Journal of applied microbiology · 2009
Phage-mediated bioluminescent detection of Bacillus anthracis.
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Schofield D A, Westwater C
Аннотация
AIMS: Bacillus anthracis, the causative agent of anthrax, is a serious human pathogen. The aim of this study was to provide the proof of principle results for the development of a 'bioluminescent' reporter bacteriophage that was capable of specifically detecting B. anthracis.
METHODS AND RESULTS: The reporter phage was engineered by integrating the bacterial luxA and luxB reporter genes into a nonessential region of the lysogenic Wbeta phage genome. This resulted in a phage that was capable of specifically infecting and conferring a bioluminescent phenotype to B. anthracis viable cells. No processing or cell preparation was required; the phage and cells were simply mixed, and the samples were analysed for bioluminescence. A bioluminescent signal was evident after 16 min postinfection of vegetative cells. The strength and time required to generate the signal was dependent on the number of cells present. Nevertheless, 10(3) CFU ml(-1) was detectable within 60 min. The utility of the bioluminescent phage was analysed using spores as the starting material. The Wbeta::luxAB phage was able to transduce a bioluminescent signal to germinating spores within 60 min.
CONCLUSIONS: This proof of principle study demonstrates that the reporter phage displays promise as a tool for the rapid detection of B. anthracis.
SIGNIFICANCE AND IMPACT OF THE STUDY: The new methodology offers the potential for the detection of viable cells from either environmental or clinical samples.
METHODS AND RESULTS: The reporter phage was engineered by integrating the bacterial luxA and luxB reporter genes into a nonessential region of the lysogenic Wbeta phage genome. This resulted in a phage that was capable of specifically infecting and conferring a bioluminescent phenotype to B. anthracis viable cells. No processing or cell preparation was required; the phage and cells were simply mixed, and the samples were analysed for bioluminescence. A bioluminescent signal was evident after 16 min postinfection of vegetative cells. The strength and time required to generate the signal was dependent on the number of cells present. Nevertheless, 10(3) CFU ml(-1) was detectable within 60 min. The utility of the bioluminescent phage was analysed using spores as the starting material. The Wbeta::luxAB phage was able to transduce a bioluminescent signal to germinating spores within 60 min.
CONCLUSIONS: This proof of principle study demonstrates that the reporter phage displays promise as a tool for the rapid detection of B. anthracis.
SIGNIFICANCE AND IMPACT OF THE STUDY: The new methodology offers the potential for the detection of viable cells from either environmental or clinical samples.