Genotype analysis of Burkholderia pseudomallei using randomly amplified polymorphic DNA (RAPD): indicative of genetic differences amongst environmental and clinical isolates

Burkholderia pseudomallei is the causative agent of melioidosis, an infecti ous disease common in the tropics. Melioidosis is most prevalent in the nor theastern part of Thailand. The diseases has diverse clinical manifestation s ranging from mild localized to fatal septicemic forms. The bacterial gene tic factors contributing to the severity of melioidosis have not been compl etely identified. We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis. Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B. pseudomal lei isolated from five patients with localized and four with septicemic mel ioidosis. The: RAPD patterns were analyzed by polyacrylamide gel electropho resis using a laser based automated fragment analyzer, GS2000. Based upon t he pattern complexity, seven pairs consisting of eight primers were chosen for further analysis. Six hundred and thirty-two samples, including duplica tes/triplicates, of B. pseudomallei isolated from melioidosis patients and the environment were analyzed. Two controls were included in each run of th e test samples. All the samples were tested and patterns analyzed by blinde d technical staff. Apparently, the method is reproducible. This is indicate d by the RAPD patterns of the two controls of between run assay. Interestin gly, some RAPD patterns were more prevalent in the clinical isolates than t he environmental specimens and vice versa. For example, Q162KKU4-0 and Q162 KKU1-0 were found 3.5 and 3.3 times more often in the clinical specimens (P < 0.025). Likewise, Q162KKU1-1 and Q167KKU4-1 were found 18 and 37 times m ore often in the environment (P < 0.770001). In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patte rns: RAPD patterns of B. pseudomallei that were found frequently in septice mic patients were less likely to be arabinose positive. The data suggest th e existence of bacterial genetic differences between the clinical and envir onmental isolates of B. pseudomallei. Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative ge nomic analysis of B. pseudomallei in accordance with the clinical data shou ld reveal genetic factors involved in severity and bacterial pathogenesis o f B. pseudomallei in melioidosis. (C) 2000 Published by Elsevier Science B. V.