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Bacteria can form single- and multispecies biofilms exhibiting diverse features based upon the microbial composition of their community and microenvironment. The study of bacterial biofilm development has received great interest in the past 20 years and is motivated by the elegant complexity characteristic of these multicellular communities and their role in infectious diseases. Biofilms can thrive on virtually any surface and can be beneficial or detrimental based upon the community's interplay and the surface. Advances in the understanding of structural and functional variations and the roles that biofilms play in disease and host-pathogen interactions have been addressed through comprehensive literature searches. In this review article, a synopsis of the methodological landscape of biofilm analysis is provided, including an evaluation of the current trends in methodological research. We deem this worthwhile because a keyword-oriented bibliographical search reveals that less than 5% of the biofilm literature is devoted to methodology. In this report, we (i) summarize current methodologies for biofilm characterization, monitoring, and quantification; (ii) discuss advances in the discovery of effective imaging and sensing tools and modalities; (iii) provide an overview of tailored animal models that assess features of biofilm infections; and (iv) make recommendations defining the most appropriate methodological tools for clinical settings.

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Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI−MB hybrids against the human pathogen methicillin-resistant Staphylococcus aureus (MRSA). Photochemical/photophysical and in vitro microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)en−MB 12 as a lead. Compound 12 showed increased uptake into S. aureus cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI−PS hybrid strategy in aPDI.