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Microscopic and molecular assessment of chlorhexidine tolerance mechanisms in Delftia acidovorans biofilms

dc.creatorRema, Taraen_US
dc.date.accessioned2016-04-06T12:00:14Z
dc.date.available2016-04-06T12:00:14Z
dc.date.created2016-03en_US
dc.date.issued2016-04-05en_US
dc.date.submittedMarch 2016en_US
dc.description.abstractOne of the most concerning characteristics of microbial biofilms is that of increased resistance to antimicrobial agents such as the commonly used biocide chlorhexidine (CHX). This can have huge impact on clinical, household and environmental settings. This is particularly alarming when it involves opportunistic pathogenic environmental organisms such as Delftia acidovorans as routine mitigation practices may fail to be effective. This thesis examines tolerance mechanisms of D. acidovorans biofilms exposed to CHX at inhibitory and sub-inhibitory concentrations. To achieve the study goals and objectives, a CHX-tolerant D. acidovorans strain (WT15), (Minimum Inhibitory Concentration; MIC-15 μg ml-1) was compared to a CHX-sensitive strain (MT51, MIC-1 μg ml-1) that was obtained by mutating the wild type strain using transposon mutagenesis. Specific morphological, structural and chemical compositional differences between the CHX-treated and untreated biofilms of wild type and mutant strains were documented using microscopic techniques including confocal laser scanning microscopy (CLSM), scanning transmission x-ray microscopy (STXM), transmission electron microscopy (TEM) and infrared (IR) spectroscopy. Molecular level changes between biofilms formed by these two strains due to CHX treatment were compared using whole-cell proteomic analysis (determined using differential in-gel electrophoresis, or DIGE) along with fatty acid methyl ester (FAME) analysis. The gene disrupted by transposon insertion that led to increased susceptibility to CHX in the mutant strain was identified as tolQ. CLSM revealed differences in biofilm architecture and thickness between the biofilms formed by strains WT15 and MT51. STXM analyses showed that WT15 biofilms contained two morpho-chemical cell variants; whereas, only one type was detected in MT51 biofilms. STXM and IR spectral analyses revealed that CHX-susceptible MT51 cells accumulated the highest levels of CHX, an observation supported by TEM wherein prominent changes in the cell envelope of CHX-susceptible MT51 cells were observed. DIGE analysis demonstrated that numerous changes in protein abundance occurred in biofilm cells following CHX exposure and that most of these proteins were associated with amino acid and lipid biosynthesis, protein translation, energy metabolism and stress-related functions. Overall, these studies indicate the probable role of the cell membrane and TolQ protein in CHX tolerance in D. acidovorans biofilms, in association with various proteins that are differentially-expressed.en_US
dc.identifier.urihttp://hdl.handle.net/10388/ETD-2016-03-2469en_US
dc.language.isoengen_US
dc.subjectChlorhexidineen_US
dc.subjectantimicrobial resistanceen_US
dc.subjectmicrobial biofilmsen_US
dc.subjectTolQen_US
dc.subjecttransposonen_US
dc.subjectDIGEen_US
dc.subjectD. acidovoransen_US
dc.titleMicroscopic and molecular assessment of chlorhexidine tolerance mechanisms in Delftia acidovorans biofilmsen_US
dc.type.genreThesisen_US
dc.type.materialtexten_US
thesis.degree.departmentFood and Bioproduct Sciencesen_US
thesis.degree.disciplineApplied Microbiologyen_US
thesis.degree.grantorUniversity of Saskatchewanen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US

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