In preschool children, otitis media with effusion often coexists with adenoid hypertrophy and eustachian tube dysfunction; recurrence or persistence can lead to conductive hearing loss and restricted speech development (Gunduz et al., 2025). In the non-surgical management phase, the core goals are to restore middle-ear ventilation and clear the effusion, and objective evaluation mainly relies on tympanogram classification and continuous parameters such as TPP, Ytm, and TW (Connolly et al., 2023; Shahnaz et al., 2023). Imbalance of nasopharyngeal microecology and colonization by pathogens are considered important upstream drivers (van den Broek et al., 2019); Streptococcus salivarius K12 is a resident upper airway bacterium with colonization and bacteriocin production, and biologically has the potential to improve middle-ear physiology through local microecological modulation (Al-Akel et al., 2024). Using lozenges to achieve sustained oropharyngeal exposure balances efficacy and accessibility, aligning with practical management needs during the observation period (Stasǩová et al., 2021). There remain key evidence gaps for this population. Conventional non-surgical interventions (e.g., nasal irrigation, autoinflation, intranasal steroids) show heterogeneous efficacy and have difficulty in reliably reversing middle-ear negative pressure in the short term (Nibhanupudy et al., 2024; Webster et al., 2023); previous probiotic studies mostly used upper respiratory infection or acute otitis media episodes as endpoints (Sarlin et al., 2023; Zhao et al., 2022), and randomized controlled trials using tympanogram classification as the primary endpoint while simultaneously quantifying microecological changes are lacking; previous studies rarely adopted ear-level correlation modeling, making it difficult to handle the structural correlation of bilateral ear outcomes (Sheng et al., 2022; Chen et al., 2022); colonization and pathogen load, tympanometric physiological indices, and audiological outcomes were often assessed separately, making it difficult to form integrated evidence for a causal chain. Clinically, a trial is still lacking that, within the context of standardized non-surgical management, centers on objective middle-ear physiological indicators and simultaneously addresses mechanism and safety to support the application of lozenge-based microecological intervention (Bourdillon and Edwards, 2021; Chen et al., 2020). This study conducted a single-center, randomized, double-blind, placebo-controlled trial to evaluate, in children aged 3–6 years with adenoid hypertrophy and otitis media with effusion undergoing non-surgical management, the effect of daily ≥1×10⁹ CFU Streptococcus salivarius K12 lozenges over 12 weeks on improvement in tympanogram classification, and to build an evidence chain linking ear physiology, microecology, and audiology using linear mixed-effects models and GEE, with systematic assessment of adherence, product viability, and safety. The trial showed that SSK12 achieved a higher probability of classification improvement than placebo, with time-dependent signals consistent with changes in TPP, Ytm, TW, audiological outcomes, and decreases in pathogen load; safety and acceptability were good, suggesting that introducing an oropharyngeal lozenge microecological intervention during the observation period has clinical translational value.

Our study focused on an objective and clinically meaningful primary endpoint, namely tympanogram improvement (Jerger type B/C→A) at week 12, in a well-defined pediatric OME population with adenoid hypertrophy (grade II–IV). Tympanogram normalization is a direct indicator of improved middle-ear ventilation and effusion resolution, making it an appropriate endpoint for assessing non-surgical interventions in this setting.

This study showed that, at the child-level primary endpoint, SSK12 achieved a higher tympanogram classification improvement rate than placebo, with consistent directions across prespecified subgroups; at the ear level, conclusions remained robust after correcting for within-child bilateral correlation using GEE; longitudinal changes in continuous parameters were also consistent, manifesting as increases over time in ΔTPP and ΔYtm and a decrease in ΔTW, with between-group differences gradually widening, forming an effect pattern that corroborated the primary endpoint. Conversion from B/C to A represents restoration of eustachian tube pressure regulation, improvement in tympanic compliance, and normalization of the pressure curve, consistent with the pathophysiological chain of “ventilation improvement—effusion absorption” (Vanneste and Page, 2019; Andresen et al., 2021); after colonization in the oropharynx/nasopharynx, SSK12 suppresses key pathogens through bacteriocin-mediated occupancy and antagonism, reduces local inflammation and mucus viscosity, and improves ciliary clearance, thereby promoting the return of negative middle-ear pressure to normal and clearance of effusion, presenting a time-dependent pathway of microecological remodeling (Tagg et al., 2023; Do et al., 2024); ear-level analyses indicated moderate correlation between the two ears within the same child due to shared nasopharyngeal environment and behavioral exposures, which was adequately controlled by the modeling. Previous randomized controlled studies mostly used acute episodes as endpoints, and evidence on objective middle-ear physiological indicators is relatively scarce (Persaud et al., 2024; Scott et al., 2019). In the context of standardized non-surgical management, this study, with a double-blind design and centrally read tympanogram indices, directly captured improvement in middle-ear ventilation and effusion status, and used ear-level models to verify the lateral consistency and robustness of efficacy, supplementing the evidence base for microecological interventions during the non-surgical management phase of OME, and aligning with the direction of audiological outcomes over the same follow-up period, with clear clinical translational significance. Maintenance of adherence and product viability, successful blinding, and consistency in measurement and reading provided sufficient internal validity support for the above efficacy determinations.

The treatment effect of SSK12 was generally consistent across prespecified age strata and adenoid hypertrophy grades, suggesting broad applicability within the studied population. However, clinical context remains important. Younger children may have greater baseline susceptibility to Eustachian tube dysfunction due to anatomical and immunological immaturity, whereas older children may experience more spontaneous resolution of OME. Similarly, children with moderate adenoid hypertrophy (grade II–III) may derive the greatest benefit from adjunctive non-surgical therapy, as microbial and inflammatory modulation may meaningfully improve Eustachian tube function. In contrast, children with severe adenoid hypertrophy (grade IV), where mechanical obstruction predominates, may be less responsive to conservative measures alone and more likely to require surgical intervention. These considerations support individualized use of probiotic therapy within established clinical decision pathways.

The beneficial effect of nasopharyngeal Streptococcus salivarius K12 (SSK12) on middle-ear pressure and tympanogram normalization is likely multifactorial, involving both pathogen suppression and host immunomodulation, rather than a purely antimicrobial mechanism. First, SSK12 colonization reduces the nasopharyngeal burden of key otopathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis, through competitive exclusion and production of bacteriocins (salivaricins). Suppression of these pathogens diminishes chronic nasopharyngeal inflammation, which is a major contributor to Eustachian tube dysfunction. Reduced mucosal edema and biofilm burden facilitate restoration of Eustachian tube patency, thereby improving middle-ear ventilation and normalizing tympanometric peak pressure. Second, SSK12 may exert local immunomodulatory effects at the nasopharyngeal mucosal surface. Probiotic colonization has been shown to modulate innate immune responses by downregulating pro-inflammatory cytokines and promoting mucosal immune homeostasis. This immunoregulatory action may reduce persistent low-grade inflammation in the Eustachian tube and middle-ear mucosa, further supporting pressure equalization and effusion resolution. Third, the observed improvement in tympanometric parameters without a proportional increase in systemic antibiotic use suggests a functional recovery mechanism, whereby microbial ecosystem stabilization restores physiological Eustachian tube function rather than merely eradicating infection. The parallel improvements in tympanogram type, DPOAE conversion, and audiometric thresholds support a coherent pathway linking nasopharyngeal microbial balance to middle-ear mechanics. Taken together, these findings support a dual mechanism in which SSK12 acts through (i) suppression of pathogenic nasopharyngeal bacteria and (ii) modulation of mucosal immune responses, leading to improved Eustachian tube function and normalization of middle-ear pressure. Future studies incorporating cytokine profiling and comprehensive microbiome analyses may further elucidate the relative contribution of these pathways.

Microecological and pathogenetic evidence together with clinical endpoints point to a coherent causal chain. SSK12 achieved a high proportion and sustained colonization in the oropharynx/nasopharynx, with load maintained during the intervention period and diverging from the placebo group over time; in parallel, loads of Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis showed continuous downregulation, with significant treatment × time interactions, indicating that a colonization–antagonism effect had occurred at the target anatomical site. The reduction in pathogen load was directionally consistent with decreases in episodes of upper respiratory infection and acute otitis media, while systemic antibiotic prescriptions did not increase, weakening the explanation of apparent improvement driven by rescue medication (Mosquera et al., 2025). Biologically, SSK12, through bacteriocin-mediated inhibition and occupancy competition, attenuates pathogenic adhesion and biofilm formation, together with reductions in local inflammation and improvement in mucus rheology (Stasǩová et al., 2021), thereby enhancing nasopharynx–eustachian tube ventilation, reducing negative middle-ear pressure, and promoting effusion clearance (Hamrang-Yousefi et al., 2020), resulting in time-dependent improvements in tympanogram classification and ΔTPP, ΔYtm, and ΔTW. The reliability of intervention exposure was supported by high adherence and stability of viable counts by batch (Visciglia et al., 2022), and consistency of central reading at the measurement level together with successful blinding reduced the likelihood of information bias and observer bias. Previous studies mostly focused on upper respiratory episodes or subjective symptoms (Merenstein et al., 2024) and lacked objective middle-ear physiological indices synchronized with pathogenetics; in the context of standardized non-surgical management, this trial integrated colonization, load, pathogen spectrum, otologic objective indices, and event outcomes, coherently presenting the pathway “colonization → pathogen decrease → ventilation restoration → classification improvement → hearing benefit,” which more closely approaches causal inference at the mechanistic level than previous evidence and provides methodological and clinical bases for introducing an oropharyngeal lozenge microecological intervention in the “observation period” scenario.

The concurrent assessment of tympanometric, audiological, and microbiological outcomes provides an integrated framework for interpreting treatment effects. Improvements in tympanogram type and middle-ear pressure were accompanied by favorable changes in hearing thresholds and otoacoustic emissions, together with modulation of nasopharyngeal microbial profiles. This convergence supports a biologically plausible pathway in which nasopharyngeal probiotic colonization contributes to improved Eustachian tube function and middle-ear ventilation, rather than reflecting an isolated physiological change. The audiological and otologic functional outcomes were aligned in a beneficial direction with the primary endpoint, with a time-dependent between-group separation in the decrease of the four-frequency pure-tone average and a higher overall probability of distortion product otoacoustic emission (DPOAE) conversion from negative to positive, indicating that restoration of the conductive pathway had translated into perceptible functional benefit; adherence remained high, daily dose exposure was stable, viable counts of the preparation and cold-chain monitoring met standards, and blinding success together with good measurement/reading consistency provided robust internal validity support, while the safety profile was comparable to placebo, rendering the risk–benefit ratio clinically acceptable. Pathophysiologically, reduction of middle-ear effusion and improvement of eustachian tube ventilation can reduce mechanical damping of the tympanic membrane–ossicular chain (Merchant and Neely, 2023; Won et al., 2020), making otoacoustic emissions generated by outer hair cells more readily transmitted through the middle ear to the external auditory canal, so that DPOAE conversion to positive and a decrease in hearing thresholds become functional extensions following improvement in tympanogram parameters (Aithal et al., 2019); after an oropharyngeal lozenge establishes stable colonization in the oropharynx, it maintains the local microecology, together with downregulation of pathogen load and alleviation of inflammation, providing sustained biological impetus for the gradual recovery of the middle-ear conductive chain. Compared with previous intervention studies that used only respiratory episodes or symptoms as endpoints (Paduchova et al., 2024; Darbandi et al., 2021), the present evidence, in the context of standardized non-surgical management, simultaneously presents objective middle-ear physiological indices and audiological outcomes, demonstrating that microecological intervention not only alters the nasopharyngeal ecology but also produces quantifiable auditory improvement within 12 weeks; lozenge administration is convenient for implementation in outpatient and home settings, and adherence data together with quality-control results suggest real-world feasibility of this regimen, strengthening its clinical translational value as a supplementary strategy during the “observation period.”

Economic considerations: The probiotic regimen evaluated in this study represents a low-cost, non-invasive intervention that can be administered in the outpatient setting with minimal resource utilization. In contrast, persistent OME and recurrent AOM are associated with substantial healthcare costs related to repeated clinic visits, antibiotic prescriptions, audiological assessments, and surgical interventions such as tympanostomy tube placement and adenoidectomy.

By improving middle-ear function and hearing during the watchful-waiting period, adjunctive SSK12 therapy may reduce the incidence of AOM episodes, limit antibiotic exposure, and decrease progression to surgery, thereby offering potential cost savings at both the patient and health-system levels. Although a formal cost-effectiveness analysis was not performed, the favorable safety profile, ease of administration, and relatively low acquisition cost of probiotic lozenges suggest that this approach could be economically attractive if clinical benefits are confirmed in larger, multicenter studies.

Limitations: This study was conducted at a single tertiary pediatric otolaryngology center, which may limit the generalizability of the findings to other healthcare settings and populations. Differences in patient demographics, microbiological ecology, and clinical practice patterns may influence treatment response. The follow-up duration was insufficient to assess recurrence, long-term hearing and language development outcomes, and it was also difficult to observe differences in surgical outcomes. Event outcomes were infrequent; although the negative binomial model reported aIRR and θ, the statistical power was limited. Information on adherence and events was mainly derived from parental diaries and weekly telephone follow-up, with potential recall and reporting bias. Microecological testing used targeted qPCR and did not characterize community structure, functional genes, or inflammatory profiles, so the causal chain remains incomplete. Eustachian tube dynamics were not directly tested, making it difficult to quantify the physiological magnitude of ventilation improvement. Future multicenter studies across diverse geographic regions and care environments are warranted to confirm the reproducibility and external validity of these findings. Future studies should be multicenter with adequate seasonal coverage and follow-up ≥6–12 months, incorporate objective ventilation indices such as wideband tympanometry and tubomanometry/sonotubometry, and combine 16S/metagenomics and nasopharyngeal inflammatory omics to link the longitudinal mediating pathway among SSK12 colonization, pathogen load, inflammation, and otologic endpoints; use electronic adherence recording and linkage to medical records to reduce bias; design dose–frequency exploration and cost–effectiveness evaluation, and adopt “time to tympanostomy tube insertion” and “long-term hearing thresholds and sustained DPOAE outcomes” as key endpoints to verify and optimize the application strategy of SSK12 during the non-surgical management period. Although the observed associations between nasopharyngeal SSK12 colonization, reductions in otopathogen burden, and improvements in tympanometric and audiological outcomes support a plausible biological mechanism, these microbiota findings are associative rather than causal. As microbial measures were not experimentally manipulated independently of the intervention, causality cannot be fully established from this trial alone. Future studies incorporating targeted microbiome manipulation, longitudinal immune profiling, and mediation analyses will be required to more definitively delineate causal pathways.

Daily oral SSK12 lozenges for 12 weeks effectively improved tympanogram classification in children aged 3–6 years with adenoid hypertrophy and otitis media with effusion undergoing non-surgical management, manifested as increased probabilities of conversion from types B/C to type A at both the child level and the ear level. Synchronous improvements in continuous tympanogram parameters and audiological function verified the physiological mechanism of restoration of middle-ear ventilation and effusion clearance. Nasopharyngeal microecology analyses showed that SSK12 achieved stable colonization and significantly suppressed common nasopharyngeal pathogens, supporting the causal chain of “SSK12 colonization—decrease in pathogen load—improvement in eustachian tube function—favorable tympanogram classification outcome.” Product viability, successful blinding, measurement consistency, and safety all met quality-control requirements, suggesting that the lozenge can serve as a low-risk, clinically feasible adjunctive intervention during the observation period of non-surgical management.