Research Article: Genetic Diversity and Microevolution of Burkholderia pseudomallei in the Environment

Date Published: February 20, 2008

Publisher: Public Library of Science

Author(s): Narisara Chantratita, Vanaporn Wuthiekanun, Direk Limmathurotsakul, Mongkol Vesaratchavest, Aunchalee Thanwisai, Premjit Amornchai, Sarinna Tumapa, Edward J. Feil, Nicholas P. Day, Sharon J. Peacock, Bart Currie

Abstract: BackgroundThe soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined.Methods and FindingsWe undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m2 of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone.ConclusionsWe discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

Partial Text: The soil dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. This organism is present in the environment across much of southeast Asia and Northern Australia and is increasingly recognised elsewhere, including parts of South America [1],[2]. Infection occurs through bacterial inoculation and contamination of wounds, and more rarely by inhalation and ingestion [1],[3]. Environmental sampling underpins efforts to define the global distribution of B. pseudomallei in soil, and the associated geographic distribution of risk to humans and livestock. Sampling is also performed during the investigation of suspected outbreaks, when bacterial genotyping is used to compare B. pseudomallei obtained from cases of melioidosis with strains from a specified environment or substance. Environmental sampling would also be required following the deliberate release of B. pseudomallei associated with bioterrorist activity. The accuracy of such studies depends on the detection of all of the B. pseudomallei genotypes present at a given site with the exception of those present at an extremely low frequency. Informed sampling strategies are also a prerequisite for meaningful comparisons between environmental isolates and those recovered from cases of disease in humans and animals, which provide an important means to identify clones with heightened virulence.

A total of 80 out of the 100 sampling points were culture positive for B. pseudomallei, of which 77 were positive from both direct plating onto Ashdown’s agar and selective enrichment broth, and 3 were positive from selective enrichment broth culture alone (Figure 1). B. thailandensis was not detected. The genetic variability of B. pseudomallei was defined and compared within and between sampling points by genotyping 200 colonies at each of three positive points (A11, D10 and E4, see Figure 1). PFGE of 600 individual primary colonies revealed 12 PFGE banding pattern types. MLST of a single random isolate of each of the 12 PFGE types revealed 9 distinct sequence types (STs) (Table 1).

This study has demonstrated marked geographic structuring of B. pseudomallei genotypes in soil. The dramatic differences in genotype frequency over such small distances are striking, but difficult to interpret. One explanation is that the numerically dominant ST at each sampling point represents a strain with superior biological fitness compared with STs present as a minority of the population. This could relate to factors such as soil type or pH, or competition with other microbial species. This would assume that adjacent foci of soil have variable microenvironments, but it seems unlikely that nearby sampling points within a confined area of disused land would differ sufficiently to support multiple, non-overlapping niches. An alternative possibility is that of local competition between clones of B. pseudomallei. Flooding or other disturbance mechanisms would provide the means for a given clone to migrate and become established within a specific plot. Once the clone has reached a certain threshold frequency, it could repel invaders either by the production of microbicides, through phage to which they themselves are resistant, or via other killing mechanisms. The presence of a clone as a minority population could represent the ability of this strain to survive at a lower level, or could represent the boundary of a point of predominance in an adjacent point or focus.



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