Real-time Monitoring of Staphylococcus Aureus Biofilm Formation Under Flow Conditions in Microfluidic Chambers

Abstract

Staphylococcus aureus (S. aureus) causes most of life-threatening infections such as endocarditis, osteomyelitis and sepsis. Since biofilm development is often a key factor in the development of an infection of indwelling medical devices, we sought to study biofilm formation in real-time using a lab-made microfluidic device with the goal of assessing the efficacy of therapeutic agents. In this study, we monitored the bacterial growth of S. aureus in side-by-side microfluidic channels. The total volume of an individual microfluidic channel was 0.148 mm3. Each independent micro-channel has three separated ports (two inlets and an outlet for waste) to allow for constant flow of both growth medium and the bacterial cells. Biofilm coverage of S. aureus was characterized under various flow conditions ranging from 0.1 to 1 μL/min. Optimal biofilm formation was evident at 0.5 μL/min, therefore that flow rate was used in all subsequent experiments. To more closely mimic the human plasma environment, the effect of fibrinogen (Fbg) supplementation was tested. We found that fibrinogen enhanced the process of biofilm formation. To determine if we were monitored true biofilm formation rather than simple bacterial grow, we infused calcofluor white (CFW) into the channels and imaged by a real-time fluorescence microscope. We verified true biofilm formation by visualizing the production of exopolysaccharides matrix by S. aureus indicated by positive CFW staining. By using this microfluidic-based model, we assessed the possibility of inhibiting the biofilm development by testing the anti-staphylococcal activity of a potential probiotic bacilli strain, called B. velezensis AP183, and the bacteriolytic activity of lysostaphin, an extracellular enzyme secreted by staphylococcus simulans strain. These results may help to better understand the biofilm formation process and discover new drugs targets for the treatment of staphylococcal infections.