Dynamic Dosing Assay Relating Real-Time Respiration Responses of Staphylococcus aureus Biofilms to Changing Microchemical Conditions Jinzi Deng, † Adit Dhummakupt, ‡ Philip C. Samson, § John P. Wikswo, §,∥ and Leslie M. Shor* ,†,⊥ † Department of Chemical & Biomolecular Engineering, University of Connecticut, Storrs, Connecticut 06269, United States ‡ Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, United States § Vanderbilt Institute for Integrative Biosytems Research and Education (VIIBRE), Vanderbilt University, Nashville, Tennessee 37235, United States ∥ Departments of Biomedical Engineering, Physics & Astronomy, and Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee 37235, United States ⊥ Center for Environmental Sciences and Engineering, University of Connecticut, Storrs, Connecticut 06269, United States * S Supporting Information ABSTRACT: Bacterial biofilms are a metabolically heteroge- neous community of bacteria distributed in an extracellular matrix comprised primarily of hydrated polysaccharides. Effective inhibitory concentrations measured under planktonic conditions are not applicable to biofilms, and inhibition concentrations measured for biofilms vary widely. Here, we introduce a novel microfluidic approach for screening respiration inhibition of bacteria in a biofilm array morphology. The device geometry and operating conditions allow antimicrobial concentration and flux to vary systematically and predictably with space and time. One experiment can screen biofilm respiratory responses to many different antimicrobial concentrations and dosing rates in parallel. To validate the assay, onset of respiration inhibition following NaN 3 exposure is determined optically using an O 2 -sensing thin film. Onset of respiration inhibition obeys a clear and reproducible pattern based on time for diffusive transport of the respiration inhibitor to each biofilm in the array. This approach can be used for high-throughput screening of antimicrobial effectiveness as a function of microbial characteristics, antimicrobial properties, or antimicrobial dosing rates. The approach may also be useful in better understanding acquired antimicrobial resistance or for screening antimicrobial combinations. B acterial biofilms are comprised of pure or mixed cultures distributed in a self-secreted hydrogel matrix. Respiration of living cells within the biofilm and the absence of bulk mixing cause microscale gradients to persist in biofilms, as typified by decreasing O 2 concentrations with depth for aerobic biofilms. 1 Persistent gradients promote phenotypic differentiation, while proximity of cells facilitates lateral transfer of antimicrobial resistance genes. 2 In addition to their importance in clinical settings, biofilms in industry reduce the efficiency of water desalination 3 and heat exchangers. 4 In environmental systems, biofilms can protect bacteria from predation, 5 they protect plant roots from pathogens, 6 and they help retain moisture in soils. 7 Biofilm-associated bacteria exist in a distinct physiological state from planktonic cells, so inhibitory concentrations measured for planktonic cultures do not apply to biofilms. Generally speaking, biofilm-associated bacteria can tolerate much higher antimicro- bial concentrations than can planktonic cultures. 8,9 For example, Anderl et al. show that bacteria growing as a “biofilm” on an agar plate exhibit a markedly different antimicrobial susceptibility than bacteria grown in liquid suspension. In their work, antimicrobial exposures that reduced the number of live planktonic cells by 4 orders of magnitude caused virtually no change in the biofilm-associated cultures. 10 Further complicating matters, antimicrobial susceptibility of biofilms varies widely, 11,12 due to differences between parent and mutant strains 13 or among phenotypes 14 or due to adaptation to experimental conditions. 15 For example, Nelson et al. reported that minimum biofilm eradication concentrations (MBEC 99.9 ) for Pseudomonas aeruginosa PA14 biofilms differ by a factor of 24. 14 The methods used to study biofilm inhibition by antimicro- bials include flow cells, diffusion cells, multiwell plates, chemostats, and microfluidic devices. These techniques can usually be classified as high-throughput or high-content. High- throughput screening of biofilm antimicrobial susceptibility is Received: December 29, 2012 Accepted: April 30, 2013 Published: April 30, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 5411 dx.doi.org/10.1021/ac303711m | Anal. Chem. 2013, 85, 5411−5419