A bacterial biofilm is a group of cells adhered to a surface and embedded in an extracellular polymeric matrix. Cells within a biofilm are at a high cell density and vary phenotypically with position inside the biofilm. Biofilm bacteria differ metabolically and physiologically from bacteria dispersed in liquid culture. Consequently, inhibitory antimicrobial concentrations tested on planktonic cells are not comparable to concentrations required to inhibit cells in biofilm morphology. Existing antibiotic screening techniques for biofilms fail to indicate real-time respiratory response to antibiotic stress. Here, we report the dynamic respiratory response of Staphylococcus aureus biofilm to constant exposure to daptomycin. In microfluidic devices, we treated biofilm arrays with daptomycin concentrations ranging from 0.031-1 mM. An oxygen-sensing film reported real-time respiratory response of the bacteria, by optically quantifying the oxygen concentration at the base of the biofilm over a 24 hour period. The results suggest that the proportion of actively-respiring bacteria in Staphylococcus aureus biofilm decreases with daptomycin concentration in a non-linear fashion. The dynamic respiratory data indicate that higher antibiotic concentrations inhibited cell respiration at a faster rate. The respiration rate of the bacteria eventually plateaus at a relatively low rate after daptomycin exposure. The final respiration rate appears to be dependent on the concentration of daptomycin used to treat the biofilm. The results may indicate that a larger portion of bacterial cells continue to respire after exposure to lower antibiotic concentrations. Even after exposure to high daptomycin concentrations, the data suggest that a small percentage of bacteria may continue to respire. We believe the bacterial cells that continue to respire after treatment could be biofilm persister cells or cells that built up genetic resistance to the antibiotic. Our results show the portion of respiring S. aureus bacteria that will be present after exposure to a particular daptomycin concentration. This study captures the dynamic nature of biofilms response to antibiotic and provides results that could be used to improve antimicrobial treatments. Understanding optimal antimicrobial doses could help avoid antimicrobial resistance built up by biofilms.
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