Using Land Runoff to Survey the Distribution and Genetic Diversity of Burkholderia pseudomallei in Vientiane, Laos

Abstract

Melioidosis is a disease of significant public health importance that is being increasingly recognized globally. The majority of cases arise through direct percutaneous exposure to its etiological agent, Burkholderia pseudomallei In the Lao People's Democratic Republic (Laos), the presence and environmental distribution of B. pseudomallei are not well characterized, though recent epidemiological surveys of the bacterium have indicated that B. pseudomallei is widespread throughout the environment in the center and south of the country and that rivers can act as carriers and potential sentinels for the bacterium. The spatial and genetic distribution of B. pseudomallei within Vientiane Capital, from where the majority of cases diagnosed to date have originated, remains an important knowledge gap. We sampled surface runoff from drain catchment areas throughout urban Vientiane to determine the presence and local population structure of the bacterium. B. pseudomallei was detected in drainage areas throughout the capital, indicating it is widespread in the environment and that exposure rates in urban Vientiane are likely more frequent than previously thought. Whole-genome comparative analysis demonstrated that Lao B. pseudomallei isolates are highly genetically diverse, suggesting the bacterium is well-established and not a recent introduction. Despite the wide genome diversity, one environmental survey isolate was highly genetically related to a Lao melioidosis patient isolate collected 13 years prior to the study. Knowledge gained from this study will augment understanding of B. pseudomallei phylogeography in Asia and enhance public health awareness and future implementation of infection control measures within Laos.IMPORTANCE The environmental bacterium B. pseudomallei is the etiological agent of melioidosis, a tropical disease with one model estimating a global annual incidence of 165,000 cases and 89,000 deaths. In the Lao People's Democratic Republic (Laos), the environmental distribution and population structure of B. pseudomallei remain relatively undefined, particularly in Vientiane Capital from where most diagnosed cases have originated. We used surface runoff as a proxy for B. pseudomallei dispersal in the environment and performed whole-genome sequencing (WGS) to examine the local population structure. Our data confirmed that B. pseudomallei is widespread throughout Vientiane and that surface runoff might be useful for future environmental monitoring of the bacterium. B. pseudomallei isolates were also highly genetically diverse, suggesting the bacterium is well-established and endemic in Laos. These findings can be used to improve awareness of B. pseudomallei in the Lao environment and demonstrates the epidemiological and phylogeographical insights that can be gained from WGS.

FIG 1

Sampling site locations across urban Vientiane Capital. Sites where B. pseudomallei was detected by culture and/or direct detection in water are indicated by red triangles. Negative sites (green triangles) are those where B. pseudomallei was not identified in water by either detection method. The base map is from ArcGIS/Esri (sources: Esri, HERE, Garmin, Intermap, increment P Corp., GEBCO, USGS, FAO, NPS, NRCAN, GeoBase, IGN, Kadaster NL, Ordnance Survey, Esri Japan, METI, Esri China [Hong Kong], OpenStreetMap contributors, and the GIS user community).

FIG 2

Generalized estimated equation (GEE) margins plots of adjusted predicted probabilities of B. pseudomallei occurrence with all other variables in the model held constant. Shown are predicted probabilities as a function of increased water TDS (A) in each of the districts surveyed and turbidity (B) or probabilities in unlined or cement-lined drains based on TDS (C) or turbidity of water sample (D). Bars indicate 95% confidence intervals (CIs).

FIG 3

Midpoint rooted maximum parsimony phylogeny of 40 Lao B. pseudomallei isolates based on 56,532 core genome SNPs and indels. Black circles indicate bootstrap values of <80.

FIG 4

Maximum-parsimony phylogeny of B. pseudomallei from Laos (n = 40, green branches) with a global set of genomes (n = 159) based on 168,934 core genome SNPs and indels. The closed Thai isolate K96243 was used as the reference strain and the tree was rooted at MSHR0668, the most ancestral B. pseudomallei strain identified in a previous study (31). Black circles indicate bootstrap values <80.

FIG 5

Map of the 18 provinces making up Lao PDR with Vientiane Capital highlighted in red. The expanded view shows Vientiane Capital comprised of its nine districts. Numbers indicate the four urban and one semiurban district where survey sites were located (1, Chanthabuly; 2, Sikhottabong; 3, Xaysetha; 4, Sisattanak; 5, Xathany).