Comparative evaluation of sample preparation and enrichment methods for culture and molecular detection of Burkholderia pseudomallei in naturally contaminated soils from Thailand over one year.

Abstract

Background Burkholderia pseudomallei is a soil-dwelling bacterium that causes melioidosis. Accurate environmental detection of B. pseudomallei is critical for surveillance strategies and for tracing the source of human infection. However, the detection of B. pseudomallei in soil samples, which are complex matrices with a high microbial background, remains difficult. This study aimed to compare the culture and the molecular detection of B. pseudomallei with or without pre-enrichment in naturally contaminated soils from Thailand. Methodology/principal findings In 2025, a total of 640 soil samples were collected from 10 sites from different environments and sampling periods in Amnat Charoen Province. The soils were evaluated for the presence of B. pseudomallei either without enrichment or following enrichment in Ashdown or ACER broths, using culture and real-time PCR detection methods. qPCR consistently identified a higher number of B. pseudomallei positive samples than culture regardless of the pre-treatment method and sampling period. ACER enrichment nearly doubled the odds of detection compared with no enrichment (OR = 1.90) or with Ashdown enrichment (OR = 2.09), with a model-estimated mean Ct difference of 13.6 between ACER enrichment and no enrichment. Overall, qPCR yielded higher detection rates than culture for B. pseudomallei in soil samples, particularly following enrichment. Conclusions/significance This study highlights the importance of pre-enrichment protocols with detection techniques to successfully identify B. pseudomallei in environmental samples. For both culture and molecular detection methods, B. pseudomallei was better detected when the soil samples were enriched in ACER broth compared to Ashdown broth and without enrichment. These findings highlight the need for combining optimal selective enrichment strategies with detection techniques for environmental surveillance. Although ACER enrichment improved the recovery of viable strains, culture recovered B. pseudomallei from only a fraction of qPCR-positive samples, highlighting the need for more selective media to facilitate isolation of B. pseudomallei.