Synergistic activity of caspofungin and posaconazole against <i>Candida</i> ( <i>Candidozyma</i> ) <i>auris</i> biofilms based on phenotypic, transcriptomic, and <i>in vivo</i> insights

<i>Candida</i> (<i>Candidozyma</i>) <i>auris</i> is an emerging multidrug-resistant fungal pathogen capable of establishing persistent skin colonization, contaminating the environment, and causing nosocomial outbreaks associated with high mortality rates. Conventional monotherapy frequently proves inadequate against biofilm-associated infections, underscoring the urgent need for novel therapeutic strategies. Therefore, we investigated the physiological and molecular responses of South Asian clade <i>C. auris</i> biofilms to treatment with a caspofungin-posaconazole combination. This regimen markedly reduced the median minimum inhibitory concentrations (4- to 32-fold for caspofungin; 8- to 64-fold for posaconazole) compared with monotherapies. Synergistic interactions were observed in all isolates, with fractional inhibitory concentration indices ranging from 0.078 to 0.31, and were further confirmed <i>in vivo</i>. Transcriptomic profiling revealed activation of multiple stress-response pathways, driving adaptive changes such as enhanced extracellular matrix production and biofilm-forming capacity, maintenance of intracellular cation homeostasis, osmotic stress response, and extensive cell wall and membrane remodeling affecting the mannan-glucan complex, chitin, sphingolipids, phosphatidylinositol-(4,5)-bisphosphate, and ergosterol content. Additional responses included activation of <i>RCT1</i> (fluconazole-inducible protein) and <i>MDR1</i> (drug efflux pump), collectively promoting survival under combined antifungal pressure. These findings demonstrate the potent synergistic activity of caspofungin and posaconazole against <i>C. auris</i> biofilms, thereby supporting the development of effective combination therapies for this high-risk pathogen.IMPORTANCE<i>Candida auris</i> is a rapidly emerging fungal pathogen that presents substantial challenges for infection control owing to its multidrug resistance, persistence in healthcare environments, and capacity to cause large-scale outbreaks. Biofilm formation on indwelling medical devices plays a pivotal role in <i>C. auris</i> outbreaks within healthcare settings and is implicated in nearly 90% of <i>C. auris</i> candidemia cases. These biofilms also exhibit pronounced tolerance to antifungal agents, thereby restricting available treatment options. Our study demonstrates that the combination of caspofungin and posaconazole exerts a strong synergistic effect against <i>C. auris</i> biofilms, both <i>in vitro</i> and <i>in vivo</i>. By elucidating the molecular mechanisms behind this synergy-including stress-response activation, cell wall and membrane remodeling, calcium signaling, and regulation of drug efflux pumps-this work provides important insights into antifungal therapeutic responses in <i>C. auris</i> and underscores combination therapy as a promising strategy to overcome biofilm-associated antifungal resistance in this high-risk pathogen.