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Bacterial resistance to biocides: development of a predictive protocol

Knapp, Laura 2014. Bacterial resistance to biocides: development of a predictive protocol. PhD Thesis, Cardiff University.
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Abstract

In the last 10 years biocides have been used increasingly and questions have been raised about their contribution to the reported increase in biocide and antibiotic resistance in pathogenic bacteria. The EU Biocidal Product Regulation (BPR) now requires information on the risk of resistance development in organisms targeted by the biocidal product. There is no current protocol available to predict the likelihood of bacteria becoming resistant to a biocidal product or biocides contained therein. This study aimed to identify useful markers of biocide resistance and develop a step-by- step protocol predictive of bacterial biocide resistance and antibiotic cross-resistance following biocide exposure. A range of experimental techniques with the potential to generate markers of biocide resistance were explored. These included minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC)/antibiotic susceptibility determination, flow cytometry, efflux activity measurements, outer membrane protein changes, real-time PCR and microarrays. Salmonella enterica serovar Typhimurium strains SL1344 and 14028S, and Burkholderia lata strain 383 were exposed to low concentrations of chlorhexidine gluconate and benzalkonium chloride as test biocides. Baseline and post-exposure data were then compared. Techniques used to understand any change in antimicrobial susceptibility were assessed in terms of practicality, cost and ease of use, and a step-by-step protocol was put together accounting for each of these factors. Increases in biocide MIC and MBC of up to 100 fold were observed in SL1344 and 14028S after exposure to both biocides. However these changes were not stable after subculture of surviving organisms in the absence of either biocide. No such dramatic changes were observed within B. lata. Up-regulation of efflux activity was observed as a result of CHG/BZC exposure and the efflux regulatory gene acrR underwent a >100 fold down-regulation in both Salmonella strains after CHG exposure. Flow cytometry experiments performed using SL1344 and 14028S indicated that at low CHG/BZC concentrations (0.0001 – 0.0004 %) greater than 50 % of the population were not killed and that these organisms could be sorted and further investigated to determine the mechanisms behind their survival. Reduction in the expression of two outer membrane proteins was observed in strain SL1344 after exposure to 0.0004 % CHG but further protein sequencing would be required to identify these. Changes in phenotype and genotype of biocide-exposed bacteria were identified using different experimental techniques. Some of these changes e.g. increased MIC/MBC values, altered antibiotic susceptibility, up-regulated efflux activity, alterations in the expression of specific genes and surviving organisms identified by flow cytometry represent useful markers of biocide resistance. A preliminary step by step protocol incorporating these techniques was successfully developed and allows for the rapid identification of biocide resistance and antibiotic cross-resistance as a result of biocide exposure, and will prove particularly useful in light of the recent changes to the BPR

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Pharmacy
Subjects: Q Science > QR Microbiology
Uncontrolled Keywords: Biocides, Resistance, Burkholderia, Salmonella, Antibiotics
Date of First Compliant Deposit: 30 March 2016
Last Modified: 19 Mar 2016 23:41
URI: http://orca-mwe.cf.ac.uk/id/eprint/60862

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