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Effective treatment remains challenging due to antifungal resistance, driven by efflux pumps, target enzyme mutations, biofilm formation, and persister cells. This study evaluated efflux pump gene expression and its role in biofilm development and resistance in persister cells of both species, as well as the effects of combined treatments\u2014fluconazole + FK506 and fluconazole + octyl protocatechuate\u2014on biofilms. Materials and Methods included microscopy, biofilm characterization during formation, treatment assays, induction of persister cells, and relative quantification of efflux pump genes\n                      <jats:italic toggle=\"yes\">AFR1<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">AFR2<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">AFR3<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      . Biofilms of both species were robust, with increased extracellular matrix linked to efflux pump overexpression. Combined treatments reduced biofilm growth and extracellular matrix production while downregulating\n                      <jats:italic toggle=\"yes\">AFR1<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">AFR2<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">AFR3<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">MDR1<\/jats:italic>\n                      , highlighting a link between efflux pumps and matrix export. Persister cells displayed low metabolic activity under amphotericin B treatment, followed by slow regrowth and reactivation of efflux pumps. These findings demonstrate multifaceted roles of efflux pumps in antifungal resistance and biofilm development, confirm the presence of persister cells in\n                      <jats:italic toggle=\"yes\">C. neoformans<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">C. gattii<\/jats:italic>\n                      , and suggest novel therapeutic strategies for cryptococcosis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Cryptococcosis is a fungal infection with mortality exceeding 75%, predominantly affecting immunocompromised individuals\u2014including transplant recipients and people living with HIV\u2014but also capable of infecting healthy people. Treatments are lengthy, toxic, and increasingly ineffective due to antifungal resistance. Two major contributors to therapeutic failure are biofilms\u2014protective fungal communities encased in a self-produced matrix\u2014and persister cells, a dormant subpopulation that survives lethal drug concentrations and reactivates infection once treatment stops. This study reveals that efflux pumps\u2014proteins that expel drugs from fungal cells\u2014are not only resistance mechanisms but also active contributors to biofilm construction. Critically, persister cells reactivate their efflux pumps upon recovery, providing a biological explanation for infection recurrence. Two drug combinations\u2014fluconazole + FK506 and fluconazole + octyl protocatechuate\u2014synergistically disrupted biofilms and reduced persister cell viability while potentially lowering drug doses and toxicity, pointing toward safer, more effective strategies for cryptococcosis.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00203-26","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T13:06:06Z","timestamp":1783947966000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Dynamics associated with the efflux pumps in biofilms and persister cells of\n                    <i>Cryptococcus neoformans<\/i>\n                    and\n                    <i>Cryptococcus gattii<\/i>"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-4372-6017","authenticated-orcid":true,"given":"Gabriel Rezende","family":"Pimenta","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00987cb86","id-type":"ROR","asserted-by":"publisher"}],"name":"Laboratory of Clinical Mycology - 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