Rapid antibiotic sensitivity testing in microwell arrays

Abstract

The widespread bacterial resistance to a broad range of antibiotics necessitates rapid antibiotic susceptibility testing before effective treatment could start in the clinic. Among resistant bacteria, Staphylococcus aureus is one of the most important, and Methicillin-resistant (MRSA) strains are a common cause of life threatening infections. However, standard susceptibility testing for S. aureus is time consuming and thus the start of effective antibiotic treatment is often delayed. To circumvent the limitations of current susceptibility testing systems, we designed an assay that enables measurements of bacterial growth with higher spatial and temporal resolution than standard techniques. The assay consists of arrays of microwells that confine small number of bacteria in small spaces, where their growth is monitored with high precision. These devices enabled us to investigate the effect of different antibiotics on S. aureus growth. We measured the Minimal Inhibitory Concentration (MIC) in less than 3 hours. In addition to being significantly faster than the 48 hours needed for traditional microbiological methods, the assay is also capable of differentiating the specific effects of different antibiotic classes on S. aureus growth. Overall, this assay has the potential to become a rapid, sensitive, and robust tool for use in hospitals and laboratories to assess antibiotic sensitivity.

Recent Advances in the Race to Design a Rapid Diagnostic Test for Antimicrobial Resistance

“…(C) Fluorescence images were taken every 30 min for 6 h of PDMS microwells containing fluorescent Staphylococcus aureus bacteria colonies. Reprinted with permission from ref . Copyright World Scientific Publishing 2017.…”

“…Cells, taken directly from urine, were stained with the fluorescent dye resazurin in order to monitor bacterial metabolic activity over time via time-lapse microscopy with “susceptible” and “resistant” determinations performed within 4.5–5.5 h of imaging . Likewise, Jalali et al created a device containing 24 viewing fields, each containing cylindrical microwells as shown in Figure C. Bacterial cells are seeded within the microwells, and bacterial populations are imaged over time .…”

“…Likewise, Jalali et al created a device containing 24 viewing fields, each containing cylindrical microwells as shown in Figure C. Bacterial cells are seeded within the microwells, and bacterial populations are imaged over time . In both cases, microscope images were captured over time and processed by in-house algorithms to give MIC readouts within a couple hours.…”

“…Bacterial cells are seeded within the microwells, and bacterial populations are imaged over time. 89 In both cases, microscope images were captured over time and processed by in-house algorithms to give MIC readouts within a couple hours. The aforementioned methods analyze cells that are part of bacterial populations or groups, but time-lapse microscopy has also been employed to monitor single cells of bacteria over time, 90 which can potentially reduce the starting volume and concentration needed for an AST assay.…”

Combinatorial Antimicrobial Susceptibility Testing Enabled by Non-Contact Printing

“…One of the commonly used tools for high-content biological tests are plates that feature arrays of small wells, micrometer to millimeter in size [35]. Microwell arrays are a popular choice because they are usually compatible with widespread optical readout methods, such as plate readers or fluorescent microscopes [36]. In each of the independent wells, a different experiment can be carried out, depending on the content of the well.…”