|About the Book|
Candida albicans is a fungal pathogen that adheres to implant materials and forms structured colonies of cells with extracellular matrices, called biofilms. There is a preponderance of evidence that biofilms are extremely resistant to drugs. ReasonsMoreCandida albicans is a fungal pathogen that adheres to implant materials and forms structured colonies of cells with extracellular matrices, called biofilms. There is a preponderance of evidence that biofilms are extremely resistant to drugs. Reasons posited for biofilm resistance range from nutrient depletion to the nature of biofilm matrix and from mass transport resistance to the antifungal to changes in the expression of efflux pump genes. Generally resistance has been understood to be a byproduct of many such factors. Recently, researchers have seen evidence for the existence of small subpopulations of cells (basal blastospores) within biofilms. These cells were found to be vastly more resistant to antifungals when compared to the rest of the biofilm.-This dissertation is centered on testing two broad hypotheses relating to understanding biofilm resistance. The first part studied the effect of mass transfer resistance to the antifungal agent within the biofilm on drug resistance. Cryo-Time of flight secondary ion mass spectrometry (ToF-SIMS) was developed for the spatial and chemical imaging of chlorhexidine digluconate (CHG), an anti-fungal agent within biofilms. The use of a novel data interpretation methodology called Maximum Autocorrelation Factor (MAF) analysis was also evaluated. Image classification using MAF analysis allowed visualization of CHG penetrating the biofilm. MAF images correlated with spectra from the original ToF-SIMS data sets. Even after the CHG had penetrated the biofilm, live basal blastospore cells were visualized, indicating that mass transport resistance does not play a role in antifungal resistance.-The second part studied molecular mechanisms underlying the resistance of biofilms by investigating the link between adhesion and resistance. Gene and protein expression studies were performed on various fractions such as the basal blastospores, bulk biofilm and stationary phase planktonic cells. Real time reverse transcriptase polymerise chain reaction was used to quantity expression of genes thought to play a putative role in both adhesion and drug resistance. Protein expression studies were performed using both one-dimensional and two-dimensional gel electrophoresis followed by protein identification. Key differentially regulated genes in important adhesion pathways were identified. This work is the first to point to a strong connection between adhesion and resistance.