Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

Abstract

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10(1) cfu g-1 soil and 10(4) cfu g-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.