved in the immunopathogenic effects of M. pneumoniae (Fan et al., 2017), highlighting its role in infection by this microbe. The P1 genotype was established in the 1990s (Dallo et al., 1990), and P1 genotyping is the earliest and most mature M. pneumoniae genotyping method. Studies from different regions have reported the dominant genotype of P1 adhesin. For example, in 2011–2012, P1-2 was the predominant genotype in Slovenia (Kogoj et al., 2015) and South Africa (Carrim et al., 2018). However, P1-1 was dominant in the same period in Germany (Dumke et al., 2015), Sweden (Gullsby et al., 2019), France (Pereyre et al., 2013), Japan (Suzuki et al., 2017), China (Sun et al., 2017), and Thailand (Whistler et al., 2017), supporting the regional characteristics of the dominant P1 genotype.

A shift in the genotype of M. pneumoniae strains has been reported in several studies (Sasaki et al., 1996; Pereyre et al., 2007; Dumke et al., 2010). These data suggested the existence of periodic changes in the P1 genotype in M. pneumoniae even in the same region (Sasaki et al., 1996; Kenri et al., 2008). Considering that mycoplasma pneumoniae infection has the characteristics of periodic prevalence, it is speculated that P1 typing may be used in epidemiological studies (Atkinson et al., 2008). The relationship between the P1 genotype and the periodic epidemic of mycoplasma pneumoniae has gradually become a hot research topic. A study (Sasaki et al., 1996) in Japan conducted P1 typing of M. pneumoniae isolates from 1976 to 1995 and found that the periodic transformation of the P1 genotype may be related to the epidemic. Dumke et al. (2004) found that host pre-infection with a certain subtype would cause subtype-specific immunity, which had a strong impact on the type of bacteria that survived the secondary infection through animal experiments. This theory explains the phenomenon that subtype shifts in outbreaks of mycoplasma pneumoniae infection. In the current study, we used a culture-independent P1 typing method to investigate the P1 genotype in clinical samples collected from 2017 to 2019. Although P1-1 remained the dominant genotype, the proportion of P1-2 strains increased over time. Meanwhile, the proportion of P1-1 strains did not differ among Beijing, Shenyang, and Baotou, consistent with the results of Xiao et al (Yan et al., 2020). This trend was also similar to that observed by Jiang et al. (2021) during the same study period, but it contradicted the findings of a later study by Guo et al (Guo et al., 2022). The P1 genotype significantly differed between adults and children, similar to the findings of Yan et al (Yan et al., 2020). Interestingly, a study from Henan Province, China, found that by 2021, the proportion of P1-2 strains increased to 50% (Li et al., 2022), a trend consistent with our study in both period and region. A subsequent study from Shanghai, China, found that the proportion of P1-1 type increased significantly in the outbreak of M. pneumoniae in children after the COVID-19 pandemic (Zhu et al., 2024). From the epidemiological point of view, these findings suggest the importance of continuous monitoring of P1 genotype changes.

P1 adhesin is an important pathogenic gene of Mycoplasma pneumoniae, which can directly damage host bronchial epithelium (Miyata and Hamaguchi, 2016) and induce intense cellular and humoral immunity to participate in the pathogenic process of M. pneumoniae (Jacobs et al., 1990; Atkinson et al., 2008). After adhesion, M. pneumoniae caused disease by producing peroxide, Superoxide anion (Atkinson et al., 2008), and community-acquired respiratory distress syndrome toxin (CARDS TX) (Peters et al., 2011), as well as activating immune cells to produce inflammatory factors (Hoek et al., 2005). P1-1 may have higher reproductive capacity in patients with lower host immune responses. Furthermore, P1-1 genotype strains infection could induce a more severe respiratory immune response, worsen respiratory symptoms, and favor opportunistic infections (You et al., 2025). Based on the processes of pathogenesis and pathogenic of M. pneumoniae, the relationship between P1 genotype and disease severity has also been extensively studied. Unfortunately, the conclusions were inconsistent.

A study from Slovenia in 2014 reported that pediatric patients carrying P1-2 genotype strains tend to experience more severe disease compared to those infected with P1-1 strains and that patients infected by carrying P1-2 strains might be more prone to central nervous system and cardiovascular complications (Rodman Berlot et al., 2018; Rodman Berlot et al., 2021). In contrast, studies from Jiangsu Province (Xu et al., 2024) and Jilin Province (You et al., 2025) in China found that P1-1 genotype strain infection was associated with more severe clinical outcomes. Additionally, a study in Switzerland from 2016 to 2020 found that M. pneumoniae genotypes do not influence clinical outcomes (Meyer Sauteur et al., 2021). These differing results highlight the uncertainty in the relation between P1 genotype and disease severity. In our study, the P1 genotype did not differ between outpatients and inpatients or between children with severe pneumonia and those with general pneumonia. Our result aligns with the findings of Meyer et al (Meyer Sauteur et al., 2021), Nilsson et al. from Sweden (Nilsson et al., 2010), and several studies from Wuhan (Xu et al., 2024), Shanghai (Chen et al., 2024), Beijing (Jiang et al., 2024), and Henan (Li et al., 2022), China, but contrasts with those of studies in Slovenia (Rodman Berlot et al., 2018; Rodman Berlot et al., 2021). Further research is needed to clarify these discrepancies.

For strains cultured from 2017 to 2019, the culture positivity rate was higher for P1-1 strains than for P1-2 strains (53.06% vs. 28.13%). At present, there is no clear evidence to confirm this phenomenon, and we speculate that it may be related to the following factors. First, the sensitivity of different P1 genotype M. pneumoniae strains and the use of antibiotics before sampling may affect the positive rate of culture. Second, the self-synthesis and reproduction different P1 genotype strains may be different. All this speculation needs further evidence. The low positivity rate in M. pneumoniae culture and the time required for results confirm the advantages of the culture-independent P1 genotype method.

Some limitations of this study should be acknowledged. First, all samples in this study were collected from tertiary hospitals. In addition, the strains were mainly isolated in Beijing, and their classification in other regions is not well understood. Second, this study only investigated the P1 genotype, and different P1-1 and P1-2 variants could not be evaluated. These shortcomings will be addressed in future studies.

In conclusion, this study found that M. pneumoniae persisted as one of the dominant etiologic causes of CAP in children in China from 2017 to 2019. During the period, P1-1 remained the dominant genotype of M. pneumoniae, nonetheless the proportion of P1-2 strains increased over time. The P1 genotype was not associated with disease severity. However, continuous monitoring of P1 genotype trends remains important for mycoplasma pneumoniae control efforts.