Bacteriophage of Burkholderia pseudomallei: friend or foe?
Other Publication ResearchOnline@JCUAbstract
Lysogenic bacteriophage carrying virulence determinants have been demonstrated to be responsible for the pathogenicity of many bacteria. Bacteriophage, or components of bacteriophage, have also been successfully used in the treatment of bacterial infections. Burkholderia pseudomallei is the causative agent of melioidosis and has been shown to carry bacteriophage. The role of bacteriophage in virulence of B. pseudomallei isolates has not yet been determined, nor have bacteriophage been examined for their potential in treatment of melioidosis. A screen for identification of bacterial isolates of interest was developed and 50 isolates were examined. Thirty-one selected isolates were then examined for bacteriophage using techniques including; transmission electron microscopy (T.E.M), mitomycin C assay, UV assay, plaque assay and restriction digestion assay. A combination of mitomycin C assay and either plaque assay or restriction digestion assay were determined to be 96.77% accurate for testing for bacteriophage in B. pseudomallei isolates. Five techniques for the concentration of bacteriophage (commercial Qiagen kit, magnesium hydroxide precipitation, PEG precipitation, zinc chloride precipitation, ultracentrifugation) were examined and ultracentrifugation determined to be the best. Two methods of DNA extraction (commercial nucleobond AX kit, phenol chloroform extraction) were compared and a phenol chloroform extraction was modified for use. A bacteriophage amplification system involving inoculation of bacteriophage into a broth of host B. pseudomallei, followed by lysis, was developed and optimised for production of lysogenic bacteriophage of B. pseudomallei. Addition of a 1:1 dose of bacteriophage to bacteria at an O.D.600nm of 0.1 in 10-100ml of broth resulted in the production of 1x1011plaque forming units (pfu)/ml of media upon lysis at 7.5 hours post-inoculation. Lysogenic bacteriophage extracted from highly virulent B. pseudomallei isolate NCTC 13178 was given the name BupsM1 and was characterised as being from the family Myoviridae with a genome 55.1kb long. This bacteriophage was then used for infection assays and molecular analysis to determine whether it played a role in virulence. Endolysin of this bacteriophage was also extracted to determine its potential for use in therapy. Four B. pseudomallei isolates tested negative for the presence of bacteriophage (#13, #69, #83, E4) and one isolate of particular interest (NAFC), were infected with BupsM1. Bacteriophage infection was found to alter colonial morphology on Ashdown agar. Infection assays in a BALB/c mouse model were carried out and no clear relationship between addition of bacteriophage BupsM1 and virulence was found. One experiment with NAFC resulted in greatly increased virulence, but this could not be repeated. All other experiments where infection with bacteriophage was successful resulted in minor upregulation or downregulation of virulence. Examination of plaque production of infected and control isolates indicated that prophage stability may play a role in survival of B. pseudomallei as addition of bacteriophage from NCTC13178 restored lysogenic stability to NAFC in several cases. Of the expected 55.1kb genome size from BupsM1, 51.3kb was sequenced with 40.9kb of this confirmed as bacteriophage. The open reading frames were determined using ORF finder and direct analysis. These open reading frames were analysed by BLASTx for putative function and several potential virulence genes were identified, as were structural, replication and lysogeny genes. Possible virulence genes include putative anaerobic dehydrogenase and oxidoreductase genes. Putative structural genes included the terminase large subunit, portal protein, head morphogenesis, tail assembly and tail fibre genes. Putative replication and lysogeny genes included transposases, insertion elements and integrase, an RNA polymerase sigma subunit, DNA cytosine methylase, Holliday junction resolvase, repressor protein, and a weak match to cro, the gene responsible for triggering lysis. Two genes of interest, the endolysin gene and a possible ADP-ribosyltransferase gene (a gene often involved in virulence) were not identified by BLASTx analysis. Techniques designed to identify genes with limited amino acid homology across species, such as identification of conserved amino acid pattern, chemo-physical comparison and phylogenetic tree analysis including bootstrap scoring, were then used to identify several open reading frames which were possible matches to these previously unidentified genes. The endolysin of BupsM1 was extracted under nine combinations of conditions from literature , using a natural host system (B. pseudomallei #4). EDTA was found to aid lysis, while chloroform was found to have no effect. Extracts were concentrated using Centricons™ and both neat and concentrated extracts were tested for their ability to lyse both killed and live B. pseudomallei #4 in broth and plate format. Neither the extracted endolysin nor its concentrate was found to lyse any of the B. pseudomallei in a form not attributable to live bacteriophage. Hence endolysin was determined not to function “from without” against B. pseudomallei. As such, this possibility for treatment of B. pseudomallei was eliminated.
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