Comparison of Rapid, Automated Ribotyping and DNA Macrorestriction Analysis ofBurkholderia pseudomallei

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Abstract
An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection ofBurkholderia pseudomalleiisolates. In a preliminary evaluation with the restriction enzymesBamHI andEcoRI, the protocol withEcoRI was more discriminating. The reproducibilities of the ribotypes obtained withEcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library ofB. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis ofXbaI digests by pulsed-field gel electrophoresis. The typeability ofB. pseudomalleibyEcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record ofB. pseudomalleiisolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release ofB. pseudomalleias a biohazard.