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Constrained by low bacterial burdens of 1\u2013100 colony\u2010forming\u2010units per ml blood (CFU\/ml), clinical diagnosis relies on lengthy culture amplification and isolation steps prior to identification and antibiotic susceptibility testing (AST). The resulting &gt;60\u2010h time to actionable treatment not only negatively impacts patient outcomes, but also increases the misuse and overuse of broad\u2010spectrum antibiotics that accelerates the rise in multidrug resistant infections. Consequently, the development of novel technologies capable of rapidly recovering bacteria from blood\u2010derived samples is crucial to human health. To address this need, we report a novel bacterial recovery technology from positive blood cultures that couples selective hemolysis with centrifugation through a sucrose cushion to perform rapid, background\u2010free cytometric ASTs without long subculturing steps. Demonstrated on the most common bloodstream infection\u2010causing bacteria: <jats:italic>Escherichia coli, Pseudomonas aeruginosa<\/jats:italic>, and <jats:italic>Staphylococcus aureus<\/jats:italic>, near\u2010pure bacteria are rapidly recovered (\u226415\u2009min) with minimal user intervention. Susceptibilities of recovered bacteria are readily performed via high throughput flow cytometry with excellent agreement with much slower, standard microbroth dilution assays. Altogether, this novel direct\u2010from\u2010positive blood culture AST technology enables susceptibility determinations within as little as 5\u00a0h, post blood culture positivity.<\/jats:p>","DOI":"10.1002\/cyto.a.24560","type":"journal-article","created":{"date-parts":[[2022,4,15]],"date-time":"2022-04-15T03:48:30Z","timestamp":1649994510000},"page":"564-576","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Rapid, label\u2010free antibiotic susceptibility determined directly from positive blood culture"],"prefix":"10.1002","volume":"101","author":[{"given":"Alexandra B.","family":"Filbrun","sequence":"first","affiliation":[{"name":"School of Chemistry and Biochemistry and Petit Institute of Bioengineering and Bioscience Georgia Institute of Technology  Atlanta Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph C.","family":"Richardson","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry and Petit Institute of Bioengineering and Bioscience Georgia Institute of Technology  Atlanta Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prakash C.","family":"Khanal","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry and Petit Institute of Bioengineering and Bioscience Georgia Institute of Technology  Atlanta Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yih\u2010Ling","family":"Tzeng","sequence":"additional","affiliation":[{"name":"Division of Infectious Disease, Department of Medicine Emory University School of Medicine  Atlanta Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0042-6194","authenticated-orcid":false,"given":"Robert M.","family":"Dickson","sequence":"additional","affiliation":[{"name":"School of Chemistry and Biochemistry and Petit Institute of Bioengineering and Bioscience Georgia Institute of Technology  Atlanta Georgia USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,4,22]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cix411"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1086\/345476"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01169-07"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13054-016-1320-7"},{"key":"e_1_2_7_6_1","doi-asserted-by":"crossref","first-page":"2019","DOI":"10.15620\/cdc:82532","volume-title":"Antibiotic resistance threats in the United States","author":"Centers for Disease Control and Prevention","year":"2019"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1377\/hlthaff.2017.1153"},{"key":"e_1_2_7_8_1","volume-title":"Antimicrobial resistance: global report on surveillance","author":"World Health Organization","year":"2014"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(17)30394-8"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics9120851"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1086\/590061"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01577-17"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1001\/jamanetworkopen.2020.2899"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.49.2.760-766.2005"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(20)30477-1"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1002\/phar.2201"},{"key":"e_1_2_7_17_1","first-page":"259","article-title":"Rapid identification and susceptibility testing using the VITEK 2 system using culture fluids from positive BacT\/ALERT blood cultures","volume":"41","author":"Chen J\u2010R","year":"2008","journal-title":"J Microbiol Immunol Infect"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.41.10.4705-4707.2003"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10096-018-03445-3"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1099\/jmm.0.000053"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-11-156"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1469-0691.2009.03006.x"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01329-17"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciaa528"},{"key":"e_1_2_7_25_1","unstructured":"Accelerate Diagnostics. 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