Abstract
Helicobacter pylori biofilms contribute to bacterial persistence and may undergo substantial remodeling under antibiotic stress produced by subinhibitory concentrations (sub-MICs). This study investigated how exposure to ¼× MIC of clarithromycin (CLR), metronidazole (MTZ), and levofloxacin (LEV) affects H. pylori biofilms using FluoSphere probes differing in size (nominally 0.2 and 1 µm) and surface functionalization (carboxylate-, amine-, and sulfate-modified). Biofilm–particle co-localizations were assessed under static conditions and subsequently analyzed in real time under microfluidic conditions using the BioFlux platform. Co-localization patterns varied with particle surface functionalization, although differences between nominal and measured sizes complicated a strict determination of size effects. Under static conditions, ¼× MIC of CLR most consistently reduced the co-localization of carboxylate-modified FluoSpheres with H. pylori biofilms, whereas co-localization with amine-modified particles remained comparatively stable across the strong biofilm-producing strains but varied in the weak biofilm-producing H. pylori Tx30a strain. Under microfluidic conditions, the most consistent reductions in co-localization were observed for carboxylate-modified FluoSpheres, particularly following exposure to ¼× MIC of CLR, whereas LEV at ¼× MIC significantly increased co-localization with amine-modified particles at specific time points. In both models, sulfate-modified FluoSpheres showed generally lower interaction, delayed antibiotic-dependent effects, and pronounced surface autoaggregation. Z-stack analysis revealed carboxylate-modified particle signals mainly in the middle biofilm layers, amine-modified signals in deeper layers, and sulfate-modified signals predominantly at the surface. Overall, our findings demonstrate that antibiotics at ¼× MIC modify H. pylori biofilm–particle co-localizations and highlight the potential of surface-functionalized fluorescent probes for investigating antibiotic-induced changes in biofilms.