Highly Sensitive Direct Detection and Quantification of Burkholderia pseudomallei Bacteria in Environmental Soil Samples by Using Real-Time PCR

Abstract

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 ؋ 10 4 genome equivalents (range, 3.65 ؋ 10 2 to 7.85 ؋ 10 5 ) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1-to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.

PCR-based Methodologies Used to Detect and Differentiate theBurkholderia pseudomalleicomplex:B. pseudomallei,B. mallei, andB. thailandensis

“…This false negative was detected by a different qPCR method (Supaprom et al, 2007;Trung et al, 2011). A total of 10 false positive soil samples were observed by the two environmental follow-up studies (Kaestli et al, 2007;Trung et al, 2011). These false positives agreed with other qPCR tests (Kaestli et al, 2007;Supaprom et al, 2007), indicating Novak et al's qPCR sensitivity beyond culture for soil samples.…”

“…The test's accuracy approached 100% (Trung et al, 2011;Kaestli et al, 2012;Price et al, 2012) with only one strain decreasing the assay's sensitivity. This false negative was a B. pseudomallei strain which had a reduced genome that lacked some virulence loci, including orf2 .…”

“…One false negative decreased the assay's sensitivity on soil samples (Trung et al, 2011). This false negative was detected by a different qPCR method (Supaprom et al, 2007;Trung et al, 2011). A total of 10 false positive soil samples were observed by the two environmental follow-up studies (Kaestli et al, 2007;Trung et al, 2011).…”

“…Kaestli et al reported 100% specificity on inoculated soil samples. One false negative decreased the assay's sensitivity on soil samples (Trung et al, 2011). This false negative was detected by a different qPCR method (Supaprom et al, 2007;Trung et al, 2011).…”

“…This false negative was a B. pseudomallei strain which had a reduced genome that lacked some virulence loci, including orf2 . Two follow-up studies evaluated the procedure's ability to be used on environmental samples (Kaestli et al, 2007;Trung et al, 2011). Kaestli et al reported 100% specificity on inoculated soil samples.…”