Cystic Fibrosis (CF) is the most common autosomal recessive genetic disorder in the UK. A mutation in the CFTR gene, alters a Cl- transporter protein resulting in dehydration at epithelial surface and a thick mucus layer that provides a nutrient rich environment ideal for opportunistic bacteria to colonise. Bronchiectasis (BR) is similar symptomatically to CF, with localised dilations and inflammation events of the bronchial tree that can be linked to lung trauma or allergy. Pseudomonas aeruginosa (Pa) is the most common opportunistic pathogen in CF that correlates to lowered lung function. Burkholderia cepacia complex (Bcc) species have been shown to be more problematic to clear than Pa, due to increased antimicrobial resistance and progression to Cepacia syndrome that can be fatal. These opportunistic bacteria and their genome plasticity allows adaptation to the lung and can correlate to pathogenicity of chronic infection which is associated with poor clinical outcomes. As adaptation to the lung environment is such a key aspect of chronic bacterial infection in the lung this study focuses on the temperate phages infecting Pa and Bcc. Temperate phages once integrated into the bacterial chromosome have been shown in other bacterial backgrounds to aid bacterial adaptation through increasing rates of recombination. They have also been previously characterised to aid positive selection by carrying genes that aid bacterial survival, aiding evolution of both the bacterium and phage. The aim of this project was to characterise temperate bacteriophages chemically induced from 94 Pa (47 associated with CF and 47 with BR patients), 47 Bcc isolates (associated with CF patients). This study focuses on 3 key areas. The first studies how phages induced from Pa and Bcc infect isolates from patients at different stages of chronic infection. All the bacterial isolates used in this study had at least one inducible lysogenic phage, in some instances polylysogens. Infectivity through Pa phage cross infection showed that adult CF phage were more infective across the Pa panel. Paediatric CF phages showed an infection profile similar to that of Pa phage induced from BR patients > 10 years after clinical diagnosis. Pa phage associated with BR patients < 10 years after clinical diagnosis showed the least infection across the Pa panel. Secondly, isolating and purifying temperate phages can be difficult and time consuming as phages that induce from the bacterial host in high numbers can mask secondary or tertiary phages. Importantly identifying individual phages can be complex, as a sensitive bacterial host is needed to isolate and propagate. This study uses novel genomic approaches to overcome this problem and separate mixed phage communities using k-mer abundance. Stratified phages have been annotated and compared for similarities that link to the clinical aetiology of the bacteria that carried that phage. Finally, this study begins to map genetic traits that may aid phage longevity in a microbial system. A model has been previously characterised with lytic phages where Ig-like domains or BAM motifs may explain how phages bind to complex carbohydrates in mucus. When evaluating the incidence of BAM, Ig-like domains in temperate phage DNA sequences isolated in this study we identify an increase in BAM motifs, that may correlating to the evolution of disease in both CF and BR. This aids the proposed BAM model and the evolution of temperate phages interacting at mucosal surfaces.

Mycobacterium tuberculosis (Mtb), the etiological agent of Tuberculosis (TB) is developing new multi drug-resistant (MDR) and extensively drug-resistant (XDR) strains to the current drugs used in therapy. Of particular concern the wide spreading of tuberculosis, the high rate of development of resistance, and the high mortality of the patients due to the lack of effective therapy against TB infections. In order to face this problem, two series of novel compounds were designed, synthesised and evaluated against a panel of mycobacterial strains. The first series of compounds includes analogues of the third line drug thioridazine (TZ). TZ is a known antipsychotic drug belonging to the phenothiazine drug group, which showed good activity against MDR-TB infections but causes severe side effects which limit its use in therapy. Among the first series of compounds, five new compounds showed anti-tubercular activity similar or higher than TZ. Moreover, two derivatives showed potent inhibition towards the whole-cell drug efflux pump activity of mycobacteria comparable to that of verapamil, and turning to be promising multi-drug resistance reversal agents. A second series of compounds consist of small molecules which have originally been designed as hybrids of the anti-tubercular drugs BM212 and SQ109. Computational studies revealed a perfect superposition of the structures of SQ109 and BM212 and showed that the two drugs share common features. Five of the resulting compounds showed micromolar anti-tubercular activity on pathogenic TB. Two of them proved to be highly active also against multi-drug resistant clinical isolates and one of these also showed minimal eukaryotic cell toxicity, and therefore would be an excellent lead candidate for preclinical trials. In parallel to the identification of novel compounds active against mycobacteria, new synthetic methodologies for the synthesis of antitubercular heterocyclic scaffolds have been developed. In particular two approaches for the synthesis of pyrrole compounds were developed. Both procedures involve an olefin or enyne metathesis reaction as a key step. The first approach involves the synthesis of 1,2,3-substituted pyrroles, through a tandem enyne cross metathesis-cyclization reaction of propargylamines with ethyl-vinyl ether. The reaction is rapid, procedurally simple and represents a facile entry to the synthetically challenging 4,5-unsubstituted pyrroles. The second methodology allows the synthesis of substituted pyrroles from diallyl-amines via a chemo-enzymatic cascade based on the combination of olefin metathesis together with monoamine oxidase (MAO) biocatalysts. These reactions were carried out in aqueous media and mild temperature leading to the formation of substituted pyrroles in a single step and in high yields.

Obstructive lung diseases are major causes of morbidity and mortality globally. The most common, chronic obstructive pulmonary disorder (COPD), is caused by smoke inhalation, whilst bronchiectasis (BR) is often caused by lung infection, and cystic fibrosis (CF) is due to the defective passage of fluids through lung epithelial cells. Whilst it is now recognised that the healthy lung harbours a microbial flora, obstructive lung diseases are characterised by periodic or persistent heavy bacterial colonization that can be determined using microbiological cultures of patient sputum as well as by novel genomic analysis, the most common species identified being Pseudomonas aeruginosa and Haemophilus influenzae. Adaptive immune responses generated against such organisms, in the form of antigen-specific antibodies and T cells, may be protective against infection but may also contribute to the inflammatory disease mechanism such as through IL-17-mediated neutrophil recruitment. This thesis asks whether such immune responses can serve as biomarkers of microbial colonization and of disease; whether levels of antibodies and T cells indicate lung infection and exacerbations frequency. However, high levels may simply indicate exposure to microbes. Thus, the aim of this study was to measure antibody and T cell responses against a range of lung-colonizing microbes in patients with BR (n=119), COPD (n=58), CF (n=30), asthma (n=14), and in healthy controls (n=28). Patients were clinically characterised in terms of exacerbations, lung function, sputum microbiology and underlying disease. Enzyme linked immunosorbent assays (ELISAs) were set up to measure specific antibodies in serum, whilst T cell responses in peripheral blood mononuclear cells (PBMCs) were measured by specific Enzyme-linked ImmunoSpot assay (ELIspot), flow cytometry and multiplex cytokine ELISA. Typical microbial colonization for the given disease was seen in the sputum-producing patients. The results showed that in BR, specific IgG responses against P. aeruginosa increased according to episodes of colonization, whilst T cell responses in the form of antigen-specific IFNY showed the opposite effect, suggesting T cell dysregulation. As well as IFNY, T cells were shown to secrete IL-17 and IL-22 in response to microbial antigens and to possess activation and homing receptors. Antibody responses were also further characterised for cross-reactivity and Ig isotype, confirming specificity and isotype switching. In CF patients that harboured P. aeruginosa, high anti-pseudomonas IgG titres were detected, and T cell IFNY responses were similar to healthy controls and were associated with greater disease stability and FEV1. In conclusion, immune responses were successfully characterised in patients with obstructive lung diseases, and specific antibody and T cells showed some associations with colonization and clinical disease, respectively, depending upon the disease and the microbe.

Rapid, sensitive and selective detection and identification of pathogens is required in the prevention and recognition of problems related to food security. Salmonella is one of the dangerous foodborne pathogens. The identification of specific volatile organic compounds (VOCs) produced by Salmonella may contribute in providing a fast and accurate detection method for Salmonella in food samples. In this study, VOCs liberated by Salmonella strains have been identified and quantified via head space-solid phase microextraction coupled to gas chromatography/mass spectrometry (HS-SPME GC/MS). The dominant chemical class of volatiles liberated from Salmonella strains was alcohol compounds. In addition, ester and ketone compounds were also detected. The most sensitive VOCs detected were ethyl octanoate (LOD = 62.0 ng/mL and LOQ = 207 ng/mL) and ethyl decanoate (LOD = 66 ng/mL and LOQ = 219 ng/mL) with the lowest LOD and LOQ when using Rappaport-Vassiliadis Soya peptone (RVS) broth media and polar SPME fiber with polar GC column. The type of culture medium was found to affect the liberated VOCs. For example, 2-heptanone was not detected when S. london and S. stanley were grown in TSB but they were detected and quantified when using BHI as growth media. Also, 1-octanol was detected and quantified in all strains when Salmonella grown in TSB and BHI, and did not detected in all strains when RVS was used as growth media. The research has been extended to include the addition of specific enzyme substrates to the culture medium (RVS). The enzyme substrates are either commercially available or have been synthesised to allow exogenous VOC detection. The specific enzymes targeted in Salmonella were α-galactosidase, C-8 esterase and pyrrolidonyl peptidase. The enzyme substrates used are phenyl α-D-galactopyranoside, 2-chlorophenyl octanoate and L-pyrrollidonyl fluoroanilide respectively. All, except pyrrolidonyl peptidase, are known to give a positive response to Salmonella. This developed methodology was initially applied to pure cultures of S. stanley to evaluate the feasibility of the approach. The developed approach shows potential for future application in food samples to detect and identify Salmonella species in food samples of a level as low as 100 CFU/mL within a 5 h incubation at 37 ºC by the detection of the liberated VOCs. Subsequently the methodology was applied to a range of food samples (milk, cheese, eggs and chicken). It was found that all food samples were Salmonella free; however, false positive was detected due to the presence of other pathogens in the food samples. Inhibition of some of these pathogens in milk and cheese samples was achieved with the addition of 5 mg/L vancomycin and 10 mg/L of novobiocin. To improve the method specificity, it was necessary to deviate from the standard method and use Salmonella selective RVS broth in pre-enrichment step than using non selective one (BPW). This results in a successful detection of Salmonella contamination on milk samples and cheddar cheese samples. However, failed in detect Salmonella in other cheeses. Inhibition of resistant pathogens (Streptococcus salivarius ssp. Thermophilus, Lactobacillus rhamnosus and Enterococcus faecalis) using another combination of selective agents (vancomycin 10 mg/L, novobiocin 10 mg/L, erythromycin 0.75 mg/L and lithium chloride 15 g/L) failed. This study highlighted the benefits of the use of specific enzyme substrates along with antibiotics into Salmonella VOC analysis to improve the specificity of Salmonella detection method. The results of VOC analysis of specific enzymes inherent within Salmonella could be extended to develop a selective portable sensor approach to be used in food production.

During the course of these studies, polyester polyurethane foam has been found to be readily degraded in soil and marine environments. The incorporation of the formulation biocide Vinyzene E.P. reduced the rate and extent of degradation in test material exposed to soil conditions, but was found to be ineffective in preventing degradation when exposed to the marine environment. As a result of these studies, a number of fungi, representing a range of genera, have been isolated, identified, and shown using Kochian principles to degrade polyester polyurethane as a sole source of carbon. A list of isolates is submitted. In addition, a number of bacteria, hitherto unreported as deteriogens of polyester polyurethane, have been isolated and also shown, again using Kochian principles, to degrade polyester polyurethane. In order to effect degradation, the bacteria were found to require supplementation of the basal mineral salts medium with non-defined, complex sources of organic nitrogen, such as yeast extract. Chemical analysis of degraded test material, using FT-IR spectroscopy has provided evidence to suggest that the ester linkage of the polymer was hydrolysed during degradation by a bacterial isolate studied, resulting in marked reductions in the physical properties recorded. No degradation, either in terms of chemical composition or in the physical properties of the polymer was recorded where the test material was inoculated with this isolate in a mineral salts medium supplemented with glucose, suggesting that the enzymes involved in the degradation process were catabolically suppressed and therefore inducible. Extracellular esterase enzyme activity was detected in cellfree supernatants where the mineral salts medium was supplemented with yeast extract, but not in the case where glucose was used. However the addition of the test material did not enhance the level of esterase activity detected. Purification/concentration of these enzymes and characterisation studies using gel filtration chromatography, SDS-PAGE and PAGE were found to support these findings. The results of the present studies suggest that the test material was degraded by the bacterial isolate as a result of co-metabolism.

Rhodococcus equi is a Gram-positive soil organism that causes an aggressive bronchopneumonia in foals and opportunistic infections in immuno-compromised humans. Virulent strains possess an 80 – 90 kb plasmid that encodes an immunogenic surface-located virulence-associated protein VapA. The virulence of the organism has been largely attributed to this protein since mutants lacking vapA are attenuated for virulence in mice. VapA is an unusual lipoprotein and existing evidence suggests its biogenesis may not involve normal lipoprotein processing. In order to understand the structure of VapA and other virulence associated proteins, their genes were cloned, expressed, purified and crystallised. VapG produce high quality crystals that diffracted to 1.8 Å. The structure was resolved to be a closed β-barrel with a long unstructured N-terminus which is similar to both VapB and VapD which have also recently been characterised.

The discovery of antibiotics in the early 20th century revolutionised medicine, but quickly bacteria began to demonstrate resistance to these agents. Antibiotic resistance is still on the increase, and soon, if this trend continues, bacterial infections may not be treatable with antibiotics. In an effort to prevent this occurring, the search for novel antibiotics is under way and new antibiotic targets are being considered. One target that has not been studied in depth is the partitioning systems of bacterial plasmids. Disruption of plasmid partition would prevent effective plasmid inheritance, which, in the case of resistance plasmids, would render the host cell antibiotic sensitive. The aim of this study was to determine whether plasmid partitioning is a viable target for a new antibiotic. A series of plasmids that contained genetically altered partitioning systems was used, which provided a range of plasmid stabilities. The plasmids all conferred antibiotic resistance on the host cell, allowing the effect of plasmid instability on a population grown in the presence of antibiotics to be determined. Several different methods of cell culture were used. Simple batch culture experiments allowed the observation that few plasmid-free cells were produced from cells containing plasmids that had a functional system. In contrast, plasmids containing a faulty system were found to be rapidly lost from the host cells. Steady-state chemostat culture was used to provide a simple model of a clinical infection. The formation of equilibria between plasmid-free and plasmid-bearing cells was observed, and the cultures contained a large proportion of plasmid-free cells when the experiments involved unstable plasmids. The slow growth rate of cultures in the chemostat was seen to dramatically affect the inheritance of plasmids relying on random distribution. Finally, cultures were subjected to washout in order to determine their maximum specific growth rate (µmax). While the results from these experiments are not entirely conclusive, there is a strong indication that the growth rate of cultures containing unstable plasmids grown in the presence of antibiotics is reduced.

This thesis is divided into two parts; the first part describing the synthesis and evaluation of novel fluorogenic enzymatic substrates for microorganism detection, and the second part investigating the synthesis of thiophenes with extended conjugation as fluorescent labels with potential applications in antimicrobial susceptibility testing. ‘Spacer’ substrates were identified as a method of incorporating a phenolic fluorophore (ArOH) into an enzymatic substrate designed at targeting aminopeptidase activity. Substrates of the general structure ArO-spacer-AA were therefore synthesised (AA = amino acid). The action of aminopeptidases on these substrates would therefore result in fragmentation, liberating the phenolic fluorophore. The spacer group of choice was para-aminobenzyl alcohol (PABA), as it has been widely used as such for the synthesis of prodrugs. After condensation of the amine group of PABA with either Boc-L-alanine or pyroglutamic acid and subsequent chlorination of the benzylic alcohol giving the corresponding benzyl chloride, 2-(2-hydroxyphenyl)benzothiazole and 2-(2-hydroxyphenyl)benzoxazole were attached using a Williamson ether synthesis to produce the Boc-protected substrates. After removal of the Boc groups, the substrates produced strongly fluorescent colonies with Gram-negative bacteria. Also investigated were a series of fluorogenic substrates, both with and without ‘spacer’ methodology, designed for the detection of nitroreductase activity but little activity was observed with bacteria. Three BODIPY-derived substrates were also prepared and evaluated for detecting nitroreductase and esterase activity. These substrates showed good activity in agar media when the agar plates were post-treated with acid resulting in the generation of strong colour and fluorescence. The second section of this thesis focuses on antimicrobial susceptibility testing. Two types of substrates possessing highly conjugated thiophene cores were synthesised: hydrazides and D-amino acids. The hydrazides are expected to label dead cells by reacting with aldehyde groups that are produced by protein carbonylation. The D-amino acid series of compounds are expected to be incorporated into the cell walls of growing bacteria, and hence live cells can be labelled. The thiophene core structures were chosen to have excitation wavelengths of approximately 488 nm because this excitation wavelength is commonly used in flow cytometry. Results from fluorescence measurement testing indicate that both sets of cell labelling substrates synthesised have an excitation wavelength of roughly 450 – 460 nm with emission wavelengths ranging from 480 nm to as high as 520 nm.

This study aims to examine the role infection plays in the pathogenesis of atherosclerosis through the molecular identification of latent bacterial species present in atherosclerotic plaque tissue from the carotid artery. Immunohistological examination of the atherosclerotic plaque tissue revealed widespread localisation of the bacterial cell wall constituent peptidoglycan (PGN). Targeted PCR amplification of the 16S rRNA gene permitted identification of a diverse collection of 16S rDNA sequences within atherosclerotic plaque samples. Thirteen of 21 (61.9%) plaques contained bacterial 16S rDNA relating to periodontal (P. gingivalis and T. forsythia), oral (Streptococcus spp.), and respiratory (Klebsiella pneumoniae and Mycobacterium tuberculosis) and commensal (Propionibacterium acnes and Staphylococcus epidermidis) species. Of the 160 isolates recovered P acnes (91%) was most frequently detected, followed by Lactobacillus spp. (3%), S. epidermidis (3%), S. mitis (3%) and S. sanguinis (1%). Challenge of THP-1 cells with a P. acnes isolate recovered from atherosclerotic plaque, resulted in significant temporal up-regulation the major cholesterogenic transcriptions factor, SREBP2 and its two target transcripts, LDLR and HMGR. Similarly, the cholesterol transporter gene ABCA1 was highly expressed in P. acnes-infected THP-1 cells in addition to inflammatory cytokines/chemokines TNFα, IL-1β, CCL3 (MIP-1α), cell adhesion molecule ICAM-1. Anti-apoptotic gene BCL2A1 showed the greatest increase in mRNA compared to all tested genes. Expression of the same panel of genes direct in atherosclerotic plaque tissue resulted in similar expression patterns.

Microbial diversity encompasses the whole of the Earth’s biosphere and is incredibly vast. The microbial diversity of three disparate micro-environments using two culture-independent techniques (denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing) were revealed. Five commercially available DNA polymerase (pol) enzymes were assessed in determining the bacterial community generated in sandy soil. The V3 region of the 16S rRNA gene was targeted for amplification by polymerase chain reaction (PCR). Using a PCR-DGGE approach, different DNA pols exhibited differences in the DGGE profiles produced. Both high-fidelity DNA pols Ex Taq™ Hot Start (HS) and Platinum® Pfx detected greater microbial diversity present within sandy soil than the other DNA polymerase enzymes. We employed Ex Taq™ HS to characterise the microbial communities present in two chronic respiratory tract diseases, non-cystic fibrosis bronchiectasis (nCFBR) and chronic obstructive pulmonary disease (COPD). Seventy individuals expectorated sputum, and using 16S and 28S rRNA PCR-DGGE polymicrobial communities were revealed. From the 70 patients investigated, 20 presented with symptoms consistent with an exacerbation, the remainder being clinically stable. Demographic and culture data were used in constrained ordination analyses to identify any significant associations between these data and changes in the sputum microbiota. The data presented indicates that bacterial lung communities in adult nCFBR patients have distinct differences between exacerbating and clinically stable episodes. Persistent colonisation by Pseudomonas aeruginosa is significantly associated with reduced lung function, and is negatively correlated with Haemophilus influenzae carriage. Bacterial communities seem to be predominantly assembled by stochastic processes. Fungal taxa present were scarce. Stable COPD populations have been previously investigated using culture-dependent techniques. Eleven clinically stable COPD patients had a bronchoalveolar lavage (BAL) fluid taken from the right lower lobe. Both 16S and 28S rRNA PCR-DGGE was performed on all clinical samples from extracted DNA. Co-migration of bands was then compared to a 16S and 28S standard ladder consisting of pure cultivars. Additionally, execution of 454-pyrosequencing and interrogation of the V3-V5 region of 16S rRNA genes resulted in 1799 unique OTUs being identified. Dominant bacterial genera identified were Streptococcus, Arthrobacter, and Staphylococcus respectively. Bacterial taxa identified were then subjected to multivariate statistical analysis to identify relationships between the microbial communities and patient phenotypes. Metagenomic analysis demonstrated that heterogeneous bacterial populations exist in all eleven individuals. This preliminary study shows that the lungs of COPD sufferers are colonised with multiple species of bacteria and demonstrate that a complex microbial community is present. Furthermore, bacterial phylotypes resolved to class-level indicated three potential drivers of community structure within the COPD lung microbiome: lung function, moderate and severe COPD progression, and smoking status in cohort. The identification of a greater number of bacterial taxa was also apparent in culture-negative patients using both PCR-DGGE and 454-pyrosequencing approaches.