Evaluating the Activity of an Anti-biofilm Agent via Imaging

The majority of human infections are considered to be due to microbes growing in complex communities called biofilms. Biofilms are often highly resistant to common antibiotics as compared to their planktonic (suspended) counterparts, yet many strategies to evaluate antimicrobials focus primarily on planktonic cells. To address the need for test methods to assess antimicrobial activity in biofilms, we previously developed an experimental framework to evaluate three cell states (planktonic, biofilm-forming and biofilm). As a demonstration of this framework, a quaternary pyridinium salt (QPS-1: 4-acetyl-1-hexadecylpyridin-1-ium iodide) was shown to be efficacious in killing planktonic and biofilm cells, as well as preventing biofilm formation of oral pathogen, Streptococcus mutans (S. mutans). Here, we present laser scanning confocal microscopy (LSCM) as an orthogonal approach to evaluate effects of QPS-1 on S. mutans biofilms. Qualitatively, QPS-1 disrupted the cell membranes, confirming the bioassay results and demonstrating QPS-1 as a promising anti-biofilm agent. Moreover, our results show LSCM is a useful platform for assessment of antimicrobials through visualization of morphological features and changes in biofilm cell membrane integrity. There remains a need for quantitative imaging of biofilms and their complex chemical, biological, and structural features to further elucidate effects of antimicrobials on biofilms.