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Title: Antimicrobial resistance of CF pathogens : mechanisms of biocide resistance and action
Author: Rose, Helen Louise
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2009
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Cystic fibrosis (CF) patients are predisposed to a number of bacterial infections, including Pseudomonas aeruginosa and the Burkholderia cepacia complex (Bcc). Both groups of bacteria have been associated with contamination of products containing biocides, leading to concerns that the over use of biocide products could select for multi drug resistant organisms. This investigation examined the susceptibility profile of panels of P. aeruginosa and Bcc strains to a range of biocides including chlorhexidine and cetylpyridinium chloride (CPC). It was found that certain epidemic strains that had spread among individuals with CF, such as the B. cenocepacia J2315 strain lineage and the P. aeruginosa Liverpool strain were less susceptible to chlorhexidine than other strains representative of the same species. Although minimum inhibitory concentration (MIC) screens gave an overall view of biocide susceptibility, minimum bactericidal concentrations (MBC) for these bacteria were higher. For CPC 27 out of 40 strains required more than 20 fold more biocide than the MIC to achieve a bactericidal effect. Suspension tests were performed on two commercial biocide formulations, a chlorhexidine-based wash, Hibiscrub and a triclosan-based hand gel Cuticura . The epidemic B. cenocepacia strain J2315 was capable of surviving in both these products after 20 minutes of exposure and viable bacteria were isolated after 60 minutes of exposure in Hibiscrub . The results from this investigation suggest that certain commercial biocides are not effective against the Bcc. Therefore to assist in the future development of biocides, three highly resistant Bcc strains were proposed as suitable reference strains to use in challenge testing for biocide efficacy. The molecular basis of biocide resistance was determined using a microarray approach to profile global gene expression of B. cenocepacia in response to chlorhexidine. B. cenocepacia J2315 was exposed to sub-inhibitory levels of chlorhexidine (5 ug/ml) and expression compared to cells not exposed to biocide. The microarray analysis demonstrated significant alterations in expression at P < 0.05, with a > 1.5 fold change with 98 up-regulated and 76 down-regulated genes. Two chlorhexidine up-regulated genes were selected for further analysis, a response regulator (BCAM 0924) potentially involved with efflux and a novel transport related gene (BCAL 2553). Site directed mutagenesis of these genes was carried out in B. cenocepacia strain K56-2 and a reduction in chlorhexidine MIC was observed for each respective mutant compared to the wildtype. 66 out of 76 (87%) of the down-regulated genes were involved with motility related functions. This led to the hypothesis that sub-inhibitory levels of chlorhexidine inhibited swarming motility and induced biofilm production. This question was tested for Bcc strains using soft- agar swarming tests and 96-well plate biofilm assays. A total of 6 of 10 strains screened exhibited both biofilm induction and swarming inhibition in response to chlorhexidine. A potential conserved regulatory binding motif was observed upstream of all gene sets down- regulated chlorhexidine in the microarray analysis. This suggested that swarming inhibition and biofilm induction in B. cenocepacia may be controlled by a coordinated regulatory pathway controlled by a two component system sensor-regulator system. A transposon mutagenesis approach was used to identify B. cenocepacia mutants that lacked the inhibitory response. The screen identified several mutations involved in the phenomenon including cheY-Uke receiver genes and a glycosyl transferase encoding gene. In conclusion, the molecular analysis of biocide resistance in Bcc bacteria demonstrated it was multifactoral, involving efflux pumps, transport related genes, membrane proteins and regulatory genes. The ability of chlorhexidine to inhibit swarming and switch Bcc to a non motile biofilm lifestyle was identified as a novel biocide survival response. With further research this regulatory pathway may be a potential target for the development of a novel biocides and therapeutics which overcome the antimicrobial resistance of these bacteria pathogens.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available