Browsing by Author "Khot, Prasanna D."

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Candida albicans viability after exposure to amphotericin B: Assessment using metabolic assays and colony forming units
(2008) Khot, Prasanna D.; Suci, Peter A.; Tyler, Bonnie J.
Metabolic assays are a preferred method for evaluation of Candida albicans viability after exposure to antimicrobial agents in cases in which the culture is a complex mixture of yeast and filamentous forms. There is a lack of published data indicating the strength of the correlation between metabolic assays and viable cell numbers determined by a standard assay such as colony forming units (CFU). We developed a kinetic metabolic assay (KMA) for quantifying viable cells which was tested on yeast cells in both exponential and stationary phase using alamarBlue and XTT as metabolic indicators. The KMA enabled quantification of the viable population over a range of 101 to 107 cells that linearly correlated (R2>0.98) with estimates made by enumeration of CFU regardless of the indicator or growth phase of the cells. Linear relationships were used to calibrate the KMA in terms of equivalent CFU. Viable cell populations were then determined after exposure to AmB. These results were compared with those obtained by direct enumeration of CFU. There were significant correlations between KMA-derived equivalent CFU and direct CFU estimates of viable cell populations for exponential-phase cells. However, the proportions of viable cells based on the KMA were consistently lower than those obtained directly by CFU. This trend was substantially more pronounced for stationary phase cells. These results show that even in the relatively simple case in which only the yeast form is present, the relationship between assessment by metabolic assays and CFU is perturbed by exposure to an antimicrobial and that, furthermore, growth phase alters the nature of the perturbation.
A small subpopulation of blastospores in Candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and ß-1,6-glucan pathway genes
(2006-09) Khot, Prasanna D.; Suci, Peter A.; Miller, R. Lance; Nelson, Raoul D.; Tyler, Bonnie J.
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 flowcell (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. 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 AmB dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes encoding for enzymes in the ergosterol (ERG1, 3, 5, 6, 9, 11, and 25) and ß-1,6-glucan (SKN1, 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 cell wall.