A horizontal gene transfer event defines two distinct groups within Burkholderia pseudomallei that have dissimilar geographic distributions

Burkholderia pseudomallei is the etiologic agent of melioidosis. Many disease manifestations are associated with melioidosis, and the mechanisms causing this variation are unknown; genomic differences among strains offer one explanation. We compared the genome sequences of two strains of B. pseudomallei: the original reference strain K96243 from Thailand and strain MSHR305 from Australia. We identified a variable homologous region between the two strains. This region was previously identified in comparisons of the genome of B. pseudomallei strain K96243 with the genome of strain E264 from the closely related B. thailandensis. In that comparison, K96243 was shown to possess a horizontally acquired Yersinia-like fimbrial (YLF) gene cluster. Here, we show that the homologous genomic region in B. pseudomallei strain 305 is similar to that previously identified in B. thailandensis strain E264. We have named this region in B. pseudomallei strain 305 the B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. We screened for these different genomic components across additional genome sequences and 571 B. pseudomallei DNA extracts obtained from regions of endemicity. These alternate genomic states define two distinct groups within B. pseudomallei: all strains contained either the BTFC gene cluster (group BTFC) or the YLF gene cluster (group YLF). These two groups have distinct geographic distributions: group BTFC is dominant in Australia, and group YLF is dominant in Thailand and elsewhere. In addition, clinical isolates are more likely to belong to group YLF, whereas environmental isolates are more likely to belong to group BTFC. These groups should be further characterized in an animal model.     

Genetic and phenotypic diversity in <Emphasis Type="Italic">Burkholderia</Emphasis>: contributions by prophage and phage-like elements

Background  

Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.     

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

Background

The facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations.

Results

B. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Nei's diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10^-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria.

Conclusion

The frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.     

Effect of acidic pH on the invasion efficiency and the type III secretion system of <Emphasis Type="Italic">Burkholderia thailandensis</Emphasis>

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.     

Recovery of a <Emphasis Type="Italic">Burkholderia thailandensis</Emphasis>-like isolate from an Australian water source

Background  

Burkholderia thailandensis, a close relative of Burkholderia pseudomallei, has previously been reported only from Southeast Asia and North America. It is biochemically differentiated from B. pseudomallei by the ability to utilize arabinose. During the course of environmental sampling for B. pseudomallei in the Northern Territory of Australia, an isolate, MSMB 43, was recovered that is arabinose positive.     

Microbial rhamnolipid production: a critical re-evaluation of published data and suggested future publication criteria

High production cost and potential pathogenicity of Pseudomonas aeruginosa, commonly used for rhamnolipid synthesis, have led to extensive research for safer producing strains and cost-effective production methods. This has resulted in numerous research publications claiming new non-pathogenic producing strains and novel production techniques many of which are unfortunately without proper characterisation of product and/or producing strain/s. Genes responsible for rhamnolipid production have only been confirmed in P. aeruginosa, Burkholderia thailandensis and Burkholderia pseudomallei. Comparing yields in different publications is also generally unreliable especially when different methodologies were used for rhamnolipid quantification. After reviewing the literature in this area, we strongly feel that numerous research outputs have insufficient evidence to support claims of rhamnolipid-producing strains and/or yields. We therefore recommend that standards should be set for reporting new rhamnolipid-producing strains and production yields. These should include (1) molecular and bioinformatic tools to fully characterise new microbial isolates and confirm the presence of the rhamnolipid rhl genes for all bacterial strains, (2) using gravimetric methods to quantify crude yields and (3) use of a calibrated method (high-performance liquid chromatography or ultra-performance liquid chromatography) for absolute quantitative yield determination.     

The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis – a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact‐dependent growth inhibition (CDI) systems of γ‐proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA‐CT/cdiI coding regions, which are predicted to encode CdiA‐CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA‐CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA‐CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA‐CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.     

Molecular phylogeny of Burkholderia pseudomallei from a remote region of Papua New Guinea

Background

The island of New Guinea is located midway between the world''s two major melioidosis endemic regions of Australia and Southeast Asia. Previous studies in Papua New Guinea have demonstrated autochthonous melioidosis in Balimo, Western province. In contrast to other regions of endemicity, isolates recovered from both environmental and clinical sources demonstrate narrow genetic diversity over large spatial and temporal scales.

Methodology/Principal Findings

We employed molecular typing techniques to determine the phylogenetic relationships of these isolates to each other and to others worldwide to aid in understanding the origins of the Papua New Guinean isolates. Multi-locus sequence typing of the 39 isolates resolved three unique sequence types. Phylogenetic reconstruction and Structure analysis determined that all isolates were genetically closer to those from Australia than those from Southeast Asia. Gene cluster analysis however, identified a Yersinia-like fimbrial gene cluster predominantly found among Burkholderia pseudomallei derived from Southeast Asia. Higher resolution VNTR typing and phylogenetic reconstruction of the Balimo isolates resolved 24 genotypes with long branch lengths. These findings are congruent with long term persistence in the region and a high level of environmental stability.

Conclusions/Significance

Given that anthropogenic influence has been hypothesized as a mechanism for the dispersal of B. pseudomallei, these findings correlate with limited movement of the indigenous people in the region. The palaeogeographical and anthropogenic history of Australasia and the results from this study indicate that New Guinea is an important region for the further study of B. pseudomallei origins and dissemination.     

The obesity and inflammatory marker haptoglobin attracts monocytes via interaction with chemokine (C-C motif) receptor 2 (CCR2)

Background

Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction.

Results

Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia.

Conclusion

We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.     

Membrane-active mechanism of LFchimera against Burkholderia pseudomallei and Burkholderia thailandensis

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265–284 and lactoferricin17–30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.     

Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

Background  

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models.     

The role of short-chain dehydrogenase/oxidoreductase, induced by salt stress, on host interaction of B. pseudomallei

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a frequently occurring disease in northeastern Thailand, where soil and water high in salt content are common. Using microarray analysis, we previously showed that B. pseudomallei up-regulated a short-chain dehydrogenase/oxidoreductase (SDO) under salt stress. However, the importance of SDO in B. pseudomallei infection is unknown. This study aimed to explore the function of B. pseudomallei SDO, and to investigate its role in interactions between B. pseudomallei and host cells.

Results

Bioinformatics analysis of B. pseudomallei SDO structure, based on homology modeling, revealed a NAD⁺ cofactor domain and a catalytic triad containing Ser149, Tyr162, and Lys166. This is similar to Bacillus megaterium glucose 1-dehydrogenase. To investigate the role of this protein, we constructed a B. pseudomallei SDO defective mutant, measured glucose dehydrogenase (GDH) activity, and tested the interactions with host cells. The B. pseudomallei K96243 wild type exhibited potent GDH activity under condition containing 300 mM NaCl, while the mutant showed activity levels 15 times lower. Both invasion into the A549 cell line and early intracellular survival within the J774A.1 macrophage cell were impaired in the mutant. Complementation of SDO was able to restore the mutant ability to produce GDH activity, invade epithelial cells, and survive in macrophages.

Conclusions

Our data suggest that induced SDO activity during salt stress may facilitate B. pseudomallei invasion and affect initiation of successful intracellular infection. Identifying the role of B. pseudomallei SDO provides a better understanding of the association between bacterial adaptation and pathogenesis in melioidosis.     

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD₅₀ via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.     

Laboratory diagnosis of melioidosis: Past,present and future

Melioidosis is an emerging, potentially fatal disease caused by Burkholderia pseudomallei, which requires prolonged antibiotic treatment to prevent disease relapse. However, difficulties in laboratory diagnosis of melioidosis may delay treatment and affect disease outcomes. Isolation of B. pseudomallei from clinical specimens has been improved with the use of selective media. However, even with positive cultures, identification of B. pseudomallei can be difficult in clinical microbiology laboratories, especially in non-endemic areas where clinical suspicion is low. Commercial identification systems may fail to distinguish between B. pseudomallei and closely related species such as Burkholderia thailandensis. Genotypic identification of suspected isolates can be achieved by sequencing of gene targets such as groEL which offer higher discriminative power than 16S rRNA. Specific PCR-based identification of B. pseudomallei has also been developed using B. pseudomallei-specific gene targets such as Type III secretion system and Tat-domain protein. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolutionary technique for pathogen identification, has been shown to be potentially useful for rapid identification of B. pseudomallei, although existing databases require optimization by adding reference spectra for B. pseudomallei. Despite these advances in bacterial identification, diagnostic problems encountered in culture-negative cases remain largely unresolved. Although various serological tests have been developed, they are generally unstandardized “in house” assays and have low sensitivities and specificities. Although specific PCR assays have been applied to direct clinical and environmental specimens, the sensitivities for diagnosis remain to be evaluated. Metabolomics is an uprising tool for studying infectious diseases and may offer a novel approach for exploring potential diagnostic biomarkers. The metabolomics profiles of B. pseudomallei culture supernatants can be potentially distinguished from those of related bacterial species including B. thailandensis. Further studies using bacterial cultures and direct patient samples are required to evaluate the potential of metabolomics for improving diagnosis of melioidosis.     

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Background

The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear.

Methodology/Principal Findings

We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour.

Conclusions/Significance

This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.     

Suppression-subtractive hybridisation reveals variations in gene distribution amongst the<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis> complex,including the presence in some strains of a genomic island containing putative polysaccharide production genes

Some strains of the Burkholderia cepacia complex, including the ET12 lineage, have been implicated in epidemic spread amongst cystic fibrosis (CF) patients. Suppression-subtractive hybridisation was used to identify genomic regions within strain J2315 (ET12 lineage; genomovar IIIA) that were absent from a non-transmissible genomovar IIIB strain. Sequence data from 15 subtracted clones were used to interrogate the genome sequence of strain J2315 and identify genomic regions incorporating the subtracted sequences. Many of the genomic regions displayed abnormally low GC content and similarity to sequences implicated in gene transfer. The distribution of three subtracted regions amongst members of the B. cepacia complex varied. A large cluster of genes with strong sequence similarity to capsular production genes from Burkholderia mallei and other bacterial pathogens was identified. This genomic island was detected in some but not all representatives of genomovar IIIA, two out of four genomovar I strains, and one of two strains of Burkholderia multivorans, but was not detected in Burkholderia stabilis, Burkholderia vietnamiensis, genomovar VI or Burkholderia. ambifaria. The polysaccharide production gene cluster of strain J2315 carries an IS 407-like sequence within the gene similar to B. mallei wcbO that is lacking in other ET12 isolates. Genes from this cluster are expressed during exponential growth in broth.