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Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant <jats:italic>Acinetobacter baumannii<\/jats:italic> (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin\/sulbactam, aztreonam, cefepime, ceftolozane\/tazobactam, doripenem, imipenem, meropenem, and piperacillin\/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim\/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensor<jats:sup>TM<\/jats:sup> Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a \u22651.5-fold increase in mode RSG fluorescence intensity (MIC<jats:sub>RSG<\/jats:sub>). Accuracy of MIC<jats:sub>RSG<\/jats:sub> was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for \u03b2-lactams (83.3% accuracy) and trimethoprim\/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours.<\/jats:p>","DOI":"10.1371\/journal.pone.0305939","type":"journal-article","created":{"date-parts":[[2024,6,24]],"date-time":"2024-06-24T19:21:18Z","timestamp":1719256878000},"page":"e0305939","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":7,"title":["Can flow cytometric measurements of reactive oxygen species levels determine minimal inhibitory concentrations and antibiotic susceptibility testing for Acinetobacter baumannii?"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6478-5888","authenticated-orcid":true,"given":"Jia Hao","family":"Yeo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia 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