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Title: Burkholderia cepacia complex bacteria and their antimicrobial activity
Author: Boaisha, Othman
ISNI:       0000 0004 2733 0923
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2012
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Burkholderia cepacia complex (Bcc) are well known for their ability to produce antifungal agents. However, the majority of past studies have screened only a limited number of isolates, which were not taxonomically well characterized. In addition, while the activity of Bcc bacteria against plant pathogenic fungal species has been characterized, very few studies have examined the interaction between Bcc and saprotrophic wood decay basidiomycete species. The overall aim of this study was to begin to the characterization of the interactions of Bcc and other Burkholderia with the model ascomycete Candida albicans and woodland basidiomycete fungi. Methods. The study made use of a large collection (397 isolates) of taxonomically well identified Burkholderia species. A systematic screening of the antifungal activity of this collection was made against C. albicans and various wood decay basidiomycetes: Bjerkandera adusta, Trametes versicolor, Hypholoma fasciculare, Resinicium bicolor and Phanerochaete velutina. A wide range of media were evaluated for Burkholderia growth, antifungal metabolite production and the ability to support the mycelial growth of basidiomycetes. An agar overlay assay was used to examine the anti-candidal activity of Burkholderia isolates, while a novel overlay assay was developed using homogenised mycelial material for the basidiomycete species. Novel and existing extraction techniques were evaluated for their ability to partially purify active Burkholderia metabolites. The purification and identification of the active Burkholderia antibiotics were performed using both preparative thin layer chromatography (TLC) and liquid chromatography combined with mass spectrometry (LC-MS). A novel TLC-bioautography assay was also developed to reveal the presence of active Burkholderia metabolites in the antimicrobial extracts. The minimum inhibitory concentration (MIC) of the semi-purified Burkholderia antifungals was also determined. The natural diversity of Burkholderia and other bacteria associated with different rhizosphere environments was evaluated by cultivating isolates from local maize crops and examining a collection of Burkholderia isolates obtained from the rainforest in Sabah, Malaysia. The genetic basis for the secretion of antifungal agents by B. ambifaria was examined using transposon mutagenesis and screening for the mutants which had lost their anti-candidal activity. Additional transposon mutants that had lost their anti-bacterial activity and had been obtained in a previous screen were also examined for alterations in antifungal activity. Finally, the Burkholderia genome database and bioinformatic techniques were used to genetically characterize these mutants and other genetic loci that had been previously implicated in antibiotic production. Results. The majority of Burkholderia isolates possessed the same antagonistic activity towards C. albicans as they did towards B. adusta. Of the 397 isolates examined, antifungal activity was the greatest in the following Burkholderia species: B. ambifaria, B. cepacia, B. cenocepacia and B. contaminans. Interestingly, no antifungal activity was observed for B. multivorans (27 isolates) and B. stabilis (18 isolates) under the experimental conditions tested. The type of medium used to study the interaction between Burkholderia and the fungi considerably effected antifungal activity, with the most suitable media for studying antagonistic activity being Sabouraud agar and an acidic minimal salts medium (BSM; pH 6) supplemented with 0.4% glycerol. Of the 397 isolates screened, 47.85% were anticandidal (190 isolates; 89.47% of these were Bcc species and 10.53% were other Burkholderia species), while 48.10% were active against B. adusta (191 isolates; 93.19% of these were Bcc species and 6.81% were other Burkholderia). Extraction using Amberlite XAD16 resin binding followed by elution with methanol was an excellent means to isolate active Burkholderia antifungals. Fractionation of these extracts using silica gel TLC and ethyl-acetate:methanol:water (20:1:0.5) as the elution solvent was optimal for the separation of the majority of the active metabolites. A TLC-bioautography assay was developed in this current study as a preliminary screening technique to detect anti-microbial components of novel Burkholderia extracts. The assay was very useful for detecting anti-candidal and anti-bacterial compounds and was adapted for use for the first time with basidiomycetes. Antifungal Burkholderia isolates produced between 1 and 10 antifungal metabolites. Several antifungal agents were identified as enacyloxin, pyrrolnitrin, bactobolin and quinolines. Novel compounds and novel bactobolin derivatives were also present. For B. ambifaria strain AMMD a number of chromosomal loci were found to be involved in the production of antifungal metabolites. Quorum sensing was essential for the expression of antifungal activity as transposon mutations in the CepI synthase-encoding gene prevented the biosynthesis of all B. ambifaria anti-candidal compounds. B. ambifaria AMMD extracts containing enacyloxin IIa also produced an inhibitory effect at low concentration against E. coli, B. multivorans and A. baumannii. Conclusions. Burkholderia are potentially a very large reservoir of known and novel antimicrobial agents, especially with nearly 50% of isolates screened exhibiting antifungal activity. Antimicrobial compounds such as enacyloxin IIa were discovered for the first time to be produced by Burkholderi and novel extraction and bioassay methods for anti-fungals were developed in this study. Overall, we now have the tools to considerably advance the isolation and characterization of antifungal Burkholderia metabolites.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology