Mycoplasma pneumoniae (M. pneumoniae) is a common pathogen responsible for community-acquired pneumonia in children (Wang et al., 2022). In recent years, there was an increase in the incidence of Mycoplasma pneumoniae pneumonia (MPP), with more than 50% of children admitted to respiratory wards having been diagnosed with MPP (Yan et al., 2024). The known pathogenic mechanisms of M. pneumoniae infection include adhesion, oxidative stress, community-acquired respiratory distress syndrome (CARDS) toxin, and immune-mediated inflammatory damage, all of which have the potential to lead to airway mucus hypersecretion (AMH) (Yao and Han, 2021). Excessive airway mucus secretion not only produces highly viscous and elastic mucus, which is difficult to remove, leading to mucus retention, even mucus plug formation. This subsequently exacerbates infections, causes airway obstruction, and airflow limitation, ultimately resulting in progressive decline in lung function (Bao and Zhang, 2018).

In children with MPP, AMH may be closely associated with the imbalance of lower respiratory tract microbiota (LRTM). Studies showed that bronchoscopy was one of the methods for evaluating airway mucus secretion, and the bronchoscopy mucus score system (BS score) for children quantified the amount and distribution of airway mucus (Chang et al., 2006; Xia and Luo, 2022). Moreover, studies on pediatric non-cystic fibrosis bronchiectasis demonstrated that the microbial growth rate in bronchoalveolar lavage fluid (BALF) increased with higher BS scores (Nursoy et al., 2021).

The LRTM not only plays a crucial role in maintaining respiratory homeostasis but may also influence the onset and progression of respiratory diseases through interactions with the host (Wang et al., 2023). Under certain conditions, bacteria that are normally symbiotic can become involved in pathogenic processes, triggering inflammatory responses (Kim et al., 2016). Conversely, pulmonary microbiota may suppress other pathogenic microorganisms (Wu and Segal, 2018) by competing for nutrients (Spiga et al., 2023) or producing metabolites (Hardy and Merrell, 2021). Changes in the structure and composition of these microbial communities may affect mucus secretion in the airways, thereby influencing the course and prognosis of MPP.

However, studies on the correlation between LRTM and AMH remain limited. Therefore, this study employed 16S rRNA amplicon sequencing to investigate the structure and compositional trends of LRTM under varying mucus secretion states in children with MPP. We analyzed the impact of characteristic bacterial genera on AMH to provide new insights and evidence for clinical interventions.

Airway mucus hypersecretion has received extensive attention in studies of chronic respiratory diseases such as asthma (Jaramillo et al., 2024), COPD (Tian and Wen, 2015), cystic fibrosis (Mall, 2016), and bronchiectasis (Chen and Zheng, 2021; Abrami et al., 2024). While AMH was also common in acute respiratory infections in children, particularly with infections like M. pneumoniae, Adenovirus (Cai et al., 2023), Influenza virus, respiratory Syncytial virus (Yan and Deng, 2024), and Bordetella pertussis (Yao and Han, 2021), research on LRTM in children with MPP and mucus hypersecretion is very limited. AMH refered to the pathophysiological process in which various pathogenic factors cause hyperplasia and hypertrophy of airway mucosal goblet cells and submucosal glands, leading to excessive mucus production (Chen and Zheng, 2021). Its severity was related to the quantity and viscosity of airway secretions and the presence of small airway mucus plugs (Chen and Zheng, 2021). This study examined the composition and structural characteristics of the LRTM from two perspectives: disease progression and mucus hypersecretion. We observed that as the pre-EOB period extended, α-diversity gradually increased, particularly in the hypersecretion group. β-diversity indicated significant spatial distribution changes in the microbial community. Importantly, the incidence of AMH increased with disease progression, suggesting a potential relationship between LRTM and AMH, beyond just M. pneumoniae infection. Interestingly, the T1 period in the hypersecretion group had the lowest Shannon index, which seems to present a potential contradiction with the sample distribution in the PCA plot at T1. The Shannon index measures species richness and evenness within a sample, reflecting the species diversity of a particular community. PCA analysis measures differences between samples, with a focus on community structure and heterogeneity between samples. The two indexes are complementary to each other.

We focused on analyzing the characteristic genera in AMH. The LRTM of children in T1 hypersecretion group, dominated by Mycoplasmoides. M. pneumoniae inhibits commensal bacteria by competing for nutrients (Chen et al., 2021) and inducing production of host cytokine, nitric oxide, antimicrobial peptides (Yang et al., 2004). It also invades host cells, depletes nutrients, and releases CARDS toxin, superoxide radicals, and hydrogen peroxide, causing direct epithelial cell damage (Hu et al., 2023). The exotoxin CARDS TX released by M. pneumoniae induces “asthma-like” histopathological changes similar to allergic diseases, characterized by elevated eosinophil levels, enhanced Th2 cell responses, increased chemokines expression, increased mucus production, and increased airway resistance (Xu et al., 2024) M. pneumoniae can promoting mucin upregulation via STAT6-STAT3 and epidermal growth factor receptor (EGFR) signaling pathways (Hao et al., 2014), and reduces ciliary clearance rate by directly damaging airway epithelial cells (Lu and Zhou, 2018), leading to mucus blockage. M. pneumoniae infection increases neutrophil counts, and the neutrophil elastase (NE) released activates the MAPK/ERK pathway and EGFR, stimulating airway epithelial cells to secrete more mucins, particularly MUC5AC and MUC5B (Chen and Tong, 2016). In T1 period, M. pneumoniae breaks down glucose through the glycolysis pathway to produce lactate. In T3 period, increased abundances of Streptococcus and Prevotella provide lactate as the preferred carbon source for the growth of Veillonella. Veillonella metabolizes lactate into short-chain fatty acids (SCFAs), such as acetate and propionate. It is well known that SCFAs have anti-inflammatory effects. However, studies have also shown that, in vitro, propionate in combination with TNF-α can synergistically increase the production of IL-6 and CXCL8 by primary human lung fibroblasts and airway smooth muscle cells (Whiteside et al., 2021). The enrichment of Streptococcus and Veillonella in airway washings from cancer patients is associated with increased inflammatory cell infiltration and upregulation of the PI3K/ERK signaling pathway in bronchial epithelium (Tsay et al., 2018). Veillonella in the oral cavity and respiratory tract forms a multispecies biofilm through symbiosis with late-colonizing pathogens, such as Streptococcus (Zhou et al., 2021). Veillonella promotes the increase of inflammatory factors through its metabolic products and pro-inflammatory signaling pathways, leading to excessive mucus secretion. The abundance of Mycoplasmoides decreased in the T3 period, while Stenotrophomonas and Chryseobacterium significantly increased. This outcome may result from a combination of microbial interactions, antibiotic use, immune responses, and prolonged disease duration. After all, increased mucin secretion directly suppress pathogen virulence, aiding in microbial coexistence (Wang B. X. et al., 2021). However, the causal relationships require further investigation.

Respiratory microbiota are influenced by migration, elimination, and microbial proliferation rates (Pérez-Cobas et al., 2023). Using a stepwise forward method, we selected clinical indicators and characteristic genera to perform a multivariate logistic regression analysis on BS grades 4 and 5 in the hypersecretion group. The results identified the peripheral neutrophil percentage, Mycoplasmoides, and Veillonella as risk factors for increased mucus secretion. Studies have shown increased neutrophil infiltration in both peripheral blood and BALF in children with MPP or refractory MPP (Wang B. X. et al., 2021; Cai et al., 2023; Abrami et al., 2024), and that MPP severity is associated with a neutrophil-mediated immune response. In MPP children with mucus plugs, higher serum IFN-γ levels were found in those with pleural effusion, which correlated positively with Veillonella abundance (Xu et al., 2020). Overabundance of Veillonella parvula induce macrophage-associated inflammation through the LPS-TLR4 pathway, impairing gut motility and further exacerbating gut dysbiosis, which promotes the development of Hirschsprung’s disease-associated enterocolitis (Zhan et al., 2022). These suggest that Veillonella may also play a role in disease progression. On the other hand, certain Prevotella species, such as Prevotella timonensis, have a multitude of predicted α/β-galactosidases, α/β-N-acetylgalactosaminidases, and α/β-N-acetylglucosaminidases, which hydrolyze glycosidic bonds in sialic acid and fucoses. Sialidases and fucosidases are key enzymes that initiate the degradation of O-glycan structures in secreted and epithelial-bound mucins, enabling further mucin degradation after the removal of these outer monosaccharides (Segui-Perez et al., 2024). This plays a regulatory role in mucus production.

Co-infection of M. pneumoniae with other pathogens is common in children with respiratory syndromes (Chiu et al., 2015). In this study, 40 co-infection cases were identified through BALF bacterial culture and pathogen DNA testing during the T3 period, with 61.9% of cases involving Streptococcus pneumoniae, 19% Haemophilus influenzae, 38.1% virus. Co-infection rates were highest in the T3 period, and elevated WBC levels compared to case with only M. pneumoniae. Additionally, the abundance of Streptococcus and Prevotella increased significantly with β-lactam antibiotics use, and Prevotella was linearly correlated with WBC. The rise of pathogenic Streptococcus and Prevotella replaced the previously dominant Stenotrophomonas. In certain environments, such as CF lung microbial communities, Prevotella has been reported to produce β-lactamase, protecting P. aeruginosa from ceftazidime. This leads to antibiotic-resistant strains dominating the microbial community (Vandeplassche et al., 2019). As the disease progresses, the likelihood of co-infection increased. This may be due to airway epithelial damage from M. pneumoniae (Zhou et al., 2022; Hu et al., 2023) and the rise of opportunistic pathogens caused by prolonged antibiotic use, leading to microbiota imbalance and increased infection risk (Grice and Segre, 2012; Wang et al., 2024). A study of bronchial mucus plugs revealed that the most prevalent genera in BALF of children with MPP were Mycoplasmoides, Prevotella, and Streptococcus (Xu et al., 2020). Several species of Prevotella are known to cause opportunistic infections (Larsen, 2017). Micro-aspiration of Prevotella during subclinical airway inflammation may occur (Huffnagle et al., 2017), though the specific mechanism linking Prevotella and inflammatory cells remains unclear and requires further study. The dominant genera that increased during the T3 period, Stenotrophomonas and Chryseobacterium, were also considered common opportunistic pathogens resistant to most β-lactam and macrolide antibiotics (Klimkaitė et al., 2023), showed a strong correlation, suggesting potential synergism. Stenotrophomonas was found to utilize various carbon sources, inhibit other microorganisms, and stimulate T lymphocytes to secrete IL-2, IFN-γ, and TNF-α, promoting lymphocyte apoptosis and immune suppression (Wang M. et al., 2021). A 9-year retrospective study found that most Chryseobacterium indologenes infections were hospital-acquired. The bacterium was found to colonize and spread via contaminated, moist medical equipment, and its prevalence has increased with the use of broad-spectrum antibiotics and invasive procedures (Zhang et al., 2021). Veillonella depends on lactate-producing bacteria such as Streptococcus and Prevotella for survival, which represents a typical metabolic symbiotic relationship. Streptococcus pneumoniae is one of the common co-infecting species in MPP patients, capable of exacerbating lung inflammation and increasing secretions and mucus production. Veillonella secretes catalase to break down the H₂O₂ produced by the metabolism of Streptococcus, providing a stable anaerobic environment that facilitates the growth of oxygen-sensitive genera (Zhou et al., 2017) such as Prevotella and Alloprevotella. This also explains the positive correlation between Veillonella and Prevotella and Alloprevotella. Stenotrophomonas and Chryseobacterium are opportunistic pathogens that are resistant to multiple broad-spectrum antibiotics. They coexist under antibiotic use, potentially forming synergistic resistance to antibiotics, which contributes to disease progression and creates competition with other anaerobic genera such as Veillonella and Prevotella The guidelines (National Health Commission of the People’s Republic of China, 2023) for the diagnosis and treatment of MPP suggest that if macrolide treatment fails to resolve fever within 72 h and clinical signs and chest imaging show no improvement or worsening, it is diagnosed as macrolide unresponsive MPP (MUMPP). In such cases, timely replacement with drugs such as doxycycline is recommended. The aim is timely intervention to reduce the risk of severe disease and complications. However, diagnosing MUMPP is not easy. From our study, it can be observed that in children with MPP, as the disease progresses and antibiotics are used, including macrolides and empirical β-lactams, airway microbial diversity begins to increase after 7 days of infection. After 10 days of infection, under conditions of mucus hypersecretion, both the likelihood of infection with drug-resistant opportunistic pathogens and common pathogens begins to rise, affecting mucus production and disease progression.

In certain environments, such as cystic fibrosis lung microbial communities, Prevotella has been reported to produce β-lactamase, protecting P. aeruginosa from ceftazidime. This leads to antibiotic-resistant strains dominating the microbial community (Vandeplassche et al., 2019). Among children with T3-period infections (n = 21), 13 (13/21) received β-lactam antibiotics for more than 7 days before EOB, with seemingly limited efficacy against Streptococcus. Additionally, 5 (5/21) received glucocorticoid treatment before EOB, with 3 using glucocorticoids for ≤3 days, all of whom experienced recurrent fever. This may be linked to dosage and duration, but also suggests heightened inflammatory responses. Stenotrophomonas maltophilia, a pathogen causing hospital-acquired infections, produces cytokines like IL-2, IFN-γ, and TNF-α, triggering systemic inflammation (Wang M. et al., 2021). TNF-α, a key fever mediator, induces fever via the hypothalamic thermoregulatory center, explaining the positive correlation between Stenotrophomonas and febrile days. Increased Stenotrophomonas abundance in sputum has been linked to poor antibiotic outcomes in acute exacerbations of COPD (Liu, 2020). In our study, Stenotrophomonas showed a negative correlation with β-lactam antibiotics, suggesting sensitivity to these drugs. As Stenotrophomonas levels declined, Prevotella and Veillonella abundance increased, with Veillonella-produced SCFAs playing an anti-inflammatory role, resulting in fewer febrile days. In later disease stages, microbial diversity increased, complicating the relationship due to antibiotic use, warranting further data analysis.

The complex use of antibiotics before admission can affect the BALF microbiota structure, and there are currently no clinical indicators and methods to correct for these factors. In the future, conducting controlled longitudinal studies with adequate sample sizes and integrating multiomics approaches could provide robust data for comprehensive statistical analyses of antibiotics and the LRTM (Pérez-Cobas et al., 2023). Due to the limitations of 16S rRNA sequencing, higher-resolution metagenomic sequencing is planned for the next phase to explore the mechanisms by which bacterial species (Bars-Cortina et al., 2024) in the LRT influence mucus secretion and host immunity.

In conclusion, we analyzed the diversity of LRTM in children with MPP from two perspectives: disease progression and mucus hypersecretion. Longer disease courses showed richer diversity, especially in the AMH conditions. We identified Mycoplasmoides, and Veillonella, along with peripheral neutrophils, as risk factors for increased mucus production. In co-infected MPP, Streptococcus and Prevotella were dominant in the LRTM, particularly under AMH conditions, and were significantly positively correlated with β-lactam antibiotic use. Prevotella was also positively correlated with WBC during this period. These suggested that characteristic genera in the lower respiratory tract of MPP children played a crucial role in influencing AMH and disease progression.