Enhancing Antimicrobial Susceptibility Testing for <i>Acinetobacter baumannii</i> Using Physiologically Relevant Culture Media and Biofilm Formation Assays

Acinetobacter baumannii is a high-risk pathogen associated with increased patient morbidity and mortality. Host-pathogen interactions amplify its virulence, in part by promoting biofilm formation-a crucial factor in antimicrobial resistance and persistence. Given the bacterium's strong propensity for acquiring resistance, antimicrobial susceptibility testing (AST) is essential for guiding effective therapeutic interventions. However, discrepancies have been observed between in vitro AST results and therapeutic outcomes, with some antimicrobials being deemed to show in vivo efficacy despite appearing ineffective in vitro. This discordance may stem from traditional AST protocols, which rely on bacteriological media such as Mueller Hinton broth (MHB) optimized for bacterial growth but not for mimicking the host environment. Moreover, conventional AST does not account for virulence traits such as biofilm formation, which further contribute to treatment failure. Incorporating physiologically relevant culture media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, alongside assessment of biofilm formation may improve the predictive value of AST. This work outlines two complementary protocols for improving AST interpretation in A. baumannii infections. Basic Protocol 1 compares minimum inhibitory concentration (MIC) values generated using MHB and RPMI. Basic Protocol 2 evaluates biofilm formation in MHB, tryptic soy broth (TSB; control), and RPMI, with and without antimicrobial exposure. Together, these approaches aim to inform alternative AST strategies that better reflect in vivo conditions and optimize therapeutic decision-making. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Comparing A. baumannii minimum inhibitory concentration (MIC) results in bacteriological (MHB) versus physiological (RPMI) media Basic Protocol 2: Comparing A. baumannii isolate(s) biofilm formation following assays completed in bacteriological culture media (MHB and control TSB) and physiological medium (RPMI).