A Small Subpopulation of Blastospores in Candida albicans Biofilms Exhibit Resistance to Amphotericin B Associated with Differential Regulation of Ergosterol and β -1,6-Glucan Pathway Genes

Abstract

The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential-or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and ␤-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.

The Transcriptional Regulator Nrg1p Controls Candida albicans Biofilm Formation and Dispersion

“…These observations have important clinical ramifications, as they demonstrate that C. albicans cells may initially attach to a surface, maintain a state of "dormancy" for extended periods of time, and then proliferate and be able to form biofilms upon the receipt of the right environmental cues. Of note, even in the absence of an extensive three-dimensional structure, attached cells already demonstrate increased levels of resistance to antifungal agents, particularly azole derivatives (9,14), and this property may be further accentuated by the change in physiological status (nongrowing) associated with this dormant state (10,12).…”

Simvastatin Inhibits Candida albicans Biofilm In Vitro

“…4A and B) indicates that at least one inhibitory mechanism is interference with ergosterol synthesis. Although genes involved in the ergosterol pathway are known to contribute to biofilm formation (37,38), the failure of ergosterol to restore biofilms treated with 5 M simvastatin suggests that concentrations Ն5 M may impair other metabolic processes as well, such as statin-dependent geranylgeranylation, farnesylation, or production of exopolysaccharide. Interestingly, as the simvastatin concentration exceeded 5 M, quantitative cultures of biofilms yielded increasing numbers of petite mutants, which represent organisms that have lost mitochondrial DNA.…”

Growth in a Biofilm Induces a Hyperinfectious Phenotype inVibrio cholerae

“…However, growth rate and metabolic state have also been proposed to contribute to biofilm-related protection from certain antimicrobials and other stresses (5,25,59,68). In addition, distinct genetic mechanisms of antibiotic resistance employed by biofilm microorganisms have been described (34,41,42).The ability to form biofilms is a virulence determinant of many microorganisms. Classic examples of biofilm infections include chronic infections of the cystic fibrosis lung by Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, uropathogenic Escherichia coli in recurrent urinary tract infections, and disease caused by microbial biofilms on a variety of indwelling medical devices (27,35,36,52,56).…”

“…However, growth rate and metabolic state have also been proposed to contribute to biofilm-related protection from certain antimicrobials and other stresses (5,25,59,68). In addition, distinct genetic mechanisms of antibiotic resistance employed by biofilm microorganisms have been described (34,41,42).…”