In this thesis mutants of K. pneumoniae resistant to BRL 41897A were isolated using TnphoA mutagenesis and used to study the mechanism of uptake of BRL 41897A by K. pneumoniae. The activity of BRL 41897A towards the parent strain (M10) was increased in iron depleted media, whereas no significant differences in the resistant (KSL) mutants were observed. Three mutants (KSL19, KSL38and KSL59) produced decreased amounts of certain iron-regulated outer membrane proteins. The uptake of 55Fe-BRL 41897A by M10 in iron-deficient medium was higher than in iron-rich medium. This result indicated the involvement of an iron transport system in the uptake of BRL 41897A by K. pneumoniae. Uptake by the KSL mutants in iron-deficient culture was higher than that by M10. This result, supported by analysis of outer membrane and periplasmic proteins of the KSL mutants, indicates that loss of one outer membrane protein can be compensated by over-expression of other outer membrane and/or periplasmic proteins. However, the increased uptake of BRL 41897A by the KSL mutants did not reflect increased activity towards these strains, indicating that there are defects in the transport of BRL 41897A resulting in failure to reach the penicillin binding protein target sites in the cytoplasmic membrane. Southern blotting of chromosomal digests and sequencing in one mutant (KSL19) showed that only one copy of TnphoA was inserted into its chromosome. A putative TnphoA inserted gene in KSL19, designated kslA, carrying a signal sequence was identified. Transformation of a fragment containing the kslA gene into KSL19 cells restored the sensitivity to BRL 41897A to that of the parent strain.

Bacterial resistance to antibiotics and biocides is a prevalent problem, which may be exacerbated by the commonplace and often unnecessary inclusion of biocides into domestic products. Addition of antimicrobials, to domestic disinfectants has raised concern about promoting microbial resistance and potential cross-resistance to therapeutic antibiotics. This study investigated the potential for resistance in Salmonella enterica serovars Enteritidis, Typhimurium, Virchow and Escherichia call 0157 to commonly used biocides, to identify mechanisms underlying resistance and whether these provided cross-resistance to antibiotics. Salmonella enterica and E. coli 0157 strains were serially exposed to sub-inhibitory. concentrations of erythromycin (ERY), benzalkonium chloride (BKC), chlorhexidine hydrochloride (CHX)and triclosan (TLN). Once resistance was achieved permeability changes in the outer membrane, including LPS, cell surface charge and hydrophobicityand the presence of,an active efflux were investigated as possible resistance candidates. Thin layer chromatography (TLC) and Gas chromatography (GC) were carried out to examine fatty acid and lipid changes in E. coli 0157 isolates with reduced susceptibility to TLN. Cross-resistance was studied by the Stoke's method and standard microdilution assays. Examination of the outer membrane proteins and LPS did not reveal any significant changes between parent and resistant strains. The hydrophobicity of the cells increased as the cells were passaged and became less. susceptible. An active efflux system was the most likely mechanism of resistance in all strains tested and a fab1 mutation was associated with E. coli 0157 resistant to TLN isolates. In all isolates investigated the resistance was stable for over 30 passages in biocide-free media. A high degree of cross-resistance was obtained in TLN-resjstant Escherichia coli 0157 strains, which repeatedly exerted decreased susceptibility to various antimicrobials, including chloramphenicol, erythromycin, imipenem, tetracycline and trimethoprirn:, as well as to various biocides. The results of this laboratory-based investigation suggest that it is possible for microorganisms to become resistant to biocides when repeatedly exposed to sublethal concentrations. This may be especially the case in the domestic environment where administration of biocides is poorly controlled. Eventually it could lead to the undesirable situation of resident strains becoming resistant to disinfection and cross resistant to other antimicrobials.

The principal aim of this project was to investigate mechanisms of drug resistance in Trypanosoma brucei, the causative agent of disease in humans (sleeping sickness) and livestock (nagana), which affects large areas of sub-Saharan Africa. By understanding the mechanisms of resistance, the useful life of current therapies (of which there are only a few) may be extended, diagnostics to identify resistant parasites could be developed and the design of novel therapies aided. We therefore developed parasites with high levels of resistance to the clinically important drug pentamidine, which is the first-line treatment for early stage West African sleeping sickness and is closely related to the main veterinary treatment diminazene aceturate (Berenil). The characterisation of this strain revealed that the resistance phenotype was at least in part due to the loss of the previously characterised high affinity pentamidine transporter (HAPT). To investigate the protein(s) responsible for HAPT activity, and to identify any other proteins contributing to the resistance phenotype, we employed a proteomic approach. The plasma membrane sub-proteome (TbPM) of long slender bloodstream form trypanosomes was defined. A number of interesting observations were made from TbPM, and it will no doubt be of benefit to the greater scientific community. One example is the positive identification of many proteins hitherto designated as putative. A quantitative approach was then employed to analyse the resistant parasites using isotope-coded affinity tagging (I-CAT) and difference gel electrophoresis (DiGE), including a novel combination of DiGE and 16-BAC protein separation technologies. Both the plasma membrane subproteome and the soluble proteome were investigated, and a number of regulated proteins identified. The role of some proteins, with potentially relevant functions, such as a kinase, adenylate cyclase and a protein involved in kinetoplast stability should be further investigated.