Evaluation of culture- and PCR-based methods for detecting Burkholderia pseudomallei in soil samples in Thailand

Abstract

BACKGROUND: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a life-threatening disease prevalent in tropical regions. Accurate detection of B. pseudomallei in the environment is essential for identifying areas that pose an infection risk. This study aimed to evaluate culture- and real-time polymerase chain reaction (PCR)-based methods for detecting B. pseudomallei directly in soil samples, and to assess its environmental prevalence in relation to antibody levels in healthy individuals from Amnat Charoen, Northeast Thailand. METHODOLOGY/PRINCIPAL FINDINGS: Initial studies used spiked soil samples to evaluate culture-based methods including Ashdown agar, phosphate-buffered acidic erythritol (ACER) agar, Threonine-basal salt solution with colistin 50 (TBSS-C50) broth, TBSS-C50 based erythritol broth, and ACER broth, and real-time PCR assays targeting BPSS1187 and TTS1-orf2. Hemolysin coregulated protein 1 (Hcp1)-specific antibodies were measured in 398 plasma samples using an enzyme-linked immunosorbent assay. Based on these antibody titers, 238 soil samples were collected from the households of individuals that were strongly positive (N = 6) and negative (N = 6) for Hcp1-specific antibodies. B. pseudomallei was isolated from 3.36% (8/238) of soil samples by direct culture on Ashdown agar and from 5.46% (13/238) after enrichment in ACER broth followed by culture on Ashdown agar. Real-time PCR assays confirmed the presence of B. pseudomallei in 2 out of 20 culture-positive soil samples. Overall, B. pseudomallei was detected in 14 of 129 samples (10.9%) from households of seropositive individuals and in 6 of 108 samples (5.5%) from households of seronegative individuals (P = 0.165). CONCLUSIONS/SIGNIFICANCE: This study demonstrates the importance of using effective methods for detection of B. pseudomallei in environmental samples. For culture-based approaches, enrichment in ACER broth followed by subculture on Ashdown agar demonstrated enhanced bacterial recovery compared to direct culture on Ashdown agar alone. There was limited detection of B. pseudomallei in soil by real-time PCR, and lack of an association between environmental detection methods and human seroprevalence. These findings highlight the need for integrated, multi-dimensional surveillance approaches.