Whole-genome sequencing unveils the outbreak of Mycoplasma pneumoniae in mainland China

The Comment published in The Lancet Microbe by Patrick M Meyer Sauteur and colleagues1Meyer Sauteur PM Beeton ML Mycoplasma pneumoniae: delayed re-emergence after COVID-19 pandemic restrictions.Lancet Microbe. 2023; 5: e100-e101Summary Full Text Full Text PDF Scopus (13) Google Scholar caught our attention for its discussion on the delayed resurgence of Mycoplasma pneumoniae post-COVID-19. Similarly, the Correspondence by Heng Li and colleagues2Li H Li S Yang H Chen Z Zhou Z Resurgence of Mycoplasma pneumonia by macrolide-resistant epidemic clones in China.Lancet Microbe. 2024; (published online Jan 17)https://doi:10.1016/S2666-5247(23)00405-6Summary Full Text Full Text PDF Google Scholar proposed that macrolide-resistant Mycoplasma pneumoniae clones might have sparked outbreaks nationwide as early as 2020, as evidenced by data on paediatric patients. Despite the lifting of COVID-19 restrictions, China's epidemiological data on M pneumoniae remain scarce. This scarcity of data could be explained by diagnostic challenges associated with M pneumoniae, as culturing this bacterium is typically not feasible due to its fastidious nature, making PCR the most reliable diagnostic method. Since mid-October 2023, an increase in respiratory illnesses in China has been noted, with regions in northern China, such as Beijing and Liaoning Province, experiencing higher numbers of influenza-like illnesses than that reported in the past 3 years. This surge might be attributed to the spread of familiar pathogens such as respiratory syncytial virus, influenza viruses, and M pneumoniae.3Parums DV Editorial: Outbreaks of post-pandemic childhood pneumonia and the re-emergence of endemic respiratory infections.Med Sci Monit. 2023; 29e943312Crossref Scopus (1) Google Scholar Analysis of PCR and serology test data for M pneumoniae from two tertiary hospitals, namely, the First Affiliated Hospital of Zhejiang University School of Medicine and Zhejiang Provincial People’s Hospital, in Zhejiang province, revealed a marked increase, with the positive rate increasing from approximately 10% to 30%, indicating a notable surge in M pneumoniae cases in 2023 compared with those in 2022 (appendix 1 p 1). However, PCR and serology tests for M pneumoniae have limitations as these methods do not allow for antimicrobial susceptibility testing.See Online for appendix 1 See Online for appendix 1 Building upon the paediatric patient data presented by Li, we collected 236 M pneumoniae-positive respiratory samples from 106 children and 130 adults across seven Chinese regions (southwest, northwest, central, south, southeast, north, and northeast regions), from July, 2021, to December, 2023, utilising metagenomic next-generation sequencing (appendix 1 p 2, appendix 2). Subsequent M pneumoniae whole-genome capture sequencing analyses were conducted on these samples, and of the 236 samples, 91 samples were deemed suitable for multilocus sequence typing (MLST) analysis, as the coverage and depth of sequencing were sufficient for housekeeping genes. The MLST analysis revealed that sequence type (ST) 3 (n=63; 69·23%) and ST14 (n=21; 23·07%) were the predominant M pneumoniae strains, followed by ST7 (n=7; 7·69%). The integration of core genome MLST and MLST analyses facilitated the typing of 149 of 236 samples, with ST3 (n=104; 69·79%) and ST14 (n=29; 19·46%; appendix 2) being the most prevalent, followed by ST7 (n=13; 8·72%). The phylogenetic analysis results indicate that the globally prevalent ST3 (P1 type 1) and ST14 (P1 type 2) M pneumoniae clones have been circulating in China since 2022 (appendix 1 pp 3–4).4Hsieh YC Li SW Chen YY et al.Global genome diversity and recombination in Mycoplasma pneumoniae.Emerg Infect Dis. 2022; 28: 111-117Crossref PubMed Scopus (3) Google Scholar Of the 236 M pneumoniae-positive samples, 168 yielded adequate sequencing data for resistance mutation analysis (appendix 1 p 3). The mutation rate for macrolide resistance-related genes in 23S rRNA was 88·10% (n=148), with A2063G being the most frequent mutation (n=148). One sample harboured a co-mutation of A2063G and C2617T. The mutation rates in the 23S rRNA domain V, which houses specific macrolide-binding sequences, varied across different regions and ST groups, with 100% (101 of 101) of ST3 isolates and 96·6% (28 of 29) of ST14 isolates harbouring resistance-related mutations. In contrast, ST7 isolates, which are predominant in Japan as macrolide-susceptible clones, showed no resistance mutations and were found in south China. In summary, our multicentre study findings indicate that M pneumoniae could have triggered outbreaks following the relaxation of COVID-19 restrictions, with macrolide-resistant M pneumoniae clones spreading across mainland China. Additionally, our research highlights the applicability of high-throughput whole-genome sequencing as a diagnostic method for epidemiological surveillance and monitoring of M pneumoniae.See Online for appendix 2 See Online for appendix 2 We declare no competing interests. YC, XL, and YF contributed equally. This study was supported by research grants, including the Key R&D Plan of the Ministry of Science and Technology of China (2022YFC2504502), the Zhejiang Province Pioneer Research and Development Project (2023C03068, 2024C03187), and the Key Research Program of the Science Technology Department of Zhejiang Province (2021C03055). The funder had no role in the writing or the decision to submit this Correspondence for publication. YC, YY, and HZ designed the experiments. YC, XL, and YF collected and analysed data. YC and HZ wrote the Correspondence. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. We thank Xu Han (Hangzhou Matridx Biotechnology, Hangzhou) for technical service and Feng Ling (Zhejiang Provincial Center for Disease Control and Prevention) for consultation with our manuscript. The study was approved by the Ethics Committee of The First Affiliated Hospital, Zhejiang University School of Medicine (IIT20231068A). Download .pdf (1.18 MB) Help with pdf files Supplementary appendix 1 Download .xlsx (.03 MB) Help with xlsx files Supplementary appendix 2