Bacterial Isolation Microwell-Plug (μWELLplug) for Rapid Antibiotic Susceptibility Testing Using Morphology Analysis

Abstract

The rapid and accurate diagnosis of infectious diseases with high morbidity rates is crucial because it can minimize the misuse and overuse of antibiotics and increase survival rates in dreadful conditions. The conventional antibiotic susceptibility test (AST) systems used to choose appropriate antibiotics require long wait times to obtain results and cannot prevent the misuse or overuse of antibiotics by clinicians who need to quickly treat patients and cannot wait to identify the underlying cause of their symptoms. Therefore, several rapid AST (rAST) methods have been developed to provide quick test results, but they are complicated to operate, require additional equipment or materials, and give less accurate results than the conventional AST methods. In this study, we propose an rAST method that can obtain precise outcomes from a simple process with a short running time using a bacterial isolation microwell-plug (μWELLplug) in a conventional 96-well plate. The specifically designed hydrogel component of the μWELLplug provides a simple process for cell isolation and the observation of bacterial growth and morphological changes induced by a variety of antibiotic concentrations. The μWELLplug is placed over each well of the 96-well plate, and then bacterial or eukaryotic cells are isolated in the microwells and treated with different antibiotic concentrations to observe their effects. Saccharomyces cerevisiae (yeast, eukaryote), Streptomyces atratus (actinomycetes, prokaryote), Escherichia coli, Staphylococcus aureus, and methicillin-resistant S. aureus were cultivated and tested using the μWELLplug. The minimum inhibitory concentration values from this system were obtained in 3−4 h and correlated well with those from the conventional AST methods whose running time is 18−24 h.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains

“…After incubation, the array could be stained and imaged to determine antimicrobial susceptibility. Similarly, hydrogel immobilization AST platforms (Figure B) have been developed to allow for automatic monitoring of microbial growth in response to antimicrobial challenge. , …”

“…Similarly, hydrogel immobilization AST platforms (Figure 1B) have been developed to allow for automatic monitoring of microbial growth in response to antimicrobial challenge. 57,58 The miniature feature sizes achievable with microfluidics have been leveraged to capture single bacteria inside cell traps. 25,59 This allows for an array of single bacteria to be monitored in response to antimicrobial challenge.…”

Accurate Prediction of Antimicrobial Susceptibility for Point‐of‐Care Testing of Urine in Less than 90 Minutes via iPRISM Cassettes

“…These include methods such as single‐cell optical imaging for tracking changes in motility and morphology, which can reduce the AST time to 30–120 min. [ 26 , 27 , 28 , 29 ] Yet, these methods rely on bacteria entrapment in microfluidic channels or hydrodynamic arrays and thus do not allow the formation of bacterial networks, [ 19 , 30 , 31 , 32 ] which may induce artifacts from a different bacteria stress response. [ 33 , 34 ] These methods also require the use of expensive equipment for high‐resolution imaging, which may restrict their routine application in a clinical laboratory setting.…”

Microfluidics for antibiotic susceptibility testing

“…A distinct use of hydrogel trapping was demonstrated recently, 94 where authors formed dried hydrogel plugs that were placed in a conventional 96-well plate. Bacteria and antibiotics were pipetted to the wells causing the dried plugs to absorb liquid and swell, causing mechanical separation of individual cells at the bottom of the well.…”