Gradient Microfluidics Enables Rapid Bacterial Growth Inhibition Testing

Abstract

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask).

Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays

“…However, these schemes often involve complicated readouts and have not been demonstrated in a format that promotes automated data collection or multiplexing for different antibiotics or antibiotic concentrations. Methods that implement gradients of antibiotics (24,25) vastly improve on multiplexing by simultaneously testing a range of antibiotic concentrations but require constant flow of antibiotics to hold the gradient profile, thereby limiting the number of simultaneous tests that can be performed.…”

Microfluidic advances in phenotypic antibiotic susceptibility testing

“…Examples of microscopic monitoring of bacterial growth (a) or death (b); All scale bars: 10 μm; (a) Bright field images of a monolayer of E. coli during the first 5 h of growth on-chip; Licensed Material 1 modified from (Li et al 2014); (b) Phase contrast (i–ii) and fluorescence (iii–iv) images for a representative methicillin-sensitive S. aureus strain within an antibiotic-containing microchannel at the start (t=1 min; i and iii) and the end of the assay (t=60 min; ii and iv); Modified and reproduced with permission (Kalashnikov et al 2014)…”

Biosensors in Antimicrobial Drug Discovery: Since Biology until Screening Platforms

“…Other methods use a Microfluidic Agarose Channel (MAC) system, determining MICs by evaluation of cell growth under microscopic observation in 3-4 hours [109]. Equally the micro channels in a microfluidic platform can be revealed using measurements of fluorescence intensity [110], or can be used for study of antimicrobial resistance induced by mechanical stress [111], which are advantages of the versatility of this platform including the decrease in evaluation time, the increased sensitivity of detection, the decrease in the number of reagents to be used [110], and the possibility to be included in a chip-based system [112].…”