Detection of Burkholderia pseudomallei in soil from Gazipur and Chattogram districts, Bangladesh using an optimized enrichment and PCR-based approach

Melioidosis is a zoonotic infectious disease caused by Burkholderia pseudomallei which can be potentially fatal, and affect both humans and certain animals. Historically, culture methods have been found to be less sensitive than molecular methods such as quantitative polymerase chain reaction (PCR) for detection of B. pseudomallei, therefore an improved method for detection of the organism from environment was sought in the present study. In the first phase of this study, optimization of processing methods for soil samples prior to analysis with a PCR assay were investigated to aid in improvement of the detection of B. pseudomallei from soil. These methods included addition of an enrichment step and testing of two methods to extract DNA from the samples. In the second phase, the optimized method was applied for identification of the organism from environmental soil. To optimize detection, sterilized and unsterilized household soil samples were spiked with B. pseudomallei (4×10⁶ to 4×10⁰ CFU/gm) and enriched in Ashdown’s selective broth. Samples were tested by culture and conventional PCR, followed by gel electrophoresis. The optimized methods were then applied to 80 soil samples from Gazipur and Chattogram districts. In first phase, PCR detected Burkholderia pseudomallei in all sterile and unsterile spiked enriched soil using a commercial DNA extraction kit, outperforming the boiling method. In sterile enriched soil, PCR showed 100% sensitivity and NPV with 100% accuracy. In unsterile enriched soil, PCR maintained 100% sensitivity but showed low specificity (14.29%) and accuracy (22.69%) due to detection of non-culturable forms missed by culture. In the second phase, application of the optimized PCR and culture techniques to 80 environmental samples from Gazipur and Chattogram districts revealed B. pseudomallei DNA in 3.3% and 16% of samples, respectively, though no isolates were recovered by culture. PCR detection of Burkholderia pseudomallei in soil samples suggests a potential environmental reservoir for melioidosis in affected districts. Higher detection rates in agricultural areas, especially rice fields, indicate that soil factors such as pH, moisture, and organic content influence its distribution. These findings support the integration of ecological data into surveillance and raise public health awareness in Bangladesh. • PCR showed higher sensitivity than culture for detecting B. pseudomallei in spiked enriched soil • False positives suggest PCR detected DNA from dead or non-culturable organisms • Culture failed to isolate B. pseudomallei in several PCR-positive samples • PCR detection rate following enrichment was 11.25% in soil samples from Gazipur and Chattogram districts in Bangladesh • Study recommends using qPCR or RNA-based methods for improved surveillance