Journal of Clinical and Diagnostic Research. 2022 Nov, Vol-16(11): DC16-DC20 16 16 DOI: 10.7860/JCDR/2022/57824.17135 Original Article Microbiology Section Successful Isolation of Burkholderia pseudomallei from Soil by Extended Incubation of Ashdown’s Agar: A Cross-sectional Study INTRODUCTION Melioidosis is an emerging infection of humans and animals caused by Burkholderia pseudomallei , a Gram negative, motile, non spore-forming environmental bacterium [1,2]. Although originally prevalent in Southeast Asia and Northern Australia, melioidosis cases are now being progressively identified in almost all other continents [3]. Based on epidemiological modelling, the global incidence of melioidosis is ~ 165,000 and in India ~52,500 cases [4]. This highly pathogenic bacterium is commonly found as a saprophyte in the soil and hence acquiring infection is likely among those exposed to such an environment. A higher incidence of melioidosis is reported during monsoon when the subterranean organisms migrate to the surface [1]. The presence of non cultivable forms as well as the existence of a non virulent, closely related species, B. thailandensis makes isolation of B. pseudomallei from the soil a challenging task [5,6]. Both conventional culture-based methods and molecular tests using PCR have been employed for this purpose [7]. Ashdown’s agar is the selective medium used for the isolation of B. pseudomallei . The presence of crystal violet and gentamicin inhibits the growth of unwanted bacteria. Glycerol helps to enhance the growth of B. pseudomallei . Both neutral red and crystal violet give B. pseudomallei colonies a characteristic purple colour [7]. Additionally, B. pseudomallei can be detected using specific PCR assay targeting a Tat domain protein [7]. Although environmental B. pseudomallei has been isolated from other parts of the world, there are not many studies reported from India. There are only three reports of successful isolation, one from Cuddalore (Tamil Nadu), the second from the Malabar coastal region of Kerala and third from Mangalore (Karnataka). Among these Tamil Nadu and Karnataka are highly endemic areas for the disease [8-10]. Prior exposure to B. pseudomallei in the study population was documented by a seroprevalence of 19.8% among the high-risk population [11]. So far, no attempts have been made to study the environmental distribution of B. pseudomallei. This information is critical in identifying high-risk zones for acquiring melioidosis. Thus, the aim of the study was to isolate B. pseudomallei from soil surrounding the residence and workplaces of patients diagnosed with melioidosis by culture. MATERIALS AND METHODS A cross-sectional study was carried out from July 2018 to January 2021 at JIPMER, Puducherry, India. This study was approved by the Institutional Ethics Committee for Human Studies, JIPMER, Puducherry, India, (JIP/IEC/2018/0230) and written informed consent was obtained from the occupants to collect the soil samples. Selection of Study Sites and Method of Sampling Seven locations in and around Puducherry were sampled between 2018 and 2021 during the dry and wet seasons [Table/Fig-1] [12]. From each site, soil samples were collected from the residence of the patients and /or their workplace [Table/Fig-2] following a standard technique depending on the total area of house/field [13]. Based on the site, ten or more sampling points were selected maintaining a distance of 2.5 m and 5 m between the sampling points, from residence and/or workplace respectively [Table/Fig-2]. With the use of protective gear (masks, gloves and rubber boots) soil samples were collected from a depth of 30 cm from the surface, using a SRUTHI RAJ 1 , SUJATHA SISTLA 2 , RAJESH AMBERPET 3 , SREERAM CHANDRA MURTHY PEELA 4 Keywords: Environment, Gram negative, Melioidosis ABSTRACT 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.