Lab on a Chip PAPER Cite this: Lab Chip, 2015, 15, 2799 Received 28th March 2015, Accepted 13th May 2015 DOI: 10.1039/c5lc00375j www.rsc.org/loc Rapid electrochemical phenotypic profiling of antibiotic-resistant bacteria† Justin D. Besant, a Edward H. Sargent* b and Shana O. Kelley* acd Rapid phenotyping of bacteria to identify drug-resistant strains is an important capability for the treatment and management of infectious disease. At present, the rapid determination of antibiotic susceptibility is hin- dered by the requirement that, in existing devices, bacteria must be pre-cultured for 2–3 days to reach detectable levels. Here we report a novel electrochemical approach that achieves rapid readout of the anti- biotic susceptibility profile of a bacterial infection within one hour. The electrochemical reduction of a redox-active molecule is monitored that reports on levels of metabolically-active bacteria. Bacteria are captured in miniaturized wells, incubated with antimicrobials and monitored for resistance. This electro- chemical phenotyping approach is effective with clinically-relevant levels of bacteria, and provides results comparable to culture-based analysis. Results, however, are delivered on a much faster timescale, with resistance profiles available after a one hour incubation period. Introduction The overuse of antibiotics and the prescription of antibiotics to which a pathogen is not susceptible contribute to rising antibiotic resistance rates – a growing threat to public health worldwide. 1 Urinary tract infections are among the most prevalent bacterial infections. 2 Gold-standard antibiotic susceptibility tests for urinary tract infections rely on culture and require 1–3 days in order to allow the bacteria to multiply to detectable levels. 3 After pre-culture of the bacteria, an additional 18 hours are typically required to perform stan- dard susceptibility tests. Reducing the time needed to deter- mine the susceptibility profile of urinary tract infections could improve clinical outcomes, especially in the case of the most severe infections that lead to urosepsis. 4 Rapid testing could also contribute to decreased unnecessary antibiotic use, 5 and could increase the efficiency of centralized diagnos- tic laboratories. Tests for antibiotic resistance that rely on enzymatic amplification of antibiotic-resistance genes reduce turn- around times compared to culture. 6–9 Unfortunately, these assays often require a pre-incubation step to allow the bacte- ria to multiply, and, further, often require several hours to amplify the genes of interest. Gene-based assays are also lim- ited by the requirement of knowing a priori which genes con- fer resistance. Dozens of constantly-evolving genes may be implicated in resistance to a given antibiotic, and it is impractical to test for all possible mutations simultaneously. 10 Assays that monitor bacterial viability in response to anti- biotics overcome the limitations of genetic tests. These tests report directly on the question of greatest clinical impor- tance: whether a given antibiotic decreases bacterial survival. New assays for antibiotic resistance include the detection of bacterial motion using AFM cantilevers, 11 electrochemical measurements of bacterial growth, 12–16 optical detection of bacterial growth, 17,18 and optical detection of redox reporters of bacterial metabolism. 19–22 In assays that detect metabolically- active pathogens, the bacteria are incubated with the antibi- otic and a redox reporter of metabolism such as resazurin or methylene blue. Metabolically-active bacteria create a reduc- ing environment and either directly or indirectly reduce the compound, and the change in redox state is read out as a change in color or fluorescence. Resistant bacteria continue to multiply and metabolize the compound, while susceptible bacteria do not. Successful detection using this type of approach hinges on the requirement that a sufficient quantity of the reduced form of the reporter compound accumulates above the detec- tion threshold, a delay that takes at least 12 hours in milliliter-scale culture. 19 Strategies have been proposed that seek to confine bacteria in microliter and nanoliter volumes with the goal of reducing the time of detection by increasing Lab Chip, 2015, 15, 2799–2807 | 2799 This journal is © The Royal Society of Chemistry 2015 a Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada. E-mail: shana.kelley@utoronto.ca b Department of Electrical and Computer Engineering, Faculty of Engineering, University of Toronto, Toronto, ON, M5S 3G4, Canada. E-mail: ted.sargent@utoronto.ca c Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2, Canada d Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, ON, M5S 1A8, Canada † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5lc00375j Published on 13 May 2015. Downloaded by University of Toronto on 29/07/2015 13:07:57. View Article Online View Journal | View Issue