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

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.