Browsing by Author "Tyler, Bonnie J."

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Action of chlorhexidine digluconate against yeast and filamentous forms in an early-stage candida albicans biofilm
(2002-11) Suci, Peter A.; Tyler, Bonnie J.
An in situ method for sensitive detection of differences in the action of chlorhexidine against subpopulations of cells in Candida albicans biofilms is described. Detection relies on monitoring the kinetics of propidium iodide (PI) penetration into the cytoplasm of individual cells during dosing with chlorhexidine. Accurate estimation of the time for delivery of the dosing concentration to the substratum was facilitated by using a flow cell system for which transport to the interfacial region was previously characterized. A model was developed to quantify rates of PI penetration based on the shape of the kinetic data curves. Yeast were seeded into the substratum, and biofilm formation was monitored microscopically for 3 h. During this period a portion of the yeast germinated, producing filamentous forms (both hyphae and pseudohyphae). When the population was subdivided on the basis of cell morphology, rates of PI penetration into filamentous forms appeared to be substantially higher than for yeast forms. Based on the model, rates of penetration were assigned to individual cells. These data indicated that the difference in rates between the two subpopulations was statistically significant (unpaired t-test, P < 0.0001). A histogram of rates and analysis of variance indicated that rates were approximately equally distributed among different filamentous forms and between apical and subapical segments of filamentous forms.
Adhesion and biofilm formation of Candida albicans on native and pluronic-treated polystyrene
(2005-01) Wesenberg-Ward, Karen E,; Tyler, Bonnie J.; Sears, Joe
Candida albicans forms part of the normal human flora whose growth is usually restricted by the normal flora bacteria and the host's immune system. It is an opportunistic fungal pathogen that causes infections in immunocompromised individuals, mechanical trauma victims and iatrogenic patients. Candida albicans can ingress the human host by adhering to a plastic surface (i.e., prosthetic devices, catheters, artificial organs, etc.) that is subsequently implanted, and forms a protective biofilm that provides a continuous reservoir of yeast to be hematogenously dispersed. In order for the medical profession to battle device-related infections, initial adhesion and biofilm formation of C. albicans needs to be better understood. There has been some skepticism as to whether the initial adhesion events bear any relationship to subsequent biofilm formation. Thus, to better comprehend the relationship between the initial adhesion rates and growth rate and biofilm formation, these events were studied on two different, well-defined culture surfaces, native polystyrene and Pluronic F127-conditioned polystyrene. The adhesion studies determined that Pluronic F127 adsorption dramatically reduced the adhesion of C. albicans to polystyrene. The biofilm growth studies, analyzed by confocal scanning laser microscopy, revealed that Pluronic F127 decreased the biofilm surface coverage, cluster group size, thickness and the presence of hyphal elements over the untreated polystyrene. These findings indicate that the effect of a material's surface chemistry on the initial adhesion process has a direct influence on subsequent biofilm formation.
Adsorption of adhesive proteins from the marine mussel, mytilus edulis, on polymer films in the hydrated state using angle dependent x-ray photoelectron spectroscopy and atomic force microscopy
(1997-10) Baty, Ace M.; Leavitt, P. K.; Siedlecki, C. A.; Tyler, Bonnie J.; Suci, Peter A.; Marchant, R. E.; Geesey, Gill G.
The adsorption of mussel adhesive protein (MAP) from the marine mussel Mytilus edulis has been investigated on polystyrene (PS) and poly(octadecyl methacrylate) (POMA) surfaces using angle dependent X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). AFM images previously published in the dehydrated state using contact mode are compared with images acquired in the hydrated state using fluid Tapping Mode to assess the contribution that hydration has on the architecture of the adsorbed proteins. To further characterize the adsorbed protein layer, XPS analysis was performed at liquid nitrogen (LN2) temperature without dehydrating the samples and at room temperature after the surfaces were dehydrated. The differences observed upon dehydration can be attributed to the strength of the interactions between MAP and the two surfaces. The AFM and XPS data indicate that adsorbed MAP is stabilized on the surface of the PS through interactions that prevent the protein layer from being disrupted upon dehydration. The adsorbed MAP on the POMA surface is representative of a loosely bound protein layer that becomes highly perturbed upon dehydration.
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.
Integration of raman microscopy, differential interference contrast microscopy, and attenuated total reflection fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in candida albicans biofilm
(2001-09) Suci, Peter A.; Geesey, Gill G.; Tyler, Bonnie J.
Factors limiting hydrogen sulfide production were identified in a two-species biofilm containing sulfate-reducing bacteria (Desulfovibrio desulfuricans) and nonsulfate-reducing bacteria (Pseudomonas fluorescens). Profiles of hydrogen sulfide (H2S) concentration, pH, local mass-transport coefficient, local flow velocity, and local relative effective diffusivity in the biofilm were measured using microelectrodes. Biofilms had a heterogeneous structure consisting of cell clusters separated by voids. Typically, the H2S concentration was lower in the voids than in the adjacent cell clusters, demonstrating that the voids acted as transport channels for removing H2S from cell clusters. The extent of biofilm heterogeneity was directly correlated with the flux of H2S from cell clusters. At flow velocities below 2 cm/s, the flux of H2S from cell clusters depended on the flow velocity. We concluded that at these flow velocities the H2S production rate was limited by the delivery rate of sulfate ions to the biofilm. At flow velocities above 2 cm/s, the H2S production rate was nearly constant and did not depend on the flow velocity. At high flow velocities (<2 cm/s) the H2S production rate was limited by metabolic reactions in the biofilm. Local intrabiofilm flow velocity profiles were influenced strongly by biofilm heterogeneity without significant pH variation within biofilms. Surprisingly, profiles of local relative effective diffusivity indicated that the biofilm was made up of two layers, which could be related to the specimen with a two-species biofilm.
Investigation of mussel adhesive protein adsorption on polystryene and poly(octadecyl methacrylate) using angle dependent xps, atr-ftir and afm
(1996-02) Baty, Ace M.; Suci, Peter A.; Tyler, Bonnie J.; Geesey, Gill G.
The irreversible adsorption of mussel adhesive proteins (MAP) from the marine mussel Mytilus edulishas been investigated on polystyrene (PS) and poly(octadecyl methacrylate) (POMA) surfaces using angle resolved X-ray photoelectron spectroscopy (XPS), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectrometry, and atomic force microscopy (AFM). Angle resolved XPS was used to quantify the elemental composition with depth of the upper 90 Å of the surface, and AFM was used to obtain the surface topography. The adsorption pattern of MAP, revealed by AFM images, is distinctly different on the two polymer surfaces and suggests that the substratum influences protein adhesion. The depth profiles of MAP, obtained from angle resolved XPS, show differences in nitrogen composition with depth for MAP adsorbed to PS and POMA. Infrared spectra of hydrated adsorbed MAP revealed significant differences in the amide III region and in two bands which may originate from residues in the tandemly repeated sequences of MAP. This data demonstrates that the chemistry of the polymer film that is present at the protein–polymer interface can influence protein–protein and protein–surface interactions.
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.