Comparative genomic analysis of clinical and environmental Burkholderia pseudomallei isolates from an endemic area of Thailand

Little is known about the genomic differences between clinical and environmental isolates of Burkholderia pseudomallei, the causative agent of melioidosis. With the advent of whole genome sequencing, the ability to sequence numerous genomes fairly inexpensively allows a thorough analysis of many genomes to be undertaken. In this study we sequenced 100 (50 clinical and 50 environmental) B. pseudomallei isolates from Thailand using Illumina’s next generation sequencing technique. While previous studies have attempted to determine differences between clinical and environmental isolates the use of a clinical isolate alone to determine probable genes could greatly limit the search. The pangenome used to produce the probes for in silico analysis in this study uses globally diverse clinical and environmental samples. Looking at the presence or absence of 2,651 genes across these genomes did not uncover any genes that could play a role in distinguishing clinical isolates from environmental. An NMDS plot based on the genes that could be potentially useful in differentiating the Thai samples illustrated groupings of clinical and environmental isolates, however, there was still a large section of overlap. When these isolates were put in a global context and an NMDS plot was produced using the larger set of differentiating genes a similar pattern was seen

No Evidence for Enzootic Plague within Black-Tailed Prairie Dog (Cynomys Ludovicianus) Populations.

Yersinia pestis, causative agent of plague, occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species, including black-tailed prairie dogs (Cynomys ludovicianus). How Y. pestis persists long-term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed, including, among others, a separate enzootic transmission cycle that maintains Y. pestis without involvement of epizootic hosts and persistence of Y. pestiswithin epizootic host populations without causing high mortality within those populations. We live-trapped and collected fleas from black-tailed prairie dogs and other mammal species from sites with and without black-tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y. pestis. Y. pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005, before and during a widespread epizootic that drastically reduced black-tailed prairie dog populations in the affected colonies. Temporal and spatial patterns of Y. pestisoccurrence in fleas and genotyping of Y. pestis present in some infected fleas suggest Y. pestis was introduced multiple times from sources outside the study area and once introduced, was dispersed between several sites. We conclude Y. pestis likely was not present in these black-tailed prairie dog colonies prior to epizootic activity in these colonies. Although we did not identify likely enzootic hosts, we found evidence that deer mice (Peromyscus maniculatus) may serve as bridging hosts for Y. pestis between unknown enzootic hosts and black-tailed prairie dogs

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

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

Detection of <it>Burkholderia pseudomallei</it> O-antigen serotypes in near-neighbor species

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei</it> is the etiological agent of melioidosis and a CDC category B select agent with no available effective vaccine. Previous immunizations in mice have utilized the lipopolysaccharide (LPS) as a potential vaccine target because it is known as one of the most important antigenic epitopes in <it>B</it>. <it>pseudomallei</it>. Complicating this strategy are the four different <it>B. pseudomallei</it> LPS O-antigen types: A, B, B2, and rough. Sero-crossreactivity is common among O-antigens of <it>Burkholderia</it> species. Here, we identified the presence of multiple <it>B. pseudomallei</it> O-antigen types and sero-crossreactivity in its near-neighbor species.</p> <p>Results</p> <p>PCR screening of O-antigen biosynthesis genes, phenotypic characterization using SDS-PAGE, and immunoblot analysis showed that majority of <it>B. mallei</it> and <it>B. thailandensis</it> strains contained the typical O-antigen type A. In contrast, most of <it>B. ubonensis</it> and <it>B. thailandensis</it>-like strains expressed the atypical O-antigen types B and B2, respectively. Most <it>B</it>. <it>oklahomensis</it> strains expressed a distinct and non-seroreactive O-antigen type, except strain E0147 which expressed O-antigen type A. O-antigen type B2 was also detected in <it>B</it>. <it>thailandensis</it> 82172, <it>B</it>. <it>ubonensis</it> MSMB108, and <it>Burkholderia</it> sp. MSMB175. Interestingly, <it>B</it>. <it>thailandensis</it>-like MSMB43 contained a novel serotype B positive O-antigen.</p> <p>Conclusions</p> <p>This study expands the number of species which express <it>B. pseudomallei</it> O-antigen types. Further work is required to elucidate the full structures and how closely these are to the <it>B. pseudomallei</it> O-antigens, which will ultimately determine the efficacy of the near-neighbor B serotypes for vaccine development.</p

Pangenome Analysis of <i>Burkholderia pseudomallei</i>: Genome Evolution Preserves Gene Order despite High Recombination Rates

<div><p>The pangenomic diversity in <i>Burkholderia pseudomallei</i> is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of <i>B</i>. <i>pseudomallei</i> and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.</p></div

Model fit parameters.

<p>Models described by Haegeman and Weitz [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140274#pone.0140274.ref010" target="_blank">10</a>]. Model A: neutral model with all genes exchanged with environment with parameter <i>θ</i>₁. Model C: Genome has a fraction (<i>λ</i>₁) of the genome that is rigid (the core), and the rest exchanges genes with the environment with parameter <i>θ</i>₁. Model D: Similar to model C except the core exchanges genes with the environment with parameter <i>θ</i>₂. The distance from the model fit to the data for <i>B</i>. <i>pseudomallei</i> is given by Δ, with smaller numbers signifying better fit.</p

Distribution of genes and fit of models described by Haegeman and Weitz [10].

<p>Open circles are data from this study, with fitted lines according to models A (red squares), C (blue filled circles), and D (black triangles). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140274#pone.0140274.t002" target="_blank">Table 2</a> and text for descriptions of models and parameters.</p