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Biofilms are found in many infections in the forms of surface-adhering aggregates on medical devices, small clumps in tissues, or even in synovial fluid. Although antibiotic resistance genes are studied and monitored in the clinic, the structural and phenotypic changes that take place in biofilms can also lead to significant changes in how bacteria respond to antibiotics. Therefore, it is important to better understand the relationship between biofilm phenotypes and resistance and develop approaches that are compatible with clinical testing. Current methods for studying antimicrobial susceptibility are mostly planktonic or planar biofilm reactors. In this work, we develop a new type of biofilm reactor—three-dimensional (3D) microreactors—to recreate biofilms in a microenvironment that better mimics those in vivo where bacteria tend to form surface-independent biofilms in living tissues. The microreactors are formed on microplates, treated with antibiotics of 1000 times of the corresponding minimal inhibitory concentrations (1000 × MIC), and monitored spectroscopically with a microplate reader in a high-throughput manner. The hydrogels are dissolvable on demand without the need for manual scraping, thus enabling measurements of phenotypic changes. Bacteria inside the biofilm microreactors are found to survive exposure to 1000 × MIC of antibiotics, and subsequent comparison with plating results reveals no antibiotic resistance-associated phenotypes. The presented microreactor offers an attractive platform to study the tolerance and antibiotic resistance of surface-independent biofilms such as those found in tissues.

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as an approximation for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays identified substitutions which conferred strong nirmatrelvir resistance and others that compromised activity. On the other hand, N142A and the P132H mutation, carried by the Omicron variant, caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.