Ryder, Victoria Joanne (2010) The mutability of staphyloccal biofilms. PhD thesis, University of Leeds.
In their natural environment, bacteria are often found as sessile populations, known as biofilms, typified by surface adherence and extracellular matrix production. This growth phase confers broad spectrum antibiotic recalcitrance, through undefined mechanisms. This work sought to investigate a role for increased mutability ofstaphylococci in biofilms, as a contributing mechanism in biofilm antibiotic recalcitrance. Initially, a novel biofilm model for use with staphylococci was established that utilised cellulose disks and incorporated human plasma to promote bacterial surface adherence. This system was validated by demonstrating antibiotic recalcitrance, dissemination by D-amino acids, and similarities with transcriptional profiles of other biofilm models. Using this novel biofilm model, and the Sorbarod flow system, mutation frequencies (MFs) were determined for Staphylococcus aureus and Staphylococcus epidermidis in biofilms and compared with those in planktonic cultures. This revealed increases in MF of up to 68-fold compared with planktonic cultures. The role of oxidative stress in biofilm mutability was investigated by addition of antioxidants to biofilms. Here, MFs were reduced up to 5-fold, suggesting a role for oxidative damage. Transcriptional profiling of biofilms revealed upregulation of several genes involved in DNA repair, compared with planktonic cultures. Genes encoding antioxidant activity were also investigated; of these only sodA was upregulated. Staphyloxanthin biosysthetic genes, however, were downregulated. During these studies, S. aureus biofilms were found to generate phenotypic variants. White variants (WVs) and large pale variants (LPVs) were mostly found amongst cells shed from biofilms. WVs had lost biofilm forming capacity and had mutations in the alternative sigma factor, SigB. LPVs retained biofilm forming capacity, but the genetic basis of their variation remains undefined. In summary, I have shown that staphylococci exhibit enhanced mutability in biofilms, accelerating the emergence of antibiotic resistance and morphological variants. This may result from oxidative stress, causing the enhanced accumulation of mutations within the genome.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of Leeds > Faculty of Biological Sciences (Leeds)|
|Depositing User:||Ethos Import|
|Date Deposited:||12 Jan 2012 14:13|
|Last Modified:||08 Aug 2013 08:47|