Rifabutin disk diffusion on chelated Mueller-Hinton agar: a robust methodological framework for accurate Acinetobacter baumannii susceptibility testing

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) is an extensively drug-resistant pathogen requiring new therapeutic strategies. Rifabutin (RBT), a U.S. Food and Drug Administration (FDA)-approved rifamycin, has demonstrated potent activity against CRAB; however, its activity is systematically underestimated by conventional antimicrobial susceptibility testing (AST) performed in conventional iron-rich Mueller-Hinton media, necessitating methods that accurately predict in vivo efficacy to guide its clinical use. Here, we describe the development of a disk diffusion method for RBT susceptibility testing against A. baumannii . Following Clinical and Laboratory Standards Institute (CLSI) M23 “Development of In Vitro Susceptibility Test Methods, Breakpoints, and Quality Control Parameters” guidance, multiple disk potencies were evaluated across standard and chelated Mueller-Hinton agar (MHA), including EDTA-supplemented media, and zone-of-inhibition (ZOI) diameters were correlated with MICs determined by agar dilution. A 20-µg RBT disk on chelated MHA, or equivalently on MHA supplemented with 100 mg/L EDTA, provided clear and reproducible discrimination of tentative wild-type and non-wild-type phenotypes, and preliminary quality control testing supported A. baumannii NCTC 13304 as a candidate quality control strain. While this investigational method has not been reviewed or approved by the CLSI, it was developed in concordance with the M23 guidance and addresses the key limitations of conventional AST, providing a robust framework for interlaboratory validation and future breakpoint determination. IMPORTANCE Antimicrobial susceptibility testing is a critical tool for guiding clinical treatment decisions. Rifabutin exhibits potent activity against carbapenem-resistant Acinetobacter baumannii (CRAB) under the iron-restricted conditions encountered in vivo ; however, its activity is systematically underestimated by standard testing methods in iron-rich Mueller-Hinton media. Here, we describe a robust, simple, and reproducible disk diffusion method using a 20-µg rifabutin disk on chelated Mueller-Hinton agar, which enables accurate assessment of rifabutin activity against A. baumannii . Our method provides a practical foundation for standardized rifabutin susceptibility testing, future breakpoint development, and broader evaluation of rifabutin against CRAB to ultimately support its implementation in clinical microbiology laboratories.