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Background: Isolation of the soil bacterium Burkholderia pseudomallei from tropical environments is important to generate a global risk map for man and animals to acquire the infectious disease melioidosis. There is increasing evidence, that the currently recommended soil culture protocol using threonine-basal salt solution with colistin (TBSS-C50) for enrichment of B. pseudomallei and Ashdown agar for subsequent subculture lacks sensitivity. We therefore investigated, if the otherwise rarely encountered erythritol catabolism of B. pseudomallei might be exploited to improve isolation of this bacterium from soil., Methodology/principal Findings: Based on TBSS-C50, we designed a new colistin-containing medium with erythritol as the single carbon source (EM). This medium was validated in various culture protocols by analyzing 80 soil samples from 16 different rice fields in Vietnam. B. pseudomallei enrichment was determined in all culture supernatants by a specific quantitative PCR (qPCR) targeting the type three secretion system 1. 51 out of 80 (63.8%) soil samples gave a positive qPCR signal in at least one of the culture conditions. We observed a significantly higher enrichment shown by lower median cycle threshold values for B. pseudomallei in a two-step culture with TBSS-C50 for 48 h followed by EM for 96h compared to single cultures in TBSS-C50 for either 48h or 144h (p<0.0001, respectively). Accordingly, B. pseudomallei could be isolated on Ashdown agar in 58.8% (30/51) of samples after subcultures from our novel two-step enrichment culture compared to only 9.8% (5/51) after standard enrichment with TBSS-C50 for 48h (p<0.0001) or 25.5% (13/51; p<0.01) after TBSS-C50 for 144h., Conclusions/significance: In the present study, we show that specific exploitation of B. pseudomallei metabolic capabilities in enrichment protocols leads to a significantly improved isolation rate of this pathogen from soil compared to established standard procedures. Our new culture method might help to facilitate the creation of environmental risk maps for melioidosis in the future., Competing Interests: The authors have declared that no competing interests exist.

The soil bacterium Burkholderia pseudomallei causes melioidosis, a potentially fatal and greatly underdiagnosed tropical disease. Detection of B. pseudomallei in the environment is important to trace the source of infections, define risk areas for melioidosis and increase the clinical awareness. Although B. pseudomallei polymerase chain reaction (PCR)-based environmental detection provides important information, the culture of the pathogen remains essential but is still a methodological challenge. B. pseudomallei can catabolize erythritol, a metabolic pathway, which is otherwise rarely encountered among bacteria. We recently demonstrated that replacing threonine with erythritol as a single carbon source in the pH-neutral threonine-basal salt solution (TBSS-C50) historically used improved the isolation of B. pseudomallei from rice paddy soils. However, further culture medium parameters for an optimized recovery of B. pseudomallei strains from soils are still ill-defined. We, therefore, aimed to design a new erythritolbased medium by systematically optimizing parameters such as pH, buffer capacity, salt and nutrient composition. A key finding of our study is the enhanced erythritol-based growth of B. pseudomallei under acidic medium conditions. Our experiments with B. pseudomallei strains from different geographical origin led to the development of a phosphate-buffered acidic erythritol (ACER) medium with a pH of 6.3, higher erythritol concentration of 1.2%, supplemented vitamins and nitrate. This highly selective medium composition shortened the lag phase of B. pseudomallei cultures and greatly increased growth densities compared to TBSS-C50 and TBSS-C50-based erythritol medium. The ACER medium led to the highest enrichments of B. pseudomallei as determined from culture supernatants by quantitative PCR in a comparative validation with soil samples from the central part of Vietnam. Consequently, the median recovery of B. pseudomallei colony forming units on Ashdown's agar from ACER subcultures was 5.4 times higher compared to TBSS-C50-based erythritol medium (p = 0.005) and 30.7 times higher than TBSS-C50 (p < 0.001). In conclusion, our newly developed ACER medium significantly improves the isolation of viable B. pseudomallei from soils and, thereby, has the potential to reduce the rate of false-negative environmental cultures in melioidosis risk areas. [ABSTRACT FROM AUTHOR]