Microfluidic Assay Relating Antimicrobial Resistance of Staphylococcus Aureus Biofilms to Changing Micro-Chemical Conditions

Abstract:

Bacterial pathogens cause more human deaths worldwide than all forms of cancer combined. The continuous adaptation of pathogens and lateral transmission of antimicrobial resistance mean new antimicrobial therapies must be continually developed. Frequently, the infections that are hardest to treat are caused by bacteria living as a biofilm. The extracellular matrix produced in the biofilm modulates the micro-chemical environment by slowing diffusion, dampening fluctuations, and sustaining micron-scale gradients. The biofilm matrix also facilitates differentiation and coordination among individual microbes within the colony. Bacteria in biofilms can tolerate antimicrobial concentrations orders of magnitude higher than free-living cells; therefore, the clinical relevance of liquid-based antimicrobial assays may be limited. Furthermore, in real infections, the time course of delivery to the site is also important, yet screening is nearly always performed at constant concentrations. Here we introduce a novel microfluidic approach for screening antimicrobial resistance of bacterial biofilms to changing micro-chemical conditions. We evaluate the effect of formalin, a model small molecule antimicrobial, on Staphylococcus aureus biofilms patterned as a 7x7 array within a microfluidic-based device with transient diffusion. The device geometry and operating conditions allow well-defined micro-chemical conditions to vary systematically with space and time within the device. Changes in microbial respiration within and between individual biofilms were measured using an oxygen-sensitive fluorescent reporting molecule embedded at the biofilm substrate. At each position and time, concentration, flux, cumulative dose, and exposure time above threshold were determined through modeling. We find antimicrobial resistance can be effectively screened by comparing onset of respiration inhibition with the associated instantaneous and time course of micro-chemical conditions as determined by modeling. As expected, biofilms experiencing slowly increasing antimicrobial flux were able to resist higher concentrations of formalin than biofilms experiencing a sudden increase in local concentration. Our approach enables antimicrobial resistance in the clinically relevant biofilm morphology to be related directly to mass transport and biokinetic fundamentals. This work illustrates how mechanisms of action might be understood for a wide variety of pathogens and candidate antimicrobials. A similar approach may be used for individualized treatment of patients with cystic fibrosis, medical implants, or a variety of life-threatening chronic infections.