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When monitoring the interactions of drugs with bacteria, it remains challenging to differentiate functionally relevant internalized drug levels from nonspecific binding. Fluorescence is a method of choice for observing dynamics of biomolecules. In order to facilitate studies involving aminoglycoside antibiotics, we have generated fluorescently labeled aminoglycoside derivatives with uptake and bactericidal activities similar, albeit with a moderate loss, to those of the parent drug. The method combines fluorescence microscopy with fluorescence-activated cell sorting (FACS) using neomycin coupled to nonpermeable cyanine dyes. Fluorescence imaging allowed membrane-bound antibiotic to be distinguished from molecules in the cytoplasm. Patterns of uptake were assigned to different populations in the FACS analysis. Our study illustrates how fluorescent derivatives of an aminoglycoside enable a robust characterization of the three components of uptake: membrane binding, EDPI, and EDPII. Because EDPI levels are weak compared to the two other types of accumulation and critical for the action of these drugs, the three components of uptake must be taken into account separately when drawing conclusions about aminoglycoside function.<\/jats:p>","DOI":"10.1021\/acsinfecdis.9b00421","type":"journal-article","created":{"date-parts":[[2020,3,20]],"date-time":"2020-03-20T12:00:20Z","timestamp":1584705620000},"page":"1008-1017","source":"Crossref","is-referenced-by-count":31,"title":["Fluorescent\nAminoglycoside Antibiotics and Methods for Accurately Monitoring Uptake\nby Bacteria"],"prefix":"10.1021","volume":"6","author":[{"given":"Mahnaz","family":"Sabeti Azad","sequence":"first","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maho","family":"Okuda","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Me\u0301lina","family":"Cyrenne","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mickael","family":"Bourge","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5232-1322","authenticated-orcid":true,"given":"Marie-Pierre","family":"Heck","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA , , ,","place":["Gif-sur-Yvette, France, 91191"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satoko","family":"Yoshizawa","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2170-3536","authenticated-orcid":true,"given":"Dominique","family":"Fourmy","sequence":"additional","affiliation":[{"name":"Universite\u0301 Paris-Saclay, CEA, CNRS , , ,","place":["Gif-sur-Yvette, France, 91198"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2020,3,20]]},"reference":[{"issue":"6","key":"2026081806115335100_cit1","doi-asserted-by":"publisher","first-page":"516","DOI":"10.1097\/QCO.0000000000000012","article-title":"Aminoglycosides:\nHow Should We Use Them in the 21st Century?","volume":"26","author":"Jackson","year":"2013","journal-title":"Curr. 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Invest."},{"issue":"27","key":"2026081806115335100_cit5","doi-asserted-by":"publisher","first-page":"10984","DOI":"10.1073\/pnas.1204073109","article-title":"Dissociation of Antibacterial Activity\nand Aminoglycoside Ototoxicity in the 4-Monosubstituted 2-Deoxystreptamine\nApramycin","volume":"109","author":"Matt","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"issue":"11","key":"2026081806115335100_cit6","doi-asserted-by":"publisher","first-page":"3763","DOI":"10.1128\/AAC.00480-05","article-title":"Pharmacokinetic-Pharmacodynamic\nRelationship of Arbekacin for Treatment of Patients Infected with\nMethicillin-Resistant Staphylococcus Aureus","volume":"50","author":"Sato","year":"2006","journal-title":"Antimicrob. 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