Morphological Alteration and Survival of Burkholderia pseudomallei in Soil Microcosms

Abstract

The resilience of Burkholderia pseudomallei, the causative agent of melioidosis, was evaluated in control soil microcosms and in soil microcosms containing NaCl or FeSO4 at 30°C. Iron (Fe(II)) promoted the growth of B. pseudomallei during the 30-day observation, contrary to the presence of 1.5% and 3% NaCl. Scanning electron micrographs of B. pseudomallei in soil revealed their morphological alteration from rod to coccoid and the formation of microcolonies. The smallest B. pseudomallei cells were found in soil with 100 μM FeSO4 compared with in the control soil or soil with 0.6% NaCl (P < 0.05). The colony count on Ashdown's agar and bacterial viability assay using the LIVE/DEAD(®) BacLight(™) stain combined with flow cytometry showed that B. pseudomallei remained culturable and viable in the control soil microcosms for at least 120 days. In contrast, soil with 1.5% NaCl affected their culturability at day 90 and their viability at day 120. Our results suggested that a low salinity and iron may influence the survival of B. pseudomallei and its ability to change from a rod-like to coccoid form. The morphological changes of B. pseudomallei cells may be advantageous for their persistence in the environment and may increase the risk of their transmission to humans.

Figure 1.

Number (log₁₀ CFU/g soil) of Burkholderia pseudomallei R3E in 100 g of the control soil microcosm and in soil microcosms containing 0.6–3% NaCl or 100–1,000 μM FeSO₄ after incubation at 30°C for 30 days. The bacteria recovered in the PEG-DOC solution (2.5% w/v polyethylene glycol 600 and 0.1% w/v sodium deoxycholate) were 10-fold serially diluted and then each dilution was spread on Ashdown's agar using the spread plate technique, after which the plates were incubated at 37°C for 48 hours. The error bars correspond to the standard deviation of the values determined in duplicate experiments. *P < 0.05 for log₁₀ CFU/g soil on the test day compared with the value determined on day 0 of each experiment.

Figure 2.

Morphology of Burkholderia pseudomallei R3E grown in the soil microcosms. The bacteria were grown in the soil microcosms for 30 days and then examined using scanning electron microscopy. Burkholderia pseudomallei R3E cells grown in the control soil (A), in soil supplemented with 0.6% NaCl (B), in soil supplemented with 100 μM of FeSO₄ (C); and in the control soil not inoculated with B. pseudomallei (D). Magnification = 5,000×.

Figure 3.

Scanning electron micrographs of Burkholderia pseudomallei R3E. Morphological characteristics of an established microcolony formed by B. pseudomallei R3E grown in a control soil microcosm for 30 days at 30°C (A) and a biofilm colony formed by cells grown on a glass slide immersed in modified Vogel–Bonner medium (MVBM) for 2 days at 37°C (B). Magnification = 5,000×.

Figure 4.

Culturability and viability of Burkholderia pseudomallei grown in the control soil microcosm. The counts of culturable bacteria determined using an Ashdown's agar plate assay (A) and the live/total cells identified using the LIVE/DEAD^® BacLight^™ viability kit in combination with flow cytometry (B) of the B. pseudomallei H777 wild-type strain (♦), its mutant M10 strain (⋄), and the B. pseudomallei R3E environmental isolate strain (▴) grown in the control soil microcosm at 30°C for 120 days. The error bars represent the standard deviation of the values obtained in duplicate experiments.

Figure 5.

Culturability and viability of Burkholderia pseudomallei grown in soil microcosms supplemented with 1.5% NaCl. The counts of culturable bacteria determined using an Ashdown's agar plate assay (A) and the live/total cells identified using the LIVE/DEAD^® BacLight^™ viability kit in combination with flow cytometry of the B. pseudomallei H777 wild-type strain (♦), its mutant M10 strain (⋄), and the B. pseudomallei R3E environmental isolate strain (▴) grown in soil supplemented with 1.5% NaCl at 30°C for 120 days. The error bars represent the standard deviation of the values obtained in duplicate experiments. *P < 0.05 for log₁₀ CFU/g soil and the live/total cells on the test day compared with the values determined on day 0 of each experiment.