Candida albicans Amphotericin B-Tolerant Persister Formation is Closely Related to Surface Adhesion

Abstract

Candida albicans persisters have so far been observed only in biofilm environment; the biofilm element(s) that trigger(s) persister formation are still unknown. In this study, we tried to further elucidate the possible relationship between C. albicans persisters and the early phases of biofilm formation, especially the surface adhesion phase. Three C. albicans strains were surveyed for the formation of persisters. We tested C. albicans persister formation dynamically at different time points during the process of adhesion and biofilm formation. The number of persister cells was determined based on an assessment of cell viability after amphotericin B treatment and colony-forming unit assay. None of the planktonic cultures contained persisters. Immediately following adhesion of C. albicans cells to the surface, persister cells emerged and the proportion of persisters reached a peak of 0.2-0.69 % in approximately 2-h biofilm. As the biofilm matured, the proportion of persisters decreased and was only 0.01-0.02 % by 24 h, while the number of persisters remained stable with no significant change. Persisters were not detected in the absence of an attachment surface which was pre-coated. Persisters were also absent in biofilms that were scraped to disrupt surface adhesion prior to amphotericin B treatment. These results indicate that C. albicans antifungal-tolerant persisters are produced mainly in surface adhesion phase and surface adhesion is required for the emergence and maintenance of C. albicans persisters.

Candida albicansbiofilms are generally devoid of persister cells

“…We encountered a technical problem at the biofilm recovery step, usually performed by scraping the cells in 1x PBS and vortexing prior to plating (8, 13, 15, 22). In our hands, the cells could not be properly resuspended and plated, as clumps of the biofilms would usually remain stranded inside the tips.…”

“…Notably, the experiments for this study were performed using in vitro biofilm model of C. albicans , developed in polystyrene 96-well plates. Following this work and relying on the protocol for persister cells isolation described therein (8), persister cells in C. albicans biofilms were described by a few other groups (13–15). However, later work by the Douglas group showed that not all Candida species and strains were able to form persister cells in laboratory-grown biofilms (16).…”

“…This was in particular the case for C. albicans strain SC5314 (17), the parental strain of almost all C. albicans strains used for functional genomics and molecular genetics studies. Unlike in the previously mentioned papers (8, 13–15), the protocol Al Dhaheri and Douglas (16) used for persisters isolation involved growing biofilms on silicone discs followed by their immersion into an antifungal solution. As the topic of C. albicans persister cells remains controversial, the main objective of this work was to reevaluate their occurrence in C. albicans biofilms.…”

Candida albicans Biofilms Are Generally Devoid of Persister Cells

“…Notably, the experiments for this study were performed using an in vitro biofilm model of C. albicans developed in polystyrene 96-well plates. Following this work and relying on the protocol for persister cells isolation described therein (8), persister cells in C. albicans biofilms were described by a few other groups (14)(15)(16). However, later work by Al-Dhaheri and Douglas showed that not all Candida species and strains were able to form persister cells in laboratory-grown biofilms (17).…”

“…This was in particular the case for C. albicans strain SC5314 (18), the parental strain of almost all C. albicans strains used for functional genomics and molecular genetics studies. Unlike in the previously mentioned papers (8,(14)(15)(16), the protocol Al-Dhaheri and Douglas (17) used for persister isolation involved growing biofilms on silicone discs, followed by their immersion into an antifungal solution. As the topic of C. albicans persister cells remains controversial, the main objective of this work was to reevaluate their occurrence in in vitro-grown C. albicans biofilms.…”