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Successful Isolation of Burkholderia pseudomallei from Soil by Extended Incubation of Ashdown’s Agar: A Cross-sectional Study

Introduction: Melioidosis is an infectious disease of humans and animals caused by an environmental saprophyte Burkholderia pseudomallei. Although the organism is associated with soil and water, environmental isolation is rarely successful which could be due to the existence of viable but non-culturable forms. Aim: To isolate B. pseudomallei from the soil to detect the environmental presence of this organism in and around Puducherry, India. Materials and Methods: A descriptive cross-sectional study was carried out from July 2018 to January 2021 at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India. A total of 473 soil samples were collected from areas surrounding the residence and workplaces of seven culture-proven melioidosis cases, from Puducherry and three districts of Tamil Nadu (Cuddalore, Nagapattinam and Villupuram) during the dry and wet seasons. Soil samples were enriched in Ashdown’s broth and cultured on Ashdown’s agar. The plates were incubated at 37°C and examined daily for seven days with a further extended period of incubation till the tenth day for samples that did not show growth. Suspected isolates were subjected to Vitek 2 system for biochemical identification. Confirmation of the isolates was carried out by antigen detection and Polymerase Chain Reaction (PCR). Results: From 473 soil samples processed, bacteria with colony morphology similar to B. pseudomallei were isolated in 56 (11.83%) samples. Only one isolate, which was detected on the tenth day of incubation was confirmed as B. pseudomallei using antigen detection and PCR. This sample was collected during the wet season (December 2020) from Endur, in the Villupuram district of Tamil Nadu, India. Conclusion: The study findings highlight the importance of extended incubation of culture plates at 37°C for up to ten days to improve the chances of isolation from the soil.