Rational design of an acidic erythritol (ACER) medium for the enhanced isolation of the environmental pathogen Burkholderia pseudomallei from soil samples

Abstract

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 erythritol-based 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.

Keywords: Burkholderia pseudomallei; culture medium; detection; environment; soil.

Figure 1

Schematic overview of the experimental setup. Twenty soil samples collected in Vietnam in October 2022 were enriched in ACER, TBSS-C50-based erythritol medium and TBSS-C50 in a single incubation step for 9  days, shaken and statically, and in a 2-step incubation for 2  days followed by 7  days under static conditions. After 9  days, 500 μL aliquots of each culture were taken for B. pseudomallei-specific qPCR and for cultivation on Ashdown agar. Samples were preserved either at −80°C as cell pellets for qPCR analyses or as glycerol stocks of 25% glycerol for cultivation.

Figure 2

Effect of pH and buffer concentration on B. pseudomallei growth in erythritol medium. (A) Growth of strain K96243 in 25 mL of TBSS-C50-based erythritol medium adjusted to pH 5.5, 6.0, 6.3, 6.5, and 7.2 at 40°C and 120 rpm. (B–D) Cultures of B. pseusomallei strain K96243 grown with either 13 or 50 mM phosphate buffer at pH 6.0 (B), 6.3 (C) and pH 7.2 (D). OD₆₀₀ data are plotted on the left axis (note the broken axis highlighted by a dashed line and different scales), pH values on the right axis. Growth curves are representative of at least two independent experiments, each experiment of which was conducted in technical duplicates. Error bars denote the standard deviation of mean from technical duplicates of a single experiment.

Figure 3

Effect of erythritol concentration and vitamins on B. pseudomallei growth. (A) B. pseudomallei K96243 cultivated in 25 mL TBSS-C50-based erythritol medium at pH 6.3 with 50 mM potassium phosphate buffer and varying erythritol concentrations (0.4, 0.8, 1.2, 1.6, and 2%) for 144 h at 40°C and 120 rpm. (B) B. pseudomallei K96243 cultivated in TBSS-C50-based erythritol medium at pH 6.3, 50 mM potassium phosphate buffer, with 1.2% erythritol and Gibco MEM Vitamin Solution diluted 1:100 and 1:50. Note the broken y-axis highlighted by a dashed line and different scales. Growth curves are representative of at least two independent experiments, each experiment of which was conducted in technical duplicates. Error bars denote the standard deviation of mean from technical duplicates of a single experiment.

Figure 4

Effect of nitrate on growth of B. pseudomallei in erythritol medium. (A) B. pseudomallei strain K96243 cultivated under shaking conditions in 50 mL falcon tubes at 120 rpm in 10 mL TBSS-C50-based erythritol medium at pH 6.3, 50 mM potassium phosphate buffer, 1.2% erythritol, Gibco MEM Vitamin Solution diluted 1:50, with and without 10 mM nitrate for 144 h at 40°C. Nitrate addition is symbolized by crosses in the growth curves. (B) B. pseudomallei strain K96243 cultivated in the same media statically in 50 mL falcon tubes. Growth curves are representative of at least two independent experiments, each of which was conducted in technical duplicates. Error bars denote the standard deviation of mean from technical duplicates of a single experiment.

Figure 5

Growth of B. pseudomallei strain K96243 in ACER medium compared to TBSS-C50 and TBSS-C50-based erythritol medium (EM) under static and shaken conditions. (A) B. pseudomallei strain K96243 was cultivated in 10 mL ACER medium, TBSS-C50 and TBSS-C50-based erythritol medium, shaken at 120 rpm in in 50 mL falcons for 144 h at 40°C. (B) B. pseudomallei strain K96243 cultivated statically in 50 mL falcons for 144 h at 40°C in 10 mL of the respective media. Note the broken y-axis highlighted by a dashed line and different scales. Growth curves are representative of at least two independent experiments, each of which was conducted in technical duplicates. Error bars denote the standard deviation of mean from technical duplicates of a single experiment.

Figure 6

Molecular detection of B. pseudomallei in supernatants from soil sample enrichments in ACER medium, TBSS-C50-based erythritol medium (EM) and TBSS-C50. Twenty soil samples collected in October 2022 were subjected to different culture procedures with TBSS-C50, TBSS-C50-based erythritol medium and ACER medium as depicted in Figure 1. Quantitative PCR results of continuous cultures for 9  days with static and shaken incubation and for a static two-step culture of 2  days followed by 7  days are shown. The C_T values with corresponding medians (horizontal line) and interquartile range are depicted in the graph. Each dot represents a single enrichment culture from a soil sample suspension performed in 10 mL medium. Samples highlighted within the grey box with C_T values below 30 were plated on Ashdown agar (Figure 7). The total number of qPCR-positive samples for each culture protocol is shown below the corresponding data points above the abscissa (*p < 0.05, **p < 0.01, and ****p < 0.0001, “ns”, not significant; Friedman test with Dunn’s correction for paired data).

Figure 7

Effects of different enrichment media on the recovery of B. pseudomallei on Ashdown agar. (A) Twenty soil samples collected in October 2022 were incubated 9  days statically at 40°C in TBSS-C50, TBSS-C50-based erythritol medium (EM) and ACER medium. Sixteen soil samples with C_T values below 30 in the three media after 9  days of static incubation were culture positive on Ashdown agar. Colony counts are log 10-transformed and depicted as bars (TBSS-C50 enrichments: plane bar; TBSS-C50-based erythritol medium: square patterned bar; ACER medium: grey bar), reflecting the median with interquartile range of all enrichments in the same medium. Recovered CFUs from single enrichments (means of duplicates cultured from each supernatant) are shown as symbols within the bars. The CFUs from TBSS-C50 are symbolized by grey triangles, from TBSS-C50-based erythritol medium by grey circles and from ACER medium by crosses (**p < 0.01 and ****p < 0.0001; Friedman test with Dunn’s correction for paired data). (B) The CFU values of all agar counts are plotted against their respective C_T values. Again, grey triangles refer to CFU on Ashdown agar derived from TBSS-C50 enrichments, circles to TBSS-C50-based erythritol medium and crosses refer to ACER cultures. Spearman’s rank correlation was performed (“r_s” Spearman’s rank correlation coefficient) to analyze the relationship between cultivated CFUs and respective C_T values. (C) The graph shows CFU values depicted as bars (left Y axis) and corresponding C_T values demonstrated as circles (right Y axis) after enrichment in all three media for every single soil sample (TBSS-C50 enrichments: plane bar; TBSS-C50-based erythritol medium: square patterned bar; ACER medium: grey bar).