Comparison of rapid, automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei.
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.
Inglis
Timothy J J
TJ
Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology, Nedlands, Australia. tim.inglis@health.wa.gov.au
O'Reilly
Lyn
L
Foster
Niki
N
Clair
Adele
A
Sampson
Judy
J
D001331:Automation; D015373:Bacterial Typing Techniques; D016957:Burkholderia pseudomallei / Q000145:classification* / Q000235:genetics; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D008554:Melioidosis / Q000382:microbiology*; D015183:Restriction Mapping / Q000379:methods*; D021521:Ribotyping
D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI