Erythritol as a single carbon source improves cultural isolation of Burkholderia pseudomallei from rice paddy soils

Abstract

BackgroundIsolation 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 findingsBased 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/significanceIn 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.

A Defined Medium for Cultivation and Exometabolite Profiling of Soil Bacteria

“…Using a defined medium also facilitates detection of microbial secreted products. Defined media designed for isolation often have a single substrate or nutrient source to select for specific organisms, which can be a complex substrate ( Davis et al, 2005 ; Trinh et al, 2019 ). Some defined media contain a synthetic carbon mixture based on soil extract or cell extracts ( Liebeke et al, 2009 ; Baran et al, 2015 ).…”

Environmental Factors Associated With Soil Prevalence of the Melioidosis Pathogen Burkholderia pseudomallei: A Longitudinal Seasonal Study From South West India

“…A period of 48 h of incubation in Ashdown broth (Ashdown, 1979) were followed by a 96 h incubation in a minimal medium based on TBSS-C50 (Galimand and Dodin, 1982) with erythritol as a single carbon source. At this time, the erythritol-based medium was under development and differed from the version published later (Trinh et al, 2019) by the N source (0.1% ammonium chloride) and the addition of a diverse set of antibiotics (32 mg/L tobramycin, 50 mg/L colistin, 16 mg/L norfloxacin, 50 mg/L gentamicin, and 50 mg/L cycloheximide) (Goodyear et al, 2013). Enrichments were subcultured in serial dilutions on Ashdown agar.…”

“…Reliance on culture protocols to identify factors associated with the abundance of B. pseudomallei in the environment could be imprecise due to the low sensitivity of soil cultures. Previous studies have clearly shown that the current consensus methods for the culture (Limmathurotsakul et al, 2013) detection of B. pseudomallei lack sensitivity, leading to false-negative results (Trung et al, 2011;Gohler et al, 2017;Trinh et al, 2019). A study from Laos showed a higher detection rate of B. pseudomallei in enrichment cultures from soil samples by PCR when compared to B. pseudomallei growth in the respective subcultures (Dance et al, 2018), which is probably due to insufficient selectivity of the culture media used.…”

Development and Validation of a Burkholderia pseudomallei Core Genome Multilocus Sequence Typing Scheme To Facilitate Molecular Surveillance

“…Environmental B. pseudomallei strains were isolated as previously described ( 13 ). For bacterial DNA isolation, the strains were cultivated on Columbia agar supplemented with 5% sheep blood overnight.…”