{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,25]],"date-time":"2026-06-25T20:59:09Z","timestamp":1782421149391,"version":"3.54.5"},"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2025,9,4]],"date-time":"2025-09-04T00:00:00Z","timestamp":1756944000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["CG21APR1011"],"award-info":[{"award-number":["CG21APR1011"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["CG21APR2005"],"award-info":[{"award-number":["CG21APR2005"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["MOH-001278-01"],"award-info":[{"award-number":["MOH-001278-01"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001349","name":"National Medical Research Council","doi-asserted-by":"publisher","award":["MOH-000018-00"],"award-info":[{"award-number":["MOH-000018-00"]}],"id":[{"id":"10.13039\/501100001349","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100019579","name":"SingHealth","doi-asserted-by":"publisher","award":["AMSGH\/03-09\/FY2024\/EX\/55-A116(a)"],"award-info":[{"award-number":["AMSGH\/03-09\/FY2024\/EX\/55-A116(a)"]}],"id":[{"id":"10.13039\/100019579","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>Early appropriate antibiotic treatment is vital in reducing patient mortality. However, current antimicrobial susceptibility testing (AST) requires 16\u201324 hours of incubation, delaying appropriate antibiotic treatment. Flow cytometry (FCM) is a rapid method in assessing fluorescence (such as fluorophores for ROS) at single-cell resolution. Reactive oxygen species (ROS) are oxygen-containing molecules, which are inducible by antibiotics and potentially bactericidal. We asked if FCM measurements of ROS in antibiotic-treated bacteria could be used in determining antibiotic MIC as an alternative to conventional AST. This study aims to develop and evaluate the feasibility of a FCM assay to determine antibiotic susceptibility accurately with a short turn-around time. MICs of amikacins, aztreonams, cephalosporins (with or without a lactamase inhibitor), carbapenems, levofloxacin, polymyxin B, trimethoprim\/sulfamethoxazole, and tigecycline were determined for six clinical carbapenem-resistant Enterobacterales isolates using conventional microbroth dilution assays and using FCM assessments in parallel. Accurate MICs determined using FCM is defined as MICs falling within 2-fold dilutions of the conventional microbroth dilution AST assay. MIC determination via ROS measurements were mostly accurate for carbapenems (22\/24; 91.7% accuracy) and trimethoprim\/sulfamethoxazole (5\/6; 83.3% accuracy). In contrary, ROS levels were less accurate in determining MICs for amikacin (4\/6; 66.7% accuracy), aztreonam (4\/6; 66.7% accuracy), cephalosporins only (5\/12; 41.6% accuracy), cephalosporin with lactamase inhibitor (11\/18; 61.1% accuracy), polymyxin-B (2\/6; 33.3% accuracy), levofloxacin (1\/6; 16.7% accuracy), and tigecycline (2\/6; 33.3% accuracy). These data support that ROS assessments using FCM is suitable for accurately determining MICs for carbapenems in Enterobacterales. Further optimisation and validation of this FCM assay with additional bacteria strains with varying antibiotic susceptibilities are warranted. Future studies include assessing other organisms and antibiotic pairs.<\/jats:p>","DOI":"10.1371\/journal.pone.0331217","type":"journal-article","created":{"date-parts":[[2025,9,4]],"date-time":"2025-09-04T17:39:11Z","timestamp":1757007551000},"page":"e0331217","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Determining minimal inhibitory concentrations and antibiotic susceptibility for Enterobacterales by flow cytometry using reactive oxygen species as a marker"],"prefix":"10.1371","volume":"20","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":"Nasren","family":"Begam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wan 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Balkhair","year":"2019","journal-title":"Int J Infect Dis"},{"issue":"2","key":"pone.0331217.ref003","doi-asserted-by":"crossref","DOI":"10.1128\/AAC.01882-17","article-title":"Epidemiology of Carbapenem-Resistant Enterobacteriaceae Infections: Report from the China CRE Network","volume":"62","author":"Y Zhang","year":"2018","journal-title":"Antimicrob Agents Chemother"},{"key":"pone.0331217.ref004","doi-asserted-by":"crossref","first-page":"127249","DOI":"10.1016\/j.micres.2022.127249","article-title":"Global spread of carbapenem-resistant Enterobacteriaceae: Epidemiological features, resistance mechanisms, detection and therapy","volume":"266","author":"J Ma","year":"2023","journal-title":"Microbiol Res"},{"issue":"1","key":"pone.0331217.ref005","doi-asserted-by":"crossref","first-page":"13","DOI":"10.3390\/microorganisms4010013","article-title":"Carbapenem Resistance in Gram-Negative Bacteria: The Not-So-Little Problem in the Little Red Dot","volume":"4","author":"JQM Teo","year":"2016","journal-title":"Microorganisms"},{"key":"pone.0331217.ref006","doi-asserted-by":"crossref","DOI":"10.1093\/cid\/cix113","article-title":"Clinical and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae Among Adult Inpatients in Singapore","volume":"64","author":"K Marimuthu","year":"2017","journal-title":"Clin Infect Dis"},{"key":"pone.0331217.ref007","article-title":"Prevalence of Healthcare-Associated Infections and Antimicrobial Use Among Adult Inpatients in Singapore Acute-Care Hospitals: Results From the First National Point Prevalence Survey","volume":"64","author":"Y Cai","year":"2017","journal-title":"Clin Infect Dis"},{"issue":"6","key":"pone.0331217.ref008","doi-asserted-by":"crossref","first-page":"1589","DOI":"10.1097\/01.CCM.0000217961.75225.E9","article-title":"Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock","volume":"34","author":"A Kumar","year":"2006","journal-title":"Crit Care Med"},{"issue":"20","key":"pone.0331217.ref009","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.1401876111","article-title":"Antibiotics induce redox-related physiological alterations as part of their lethality","volume":"111","author":"DJ Dwyer","year":"2014","journal-title":"Proc Natl Acad Sci U S A"},{"key":"pone.0331217.ref010","doi-asserted-by":"crossref","first-page":"622534","DOI":"10.3389\/fmicb.2020.622534","article-title":"Reactive Oxygen Species in Pathogen Clearance: The Killing Mechanisms, the Adaption Response, and the Side Effects","volume":"11","author":"H Li","year":"2021","journal-title":"Front Microbiol"},{"key":"pone.0331217.ref011","doi-asserted-by":"crossref","first-page":"567","DOI":"10.2147\/IDR.S142397","article-title":"Antimicrobial use of reactive oxygen therapy: current insights","volume":"11","author":"MY Memar","year":"2018","journal-title":"Infect Drug 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One"},{"issue":"2","key":"pone.0331217.ref018","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1177\/096228029900800204","article-title":"Measuring agreement in method comparison studies","volume":"8","author":"JM Bland","year":"1999","journal-title":"Stat Methods Med Res"},{"issue":"2","key":"pone.0331217.ref019","doi-asserted-by":"crossref","first-page":"141","DOI":"10.11613\/BM.2015.015","article-title":"Understanding Bland Altman analysis","volume":"25","author":"D Giavarina","year":"2015","journal-title":"Biochem Med (Zagreb)"},{"issue":"5","key":"pone.0331217.ref020","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1016\/j.celrep.2015.09.059","article-title":"Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage","volume":"13","author":"P Belenky","year":"2015","journal-title":"Cell Rep"},{"key":"pone.0331217.ref021","article-title":"Genetic resisters to antibiotics in Escherichia coli arise from the antibiotic-surviving population containing three reactive oxygen species","volume":"368","author":"A Paul","year":"2022","journal-title":"FEMS Microbiol Lett"},{"issue":"6","key":"pone.0331217.ref022","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.1093\/jac\/dkl403","article-title":"Serum, tissue and body fluid concentrations of tigecycline after a single 100\u2009mg dose","volume":"58","author":"KA Rodvold","year":"2006","journal-title":"J Antimicrob Chemother"},{"issue":"10","key":"pone.0331217.ref023","doi-asserted-by":"crossref","first-page":"1216","DOI":"10.1592\/phco.22.15.1216.33484","article-title":"Pharmacokinetics of intravenous and oral levofloxacin in critically ill adults in a medical intensive care unit","volume":"22","author":"JA Rebuck","year":"2002","journal-title":"Pharmacotherapy"},{"key":"pone.0331217.ref024","year":"2020"},{"issue":"6","key":"pone.0331217.ref025","doi-asserted-by":"crossref","DOI":"10.1093\/ofid\/ofz194","article-title":"Antimicrobial Resistance or Delayed Appropriate Therapy-Does One Influence Outcomes More Than the Other Among Patients With Serious Infections Due to Carbapenem-Resistant Versus Carbapenem-Susceptible Enterobacteriaceae?","volume":"6","author":"TP Lodise","year":"2019","journal-title":"Open Forum Infect Dis"},{"issue":"2","key":"pone.0331217.ref026","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1038\/nprot.2007.521","article-title":"Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances","volume":"3","author":"I Wiegand","year":"2008","journal-title":"Nat Protoc"},{"issue":"7","key":"pone.0331217.ref027","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.7150\/thno.19217","article-title":"Current and emerging techniques for antibiotic susceptibility tests","volume":"7","author":"K Syal","year":"2017","journal-title":"Theranostics"},{"issue":"1","key":"pone.0331217.ref028","doi-asserted-by":"crossref","first-page":"456","DOI":"10.3390\/ijms22010456","article-title":"Emerging Options for the Diagnosis of Bacterial Infections and the Characterization of Antimicrobial Resistance","volume":"22","author":"S Rentschler","year":"2021","journal-title":"Int J Mol Sci"},{"issue":"1","key":"pone.0331217.ref029","doi-asserted-by":"crossref","first-page":"9719","DOI":"10.1038\/s41467-024-53930-x","article-title":"Next-generation rapid phenotypic antimicrobial susceptibility testing","volume":"15","author":"G Reszetnik","year":"2024","journal-title":"Nat Commun"},{"issue":"2","key":"pone.0331217.ref030","doi-asserted-by":"crossref","first-page":"165","DOI":"10.3390\/pathogens10020165","article-title":"The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance","volume":"10","author":"B Kowalska-Krochmal","year":"2021","journal-title":"Pathogens"},{"issue":"11","key":"pone.0331217.ref031","doi-asserted-by":"crossref","DOI":"10.1016\/j.cmi.2020.08.019","article-title":"Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii","volume":"26","author":"D Fonseca E Silva","year":"2020","journal-title":"Clin Microbiol Infect"},{"issue":"12","key":"pone.0331217.ref032","doi-asserted-by":"crossref","first-page":"3183","DOI":"10.1093\/jac\/dkab328","article-title":"Rapid high-resolution detection of colistin resistance in Gram-negative bacteria using flow cytometry: a comparison with broth microdilution, a commercial screening test and WGS","volume":"76","author":"O Ekelund","year":"2021","journal-title":"J Antimicrob Chemother"},{"issue":"1","key":"pone.0331217.ref033","doi-asserted-by":"crossref","first-page":"1903","DOI":"10.1038\/s41598-017-02009-3","article-title":"Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae","volume":"7","author":"KT Mulroney","year":"2017","journal-title":"Sci Rep"},{"issue":"4","key":"pone.0331217.ref034","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1111\/jam.14704","article-title":"Antimicrobial susceptibility testing: currently used methods and devices and the near future in clinical practice","volume":"129","author":"M Benkova","year":"2020","journal-title":"J Appl Microbiol"},{"issue":"9","key":"pone.0331217.ref035","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1007\/s11274-022-03332-2","article-title":"Determination of antibiotic susceptibility of bacteria by flow cytometric method","volume":"38","author":"H Hatipoglu","year":"2022","journal-title":"World J Microbiol Biotechnol"},{"issue":"10","key":"pone.0331217.ref036","doi-asserted-by":"crossref","first-page":"1675","DOI":"10.1007\/s10096-014-2124-9","article-title":"Combination antibiotic treatment versus monotherapy for multidrug-resistant, extensively drug-resistant, and pandrug-resistant Acinetobacter infections: a systematic review","volume":"33","author":"P Poulikakos","year":"2014","journal-title":"Eur J Clin Microbiol Infect Dis"},{"key":"pone.0331217.ref037","doi-asserted-by":"crossref","first-page":"80","DOI":"10.3389\/fmicb.2019.00080","article-title":"Infections Caused by Carbapenem-Resistant Enterobacteriaceae: An Update on Therapeutic Options","volume":"10","author":"C-C Sheu","year":"2019","journal-title":"Front Microbiol"},{"issue":"2","key":"pone.0331217.ref038","doi-asserted-by":"crossref","first-page":"149","DOI":"10.3109\/03009734.2014.899279","article-title":"Combination antibiotic therapy for multidrug-resistant Gram-negative bacteria","volume":"119","author":"T T\u00e4ngd\u00e9n","year":"2014","journal-title":"Ups J Med Sci"},{"key":"pone.0331217.ref039","doi-asserted-by":"crossref","first-page":"380","DOI":"10.3389\/fimmu.2015.00380","article-title":"An Introduction to Automated Flow Cytometry Gating Tools and Their Implementation","volume":"6","author":"CP Verschoor","year":"2015","journal-title":"Front Immunol"},{"key":"pone.0331217.ref040","doi-asserted-by":"crossref","first-page":"975066","DOI":"10.3389\/fphar.2022.975066","article-title":"Pharmacokinetics\/pharmacodynamics of polymyxin B in patients with bloodstream infection caused by carbapenem-resistant Klebsiella pneumoniae","volume":"13","author":"Z Yu","year":"2022","journal-title":"Front Pharmacol"},{"issue":"6","key":"pone.0331217.ref041","doi-asserted-by":"crossref","first-page":"1385","DOI":"10.1093\/jac\/dkae114","article-title":"Iron efflux by IetA enhances \u03b2-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer","volume":"79","author":"M Liu","year":"2024","journal-title":"J Antimicrob Chemother"},{"issue":"1","key":"pone.0331217.ref042","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1186\/s13613-017-0334-x","article-title":"Piperacillin-tazobactam as alternative to carbapenems for ICU patients","volume":"7","author":"B Pilmis","year":"2017","journal-title":"Ann Intensive Care"},{"issue":"6140","key":"pone.0331217.ref043","doi-asserted-by":"crossref","first-page":"1583","DOI":"10.1126\/science.1238328","article-title":"Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway","volume":"340","author":"B Ezraty","year":"2013","journal-title":"Science"},{"issue":"6124","key":"pone.0331217.ref044","doi-asserted-by":"crossref","first-page":"1213","DOI":"10.1126\/science.1232688","article-title":"Killing by bactericidal antibiotics does not depend on reactive oxygen species","volume":"339","author":"I 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