Decoding Biofilm Complexity: Artificial Intelligence, Advanced Imaging, and Multi-Omics for Precision Antibiofilm Strategies

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Microbial biofilms are highly structured multicellular communities that exhibit remarkable tolerance to antimicrobial agents and host defenses, contributing to persistent infections, industrial biofouling, food contamination, and environmental challenges. Conventional microbiological approaches have greatly advanced our understanding of biofilm formation but remain insufficient to resolve the spatial, molecular, and functional heterogeneity that underpins biofilm resilience. This review discusses how recent advances in high-resolution imaging, multi-omics technologies, single-cell and spatially resolved analyses, and artificial intelligence (AI) are transforming biofilm research from descriptive observation to precision microbiology. We critically examine state-of-the-art analytical platforms, including confocal and super-resolution microscopy, electron microscopy, Raman-based imaging, genomics, transcriptomics, proteomics, metabolomics, and emerging spatial multi-omics approaches, highlighting their complementary roles in deciphering biofilm architecture, regulatory networks, and metabolic specialization. AI/ML approaches are increasingly being applied to image analysis, multimodal data integration, predictive modeling, biomarker identification, and systems-level interpretation of biofilm biology. Furthermore, we discuss how these technological advances are enabling mechanism-based precision antibiofilm strategies, including extracellular polymeric substance-targeting enzymes, quorum-sensing inhibitors, nanotechnology-enabled therapeutics, bacteriophage-based interventions, and synthetic biology approaches. We highlight the major translational challenges, including data standardization, explainable AI, multimodal integration, benchmark datasets, and clinical validation, and outline future directions for developing clinically relevant, data-driven antibiofilm therapies. This review provides an integrated framework connecting advanced analytical technologies with next-generation precision antibiofilm interventions and offers perspectives for accelerating their translation into clinical and industrial applications.