CRISPR/Cas9-Mediated Genome Editing of T4 Bacteriophage for High-Throughput Antimicrobial Susceptibility Testing
Yawen He, Juhong Chen
Abstract
High Resolution Image Download MS PowerPoint Slide The accurate and effective determination of antimicrobial resistance is essential to limiting the spread of infectious diseases and ensuring human health. Herein, a simple, accurate, and high-throughput phage-based colorimetric sensing strategy was developed for antimicrobial susceptibility testing (AST). Taking advantage of the CRISPR/Cas9 system, the genome of the T4 phage was modularly engineered to carry lacZ-α (lacZa), a marker gene encoding the α-fragment of β-galactosidase (β-gal). T4 lacZa phages were identified by blue-white selection and then used for a biosensing application. In this strategy, the bacterial solution is exposed to the T4 lacZa phage, causing target bacteria to overexpress β-gal. Upon the addition of a colorimetric substrate, the β-gal initiates an enzymatic reaction, resulting in a solution color change from yellow to red. This sensing strategy offers a visual way to monitor bacterial growth in the presence of antibiotics, enabling the determination of bacterial antimicrobial susceptibility. As a proof of concept, our developed sensing strategy was successfully applied to identify 9 different multidrug-resistant Escherichia coli ( E. coli ) in urine samples with 100% specificity. Compared with conventional disk diffusion susceptibility tests, the engineered phage-based sensing strategy can shorten the detection time by at least half without losing detection sensitivity, providing an alternative high-throughput method for AST in clinical diagnosis.