Adhesion of Probiotic Bacteria to Caco-2/HT-29 Co-Culture and Molecular Modeling of SlpA Interactions with MUC2 Mucin Domains

Introduction. Adhesion to the intestinal mucosa is a key criterion for probiotic selection. A short-term Caco-2/HT-29 co-culture was used, combining enterocyte-like and HT-29 cells. Mucin production was not experimentally validated in this model. The aim was to assess the adhesion and cytotoxicity of the strains in this model, analyze SlpA interactions with MUC2 domains, and evaluate the feasibility of automated nuclei detection in the co-culture. Methods. The morphology of the co-culture and bacteria was examined by confocal microscopy. MTT-reduction activity of the bacteria-exposed co-culture was measured relative to untreated cellular controls, while cell membrane integrity was assessed using the trypan blue exclusion assay. Adhesion after 2 h was evaluated by cell counting and colony-forming unit (CFU) percentage. Caco-2/HT-29 nuclei were detected using Faster R-CNN and YOLO11 models. SlpA complexes with the D1 and D3 domains of MUC2 were analyzed by docking, molecular dynamics, and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Results. Monolayer integrity was preserved. Both nuclei detection models showed comparable performance (mAP@50 = 0.97; mAP 0.50–0.95 = 0.92 and 0.90). The relative MTT-reduction signal was higher after exposure to Bifidobacterium animalis subsp. lactis KB-54 (81.05 ± 33.21%), Bacillus subtilis KATMIRA 1933 (72.91 ± 31.26%), and Bacillus amyloliquefaciens B-1895 (59.47 ± 17.93%) and lower after exposure to B. animalis subsp. lactis KB-16 (13.53 ± 9.44%) and L. acidophilus KB-41 (7.35 ± 5.16%). A similar pattern was observed for cell viability. KATMIRA 1933 showed the highest number of adherent cells (12.72 ± 2.16); among the probiotic strains, higher values were observed for KB-41 and KB-16. The highest adhesion percentages were observed for KATMIRA 1933 (5.32%) and KB-16 (4.93%), whereas KB-54 and KB-41 showed the lowest values. In silico, the SlpA complex with the deglycosylated D1 domain of MUC2 showed the most favorable interaction energy (−10.63 ± 6.03 kJ/mol). Conclusions. The short-term Caco-2/HT-29 co-culture is suitable for the combined assessment of bacterial adhesion, cellular metabolic activity, and membrane integrity under the tested conditions, while nuclei can be quantified automatically. High adhesion was not always associated with preservation of cell status, and SlpA-MUC2 interactions depended on glycosylation.