High Throughput Screening the Effects of Antibiotic Delivery Rates On Biofilm Antibiotic Resistance

Bacterial biofilms are a heterogeneous community of bacteria distributed in a matrix comprised primarily of hydrated polysaccharides. Compared to bacteria dispersed in liquid cultures, biofilm-associated bacteria can tolerate antimicrobial concentrations orders of magnitude higher. Phenotypic differentiation and lateral gene transfer of antibiotic resistance genes in biofilm promotes antibiotic resistance in biofilm. High throughput technologies have been widely used to screen effective antimicrobial concentrations, where biofilms are usually exposed in constant antibiotic concentration or fixed mass of antibiotic. However, biofilms in nature are usually challenged with changing micro-chemical environment due to diffusion limitations and periodic dosing regimes. The effect of antimicrobial delivery rates on antimicrobial resistance in biofilms is infrequently measured. Here we introduce a novel microfluidic approach for high-throughput screening of respiration inhibition of bacteria in a biofilm array morphology (Figure 1).  The device geometry and operating conditions allow antimicrobial concentrations and fluxes to vary systematically and predictably with space and time. In effect, one experiment can screen biofilm respiratory responses to many different antimicrobial delivery rates. Our results demonstrate the effect of antibiotic concentration on biofilm respiration inhibition.  Biofilm experiencing slowly increasing antibiotic concentrations can withstand higher total concentrations compared with bacteria exposed to rapidly increasing concentrations.  We anticipate our approach can be used as a starting point for investigating the effects of antimicrobial delivery rate on development of antimicrobial resistance in biofilms. Longer term, the work may help direct clinical work on effective antimicrobial therapies for the treatment of biofilm-associated diseases.

Figure 1:

Figure 1. Schematic of the microfluidic diffusion device with biofilm array. (a) The microfluidic device consists of oxygen sensing film on a glass slide, a PDMS layer with a biofilm array patterned on top, and a PDMS microfluidic device bonded on top. (b) Photograph of an array enclosed in a microfluidic diffusion device, with red dye loaded in a perimeter source well.