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Journal of Clinical Microbiology, September 2002, p. 3198-3203, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3198-3203.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Timothy J. J. Inglis,1,2* Lyn O'Reilly,1 Niki Foster,2 Adele Clair,1 and Judy Sampson1,{dagger}

Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology and Medical Research,1 Department of Microbiology, University of Western Australia, Nedlands, Western Australia, Australia2

Received 28 February 2002/ Returned for modification 5 April 2002/ Accepted 23 May 2002

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (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 of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI 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 of B. pseudomallei isolates 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 of B. pseudomallei as a biohazard.

* Corresponding author. Mailing address: Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology and Medical Research, Locked Bag 2009, Nedlands, W.A. 6009, Australia. Phone: 618-9346-3461. Fax: 618-9381-7139. E-mail: tim.inglis{at}health.wa.gov.au.

{dagger} Deceased.

Journal of Clinical Microbiology, September 2002, p. 3198-3203, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3198-3203.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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