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dc.contributor.authorLangley, Ross Johnen
dc.date.accessioned2018-03-29T12:18:08Z
dc.date.available2018-03-29T12:18:08Z
dc.date.issued2005
dc.identifier.urihttp://hdl.handle.net/1842/29213
dc.description.abstracten
dc.description.abstractPulmonary bacterial infections account for 95% of morbidity and mortality in cystic fibrosis (CF) patients, and include a limited spectrum of bacteria; Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and members of the Burkholderia cepacia complex (Bcc). "B. cepacia" was first recognised in the late 1970's as a cause of life threatening respiratory infections in CF. Initial clinical observations noted that 20% of colonised CF patients developed "cepacia syndrome", a rapid and fatal necrotising pneumonia. In addition, epidemiological evidence highlighted the potential spread of certain "B. cepacia''' strains, that most isolates were highly resistant to conventional antibiotics and consequently, Bcc infections are untreatable. Subsequent taxonomic studies have identified the Bcc that contains ten distinct species ofbacteria previously termed "5. cepacia". Clinical distribution ofBcc species in CF is restricted to mainly B. cenocepacia (50%), B. multivorans (38%) and B. vietnamiensis (7%)en
dc.description.abstractThe aim of this study was to investigate novel antimicrobial agents against the Bcc and other problematic and emerging CF associated bacterial pathogens including multi-resistant epidemic P. aeruginosa strains, methicillin resistant S. aureus (MRSA), and Stenotrophomonas maltophilia. The novel antimicrobial strategies examined were based on three main themes: First, the use of natural honey, second, the potential use of bacteriophage and their associated lytic enzymes and third, novel mammalian cationic ß-defensins. The project utilised a vast collection of bacterial isolates and included relevant clinical, environmental and epidemic strains. The susceptibility to conventional antibiotics was measured, and resistance was shown to vary across the Bcc. In general, clinical isolates were statistically more resistant to conventional antibiotics than environmental isolates.en
dc.description.abstractMembers of the Bcc, and an extended panel ofresistant organisms were shown to be sensitive to New Zealand manuka honey (NZMh). The MICs ranged from 9 to 17% (w/v), and the MBCs ranged from 9 to 20% (w/v). The antimicrobial component of NZMh was investigated, and focussed on osmolality, pH and H₂O₂. All were found to contribute to the antibacterial activity, although none were solely responsible for the activity. Killing-curves suggested that NZMh kills within 24 hours. The NZMh preparation was applied to a CF patient infected with B. cenocepacia J2315, and clinical data highlighted possible benefits to the patient.en
dc.description.abstractNovel Bcc specific bacteriophages were identified from environmental samples and from lysogeny studies. The spectrum of activity of the novel bacteriophages, and previously reported Bcc bacteriophage (NS1 and NS2), was determined using a panel comprising 66 isolates of the Bcc, 55 isolates representing other pseudomonads, and 40 B. pseudomallei strains. The novel phages were shown to be very promiscuous and had activity across the Bcc, with some active against P. aeruginosa, B. gladioli, and B. pseudomallei. The wide spectrum of activity was detrimental to therapeutic use, therefore, the phage-encoded lytic enzymes were the focus for further study: bacteriophage therapy with a novel twist. Two enzymes were investigated: the B. cepacia bacteriophage Bcep781 endolysin and the P. aeruginosa phage D3 endolysin. The Bcep781 phage and phage DNA was not available, therefore the endolysin gene was synthesized using recursive PCR. Briefly, twenty-two overlapping oligonucleotides encoding the entire gene were synthesized and constructed into the endolysin gene using a single PCR reaction. Both genes were cloned into an expression plasmid and the enzymes were recombinantly expressed as 6-His fusion proteins in BL21 E. coli cells. Bcep781 endolysin was purified using nickel-affinity chromatography, and the D3 lysin was purified using a Resource S® purification protocol. High-resolution mass spectrometry analysis highlighted discrepancies in both lysins, and neither proved to active against relevant bacteria tested.en
dc.description.abstractThe activity of cationic antimicrobial peptides (CAMPs) including: a synthetic novel murine ß-defensin (Defrl) with 5-cysteine residues, which forms a covalently bound dimer; its 6-cysteine analog (Def-cys); a chemically reduced Defrl; polymyxin B and colistin; were assessed against the Bcc, as well as several multi-resistant bacterial CF pathogens. Two Bcc isolates, B. cepacia type strain ATCC 25416 and B. cenocepacia type strain J2315, were found to be inherently resistant to all CAMPs utilised in this study. Epidemic P. aeruginosa isolates were found to have a MIC of 6 μg/ml for Defrl and a MIC of 50-100 μg/ml for Def-cys, suggesting a possible relationship between defensin structure and function. Similarly, the MIC of 6 μg/ml was also noted for S. maltophilia and Ralstonia sp. that were found to be resistant to polymyxin B and colistin. The recombinant production of Defrl was also attempted in a number of expression systems in E. coli. However, although Defrl was successfully expressed, the recombinant proteins were highly insoluble. This study showed that resistance varies within the Bee. However, the data show that NZMh exerts a bactericidal effect on members of the Bcc, including B. cenocepacia J2315, and that such activity may be utilised clinically. The novel Bcc bacteriophage may prove to be a useful panel for further study, either as vectors for horizontal gene transfer or as therapeutic agents. The data confirm previous observations that the Bcc are inherently resistant to CAMPs, including a novel 5- cysteine defensin. Despite this finding, synthetic Defrl was shown to be active against a panel of multi-resistant pathogens associated with infections in CF. Further research is required to optimise the recombinant expression of Bcep781 endolysin, D3 lysin, and Defrl, to enable their use in the treatment of multiply resistant infections in CF and the wider hospital environment.en
dc.publisherThe University of Edinburghen
dc.relation.ispartofAnnexe Thesis Digitisation Project 2018 Block 17en
dc.relation.isreferencedbyAlready catalogueden
dc.titleNovel agents with inhibitory activity against the Burkholderia cepacia complexen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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