{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T16:08:36Z","timestamp":1790698116672,"version":"4.1.0"},"reference-count":45,"publisher":"American Chemical Society (ACS)","issue":"38","license":[{"start":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T00:00:00Z","timestamp":1789430400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"SIUE Department of Chemistry"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Bacterial populations that appear uniform can hide striking differences in how individual cells respond to antimicrobial stress. This hidden heterogeneity is especially important for antimicrobial photodynamic treatment (aPDT), where population-averaged assays report whether bacteria recover but cannot reveal when individual cells become damaged or why some cells respond later than others. Here, we developed an agarose-pad single-cell imaging platform to track membrane permeabilization during aPDT mediated by Br2B, a brominated boron dipyrromethene (BODIPY) dye. Using extraintestinal pathogenic Escherichia coli UMN026 and Staphylococcus aureus SA113 as model pathogens, time-resolved propidium iodide (PI) fluorescence trajectories were fitted for individual bacteria to extract the onset of PI entry (ti), final PI-positive transition time (tf), and transition duration (\u0394t = tf \u2013 ti). This approach revealed distinct species-specific response architectures that were not apparent from bulk measurements alone. In E. coli, photodynamic response heterogeneity was distributed across both delayed PI-entry onset and prolonged transition duration, particularly under nutrient-rich LB conditions and in mixed-founder populations. In contrast, S. aureus displayed a compressed onset phase, with heterogeneity emerging mainly after PI entry had begun. Br2B uptake was consistently higher in S. aureus than in E. coli, supporting a model in which envelope architecture and photosensitizer access determine where heterogeneity appears during photodynamic damage. Post-aPDT regrowth assays further showed that recovery kinetics were species- and population-dependent, linking single-cell membrane-permeabilization dynamics with later population-level outgrowth. Finally, mutation-rate estimates indicated that colony-derived inocula are not genetically identical, but that mutation-derived diversity is unlikely to be the dominant source of the observed timing patterns. Together, these results demonstrate that aPDT response is not a single synchronized event but a structured, species-dependent process shaped by photosensitizer access, nutrient context, founder-lineage history, and physiological heterogeneity. This work establishes single-cell PI-entry kinetics as a powerful framework for uncovering hidden antimicrobial response architectures during photodynamic treatment.<\/jats:p>","DOI":"10.1021\/acsomega.6c09003","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T14:01:46Z","timestamp":1789480906000},"page":"58330-58342","source":"Crossref","is-referenced-by-count":0,"title":["Single-Cell Membrane-Permeabilization\nKinetics Reveal\nSpecies-Specific Responses to Antimicrobial Photodynamic Treatment"],"prefix":"10.1021","volume":"11","author":[{"given":"Omnia","family":"Ahmed","sequence":"first","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sol R.","family":"Marti\u0301nez","sequence":"additional","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taufiq","family":"Khan","sequence":"additional","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nitika","family":"Paudyal","sequence":"additional","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yusra","family":"Amena","sequence":"additional","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mahmoud","family":"Abouelyazid","sequence":"additional","affiliation":[{"name":"Texas A&M University , , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7898-4033","authenticated-orcid":true,"given":"Andre\u0301s M.","family":"Durantini","sequence":"additional","affiliation":[{"name":"Southern Illinois University Edwardsville , , , ,","place":["Edwardsville, Illinois, United States, 62026"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,15]]},"reference":[{"key":"2026092911104897600_cit1","doi-asserted-by":"publisher","first-page":"894","DOI":"10.1080\/13696998.2023.2232256","article-title":"Chronic wound prevalence and the associated cost of\ntreatment in Medicare beneficiaries: changes between 2014 and 2019","volume":"26","author":"Carter","year":"2023","journal-title":"J. Med. Econ."},{"key":"2026092911104897600_cit2","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1089\/wound.2021.0026","article-title":"Human wound\nand its burden: Updated 2020 Compendium of Estimates","volume":"10","author":"Sen","year":"2021","journal-title":"Adv. Wound Care"},{"key":"2026092911104897600_cit3","doi-asserted-by":"publisher","first-page":"441","DOI":"10.1038\/s41579-019-0196-3","article-title":"Definitions\nand guidelines for research on antibiotic persistence","volume":"17","author":"Balaban","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"2026092911104897600_cit4","doi-asserted-by":"publisher","first-page":"255","DOI":"10.1016\/j.molcel.2019.09.028","article-title":"Bacterial\nheterogeneity and antibiotic\nsurvival: understanding and combatting persistence and heteroresistance","volume":"76","author":"Dewachter","year":"2019","journal-title":"Mol. Cell"},{"key":"2026092911104897600_cit5","doi-asserted-by":"publisher","first-page":"1215","DOI":"10.1021\/jacs.5b10288","article-title":"Reactive oxygen species mediated\nactivation of a dormant singlet\noxygen photosensitizer: from autocatalytic singlet oxygen amplification\nto chemicontrolled photodynamic therapy","volume":"138","author":"Durantini","year":"2016","journal-title":"J.\nAm. Chem. Soc."},{"key":"2026092911104897600_cit6","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.jphotobiol.2009.04.001","article-title":"Photodynamic\ntherapy (PDT): A short\nreview on cellular mechanisms and cancer research applications for\nPDT","volume":"96","author":"Robertson","year":"2009","journal-title":"J. Photochem. Photobiol., B"},{"key":"2026092911104897600_cit7","doi-asserted-by":"publisher","first-page":"436","DOI":"10.1039\/b311900a","article-title":"Photodynamic therapy:\na new antimicrobial approach to infectious\ndisease?","volume":"3","author":"Hamblin","year":"2004","journal-title":"Photochem. Photobiol. Sci."},{"key":"2026092911104897600_cit8","doi-asserted-by":"publisher","first-page":"642609","DOI":"10.3389\/fmed.2021.642609","article-title":"Factors determining\nthe susceptibility of bacteria to antibacterial photodynamic inactivation","volume":"8","author":"Rapacka-Zdon\u0301czyk","year":"2021","journal-title":"Front. Med."},{"key":"2026092911104897600_cit9","doi-asserted-by":"publisher","first-page":"174","DOI":"10.1038\/s41392-024-01866-5","article-title":"Bacterial\npersisters: molecular mechanisms and therapeutic\ndevelopment","volume":"9","author":"Niu","year":"2024","journal-title":"Signal Transduction Targeted Ther."},{"key":"2026092911104897600_cit10","doi-asserted-by":"publisher","first-page":"1228","DOI":"10.1038\/s42003-024-06894-z","article-title":"Single-cell phenotypic profiling and backtracing exposes\nand predicts clinically relevant subpopulations in isogenic Staphylococcus\naureus communities","volume":"7","author":"Hira","year":"2024","journal-title":"Commun. Biol."},{"key":"2026092911104897600_cit11","doi-asserted-by":"publisher","first-page":"2283","DOI":"10.1128\/AAC.00123-09","article-title":"A module located at\na chromosomal\nintegration hot spot is responsible for the multidrug resistance of\na reference strain from Escherichia coli clonal group\nA","volume":"53","author":"Lescat","year":"2009","journal-title":"Antimicrob. Agents Chemother."},{"key":"2026092911104897600_cit12","doi-asserted-by":"publisher","first-page":"E2774","DOI":"10.1073\/pnas.1210309109","article-title":"Rate and molecular\nspectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing","volume":"109","author":"Lee","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026092911104897600_cit13","doi-asserted-by":"publisher","first-page":"693","DOI":"10.1038\/nature18313","article-title":"Rates and mechanisms of bacterial\nmutagenesis from maximum-depth sequencing","volume":"534","author":"Jee","year":"2016","journal-title":"Nat."},{"key":"2026092911104897600_cit14","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1186\/s12864-019-5604-6","article-title":"Rapid cell\ndivision of Staphylococcus aureus during colonization\nof the human nose","volume":"20","author":"Szafran\u0301ska","year":"2019","journal-title":"BMC Genomics"},{"key":"2026092911104897600_cit15","doi-asserted-by":"publisher","first-page":"5677","DOI":"10.1021\/acsami.3c17916","article-title":"In situ single-cell\nbacterial imaging provides mechanistic insight into the photodynamic\naction of photosensitizer-loaded hydrogels","volume":"16","author":"Ortega","year":"2024","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"2026092911104897600_cit100","doi-asserted-by":"publisher","first-page":"15046","DOI":"10.1021\/acsami.6b02605","article-title":"Spatiotemporally Resolved Tracking of Bacterial Responses\nto ROS-Mediated Damage at the Single-Cell Level with Quantitative\nFunctional Microscopy","volume":"8","author":"Barroso","year":"2016","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"2026092911104897600_cit16","doi-asserted-by":"publisher","first-page":"1624","DOI":"10.1021\/acsinfecdis.9b00185","article-title":"Phenotypic\nresistance in photodynamic inactivation unravelled at the single bacterium\nlevel","volume":"5","author":"Marti\u0301nez","year":"2019","journal-title":"ACS Infect. Dis."},{"key":"2026092911104897600_cit17","doi-asserted-by":"publisher","first-page":"6","DOI":"10.1093\/infdis\/jiv429","article-title":"extraintestinal pathogenic escherichia\ncoli, a common\nhuman pathogen: challenges for vaccine development and progress in\nthe field","volume":"213","author":"Poolman","year":"2016","journal-title":"J. Infect. Dis."},{"key":"2026092911104897600_cit18","doi-asserted-by":"publisher","first-page":"603","DOI":"10.1128\/CMR.00134-14","article-title":"Staphylococcus aureus infections: epidemiology, pathophysiology,\nclinical manifestations, and management","volume":"28","author":"Tong","year":"2015","journal-title":"Clin.\nMicrobiol. Rev."},{"key":"2026092911104897600_cit19","doi-asserted-by":"publisher","first-page":"557","DOI":"10.3390\/antibiotics12030557","article-title":"Clinical impact of\nstaphylococcus aureus skin and soft tissue infections","volume":"12","author":"Linz","year":"2023","journal-title":"Antibiotics"},{"key":"2026092911104897600_cit20","doi-asserted-by":"publisher","first-page":"749","DOI":"10.1016\/j.bbamem.2017.12.003","article-title":"Surface proteins\nand the formation of biofilms by Staphylococcus aureus","volume":"1860","author":"Kim","year":"2018","journal-title":"Biochim. Biophys. Acta, Biomembr."},{"key":"2026092911104897600_cit21","doi-asserted-by":"publisher","first-page":"a000414","DOI":"10.1101\/cshperspect.a000414","article-title":"The bacterial cell\nenvelope","volume":"2","author":"Silhavy","year":"2010","journal-title":"Cold Spring Harbor Perspect. Biol."},{"key":"2026092911104897600_cit22","doi-asserted-by":"publisher","first-page":"3966","DOI":"10.1021\/acs.biochem.8b00495","article-title":"Peptidoglycan and teichoic acid levels\nand alterations\nin staphylococcus aureus by cell-wall and whole-cell nuclear magnetic\nresonance","volume":"57","author":"Romaniuk","year":"2018","journal-title":"Biochem."},{"key":"2026092911104897600_cit23","doi-asserted-by":"publisher","first-page":"108","DOI":"10.2174\/1574891x113089990012","article-title":"Antimicrobial photodynamic\ntherapy\nto kill gram-negative bacteria","volume":"8","author":"Felipe","year":"2013","journal-title":"Recent Pat.\nAnti-Infect. Drug Discovery"},{"key":"2026092911104897600_cit24","doi-asserted-by":"publisher","first-page":"6483","DOI":"10.1038\/s41598-019-42906-3","article-title":"Propidium\niodide staining underestimates\nviability of adherent bacterial cells","volume":"9","author":"Rosenberg","year":"2019","journal-title":"Sci.\nRep."},{"key":"2026092911104897600_cit25","doi-asserted-by":"publisher","first-page":"79","DOI":"10.1016\/j.mimet.2017.09.011","article-title":"Propidium ion enters viable cells\nwith high membrane\npotential during live-dead staining","volume":"142","author":"Kirchhoff","year":"2017","journal-title":"J. Microbiol.\nMethods"},{"key":"2026092911104897600_cit26","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1146\/annurev.micro.58.030603.123818","article-title":"Stationary-phase\nphysiology","volume":"58","author":"Nystro\u0308m","year":"2004","journal-title":"Annu. Rev. Microbiol."},{"key":"2026092911104897600_cit27","doi-asserted-by":"publisher","first-page":"8746","DOI":"10.1128\/JB.01368-07","article-title":"Escherichia coli physiology in Luria-Bertani\nbroth","volume":"189","author":"Sezonov","year":"2007","journal-title":"J. Bacteriol."},{"key":"2026092911104897600_cit28","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003862","article-title":"Nutrient limitation governs staphylococcus\naureus metabolism and niche adaptation in the human nose","volume":"10","author":"Krismer","year":"2014","journal-title":"PLoS Pathog."},{"key":"2026092911104897600_cit29","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0165300","article-title":"Growth-environment dependent modulation\nof staphylococcus aureus branched-chain to straight-chain fatty acid\nratio and incorporation of unsaturated fatty acids","volume":"11","author":"Sen","year":"2016","journal-title":"PLoS One"},{"key":"2026092911104897600_cit30","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1016\/j.jphotobiol.2004.11.020","article-title":"Effect of albumin on the photodynamic\ninactivation of microorganisms by a cationic porphyrin","volume":"79","author":"Lambrechts","year":"2005","journal-title":"J. Photochem. Photobiol., B"},{"key":"2026092911104897600_cit31","doi-asserted-by":"publisher","first-page":"2163","DOI":"10.1128\/AEM.72.3.2163-2169.2006","article-title":"kinetics of single\ncells: observation and modeling\nof a stochastic process","volume":"72","author":"Pin","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"2026092911104897600_cit32","unstructured":"American Type Culture Collection (ATCC).\n          .  Staphylococcus aureus subsp.\naureus (ATCC\u00ae 35556): Genome Assembly Summary; ATCC Genome Portal. Published March 27, 2020. accessed June 22, 2026."},{"key":"2026092911104897600_cit33","doi-asserted-by":"publisher","first-page":"D750","DOI":"10.1093\/nar\/gkp889","article-title":"BioNumbers\u2500the\ndatabase of key numbers in molecular and cell biology","volume":"38","author":"Milo","year":"2010","journal-title":"Nucleic Acids Res."},{"key":"2026092911104897600_cit34","doi-asserted-by":"publisher","first-page":"5239","DOI":"10.3390\/molecules25225239","article-title":"Design of\nphotosensitizing agents\nfor targeted antimicrobial photodynamic therapy","volume":"25","author":"Klausen","year":"2020","journal-title":"Molecules"},{"key":"2026092911104897600_cit35","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1016\/j.cell.2008.09.038","article-title":"Mistranslation\nof membrane proteins and two-component system activation trigger antibiotic-mediated\ncell death","volume":"135","author":"Kohanski","year":"2008","journal-title":"Cell"},{"key":"2026092911104897600_cit36","doi-asserted-by":"publisher","first-page":"482","DOI":"10.1016\/j.mib.2009.06.018","article-title":"Role of reactive\noxygen species in\nantibiotic action and resistance","volume":"12","author":"Dwyer","year":"2009","journal-title":"Curr. Opin.\nMicrobiol."},{"key":"2026092911104897600_cit37","doi-asserted-by":"publisher","first-page":"1456","DOI":"10.1111\/php.13470","article-title":"Photosensitization\nreactions of biomolecules: definition, targets and mechanisms","volume":"97","author":"Baptista","year":"2021","journal-title":"Photochem. Photobiol."},{"key":"2026092911104897600_cit38","doi-asserted-by":"publisher","first-page":"598","DOI":"10.1038\/s41579-024-01054-w","article-title":"ESKAPE\npathogens: antimicrobial resistance, epidemiology,\nclinical impact and therapeutics","volume":"22","author":"Miller","year":"2024","journal-title":"Nat. Rev.\nMicrobiol."},{"key":"2026092911104897600_cit39","doi-asserted-by":"publisher","first-page":"315","DOI":"10.1126\/science.1219192","article-title":"Oxidation\nof the guanine nucleotide pool underlies cell death by bactericidal\nantibiotics","volume":"336","author":"Foti","year":"2012","journal-title":"Sci."},{"key":"2026092911104897600_cit40","doi-asserted-by":"publisher","first-page":"443","DOI":"10.1038\/nrmicro3032","article-title":"The molecular\nmechanisms and physiological consequences of oxidative stress: lessons\nfrom a model bacterium","volume":"11","author":"Imlay","year":"2013","journal-title":"Nat. Rev. Microbiol."},{"key":"2026092911104897600_cit41","doi-asserted-by":"publisher","first-page":"15470","DOI":"10.1038\/s41598-022-19928-5","article-title":"Selection\nin a growing colony biases\nresults of mutation accumulation experiments","volume":"12","author":"Mahilkar","year":"2022","journal-title":"Sci. Rep."},{"key":"2026092911104897600_cit42","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2526088123","article-title":"Plasmid mutation rates scale with\ncopy number","volume":"123","author":"Ramiro-Marti\u0301nez","year":"2026","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026092911104897600_cit43","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1038\/s41598-016-0005-4","article-title":"Generation of mutation hotspots in ageing bacterial\ncolonies","volume":"6","author":"Sekowska","year":"2016","journal-title":"Sci. Rep."},{"key":"2026092911104897600_cit44","doi-asserted-by":"publisher","first-page":"4442","DOI":"10.1128\/AEM.00722-15","article-title":"Rich medium composition\naffects escherichia coli survival,\nglycation, and mutation frequency during long-term batch culture","volume":"81","author":"Kram","year":"2015","journal-title":"Appl. Environ. Microbiol."}],"container-title":["ACS Omega"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsomega.6c09003","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/38\/58330\/68512426\/acsomega.6c09003.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/38\/58330\/68512426\/acsomega.6c09003.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T15:51:00Z","timestamp":1790697060000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acsodf\/article\/11\/38\/58330\/5430078\/Single-Cell-Membrane-Permeabilization-Kinetics"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,15]]},"references-count":45,"journal-issue":{"issue":"38","published-online":{"date-parts":[[2026,9,15]]},"published-print":{"date-parts":[[2026,9,29]]}},"URL":"https:\/\/doi.org\/10.1021\/acsomega.6c09003","relation":{},"ISSN":["2470-1343"],"issn-type":[{"value":"2470-1343","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,15]]}}}