Reconsidering outcome frameworks and ecological windows in oral probiotic trials

To the Editor,

We read with interest the secondary microbiome analysis by Bujaldón et al. evaluating adjunctive Limosilactobacillus reuteri supplementation during non-surgical periodontal therapy in patients with diabetes [1]. Using high-resolution 16S rRNA sequencing, the authors report no detectable changes in subgingival microbial composition, adding rigorously generated evidence to ongoing discussions on the microbiological effects of oral probiotics. These findings invite reflection on how probiotic efficacy in the oral cavity is defined and evaluated.

First, the analysis is implicitly framed around the premise that biologically effective probiotic interventions should be accompanied by measurable remodelling of subgingival community composition. While common in microbiome research, this structure-centric assumption may be insufficiently aligned with the functional modes through which probiotics plausibly act. Probiotic effects may operate through modulation of host–microbial interactions, inflammatory thresholds, or biofilm behaviour without inducing stable taxonomic displacement [2]. Within this framework, null findings in alpha or beta diversity should not be interpreted as evidence of biological inactivity, but rather as an indication that structure-based readouts may be weakly coupled to mechanisms that determine clinical response. The more consequential question is therefore not whether subgingival taxa changed, but whether treatment altered microbial functions or host-response pathways that are causally linked to therapeutic benefit [3]. Without such function-aligned endpoints, negative compositional results risk being over-extended and may offer limited guidance for clinical inference or evidence synthesis.

Second, restricting microbiome assessment to deep subgingival sites may have further constrained sensitivity to detect probiotic-related modulation, as the compartment most relevant to disease expression is not necessarily the compartment most responsive to probiotic exposure [4]. Deep periodontal pockets represent highly stable and colonisation-resistant habitats maintained by mature biofilms and strong ecological inertia, whereas probiotic contact, persistence, and clearance are largely governed by upstream oral compartments responsible for exposure dynamics. This exposure–disease compartment misalignment complicates interpretation of null subgingival compositional findings, which may reflect niche insensitivity rather than absence of probiotic-related modulation across the oral ecosystem [5]. When inference is restricted to the most colonisation-resistant compartment, negative findings may systematically bias conclusions toward ecological stability, with direct implications for how probiotic efficacy is judged and translated. Designs incorporating multi-compartment oral profiling and explicitly linking upstream microbial shifts to downstream ecological or clinical trajectories may therefore provide a more reliable basis for mechanistic interpretation and translational inference.

Together, these considerations suggest that negative subgingival compositional findings should be interpreted within a broader evaluative framework rather than as evidence against probiotic utility. This rigorously conducted study is best viewed as a prompt to refine endpoint selection and sampling strategies in oral probiotic trials. Compartment-aware, function-aligned designs may enhance mechanistic interpretability, support more targeted clinical application, and help define evidence thresholds for clinical guidance and policy recommendations in diabetes-associated periodontitis.

Funding Statement

National Natural Science Foundation of China (82203056).

Disclosure statement

No potential conflict of interest was reported by the author(s).