Characterisation of the synergistic vancomycin-furazolidone action against Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Masters in Biochemistry at Massey University, Manawatū, New Zealand. EMBARGOED until 23 February 2019
The use of antibiotic combinations is garnering increased interest in the recent years due to the spread of antibiotic-resistant bacteria. The shortage of antibacterial therapy options is particularly severe for infections caused by Gram-negative bacteria, due to the formidable barrier to molecules > 600 Da imposed by the outer membrane. Vancomycin is a large glycopeptide antibiotic to which the outer membrane is poorly permeable, hence the minimal inhibitory concentration of this antibiotic for Escherichia coli is very high (~500 mg/L). Due to the resistance of E. coli and other Gram-negative pathogens to an increasing number of < 600 Da antibiotics including beta lactams, aminoglycosides and quinolones, enabling vancomycin use on Gram-negative bacteria would be valuable. Furazolidone was reported to increase sensitivity of E. coli to vancomycin, and this interaction has been investigated in this thesis in order to explore the potential of the vancomycin-furazolidone combination for clinical applications. The initial analysis of the vancomycin-furazolidone synergy demonstrated that their interaction is synergistic rather than merely additive. Furthermore, effectiveness of this combination for growth inhibition and eradication of E. coli biofilm was investigated. However, despite the synergy between vancomycin and furazolidone, the concentration of vancomycin in combinations required for growth inhibition and killing of E. coli in a planktonic mode and as a biofilm was above the nephrotoxicity (toxicity in the kidneys) threshold and therefore too high to treat infections with this organism systemically. The mechanism of synergy in the planktonic mode of growth was investigated through the analysis of E. coli gene-knock-out mutants and it was observed that TolC, the outer membrane channel common to a number of efflux systems (exporting enterobactin, xenobiotics and metabolites) is likely to be involved in vancomycin-furazolidone synergy. However, it was not possible to reliably pinpoint any particular efflux pump or enterobactin accumulation as factors in synergy. Using the genetic approach, it was found that DNA excision repair endonuclease UvrABC was ruled out as a factor involved in synergy. Overall this study characterised the synergy between vancomycin and furazolidone, initiated the enquiry into the mechanisms of interaction between these two antibiotics and examined its effectiveness against biofilms.