Multiple phylogenetically-diverse, differentially-virulent Burkholderia pseudomallei isolated from a single soil sample collected in Thailand.

Chandler Roe,Gumphol Wongsuwan,Chris J Allender,Roxanne D Nottingham,Adina Doyle,Direk Limmathurotsakul,Apichai Tuanyok,Paul Keim,Adam J Vazquez,David M Wagner,Vanaporn Wuthiekanun,Richard A Bowen,Jason W Sahl,Paul D Phillips,Sharon Peacock

doi:10.1371/journal.pntd.0010172

Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796-1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1-2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei.

Highlights

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia where it is the causative agent of melioidosis, a potentially fatal disease in humans [1]
Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia
We surveyed the genomic diversity of 169 isolates collected from a single soil sample in Ubon Ratchathani Province, Thailand

Summary

Introduction

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia where it is the causative agent of melioidosis, a potentially fatal disease in humans [1]. One study found 4 distinct sequence types (STs) within a single soil sample in Ubon, Thailand [12], the resulting data did not identify genomic and phylogenetic differences within each ST. Another study surveyed genotypic diversity with sub-genomic methods in Northeastern Thailand and found 7 distinct STs across 11 soil samples [13]. A study on MLST types in Australia identified that the diversity of B. pseudomallei populations increased with sampling area, suggesting localized adaptation [15]. A recent study used whole genome sequencing to identify genomic signatures that differ between clinical and environmental B. pseudomallei isolates in Northeastern Thailand [16]; the focus of this study was the overlap between clinical and environmental isolates and not the within-ST diversity of B. pseudomallei

Objectives

Methods

Results

Discussion

Conclusion