Survival of Burkholderia pseudomallei in Water

<p>Abstract</p> <p>Background</p> <p>The ability of <it>Burkholderia pseudomallei </it>to survive in water likely contributes to its environmental persistence in endemic regions. To determine the physiological adaptations which allow <it>B. pseudomallei </it>to survive in aqueous environments, we performed microarray analyses of <it>B. pseudomallei </it>cultures transferred from Luria broth (LB) to distilled water.</p> <p>Findings</p> <p>Increased expression of a gene encoding for a putative membrane protein (BPSL0721) was confirmed using a <it>lux</it>-based transcriptional reporter system, and maximal expression was noted at approximately 6 hrs after shifting cells from LB to water. A BPSL0721 deficient mutant of <it>B. pseudomallei </it>was able to survive in water for at least 90 days indicating that although involved, BPSL0721 was not essential for survival. BPSL2961, a gene encoding a putative phosphatidylglycerol phosphatase (PGP), was also induced when cells were shifted to water. This gene is likely involved in cell membrane biosynthesis. We were unable to construct a PGP mutant suggesting that the gene is not only involved in survival in water but is essential for cell viability. We also examined mutants of polyhydroxybutyrate synthase (<it>phb</it>C), lipopolysaccharide (LPS) oligosaccharide and capsule synthesis, and these mutations did not affect survival in water. LPS mutants lacking outer core were found to lose viability in water by 200 days indicating that an intact LPS core provides an outer membrane architecture which allows prolonged survival in water.</p> <p>Conclusion</p> <p>The results from these studies suggest that <it>B. pseudomallei </it>survival in water is a complex process that requires an LPS molecule which contains an intact core region.</p

Molecular Basis of Rare Aminoglycoside Susceptibility and Pathogenesis of Burkholderia pseudomallei Clinical Isolates from Thailand

Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments

Molecular Investigations of a Locally Acquired Case of Melioidosis in Southern AZ, USA

Melioidosis is caused by Burkholderia pseudomallei, a Gram-negative bacillus, primarily found in soils in Southeast Asia and northern Australia. A recent case of melioidosis in non-endemic Arizona was determined to be the result of locally acquired infection, as the patient had no travel history to endemic regions and no previous history of disease. Diagnosis of the case was confirmed through multiple microbiologic and molecular techniques. To enhance the epidemiological analysis, we conducted several molecular genotyping procedures, including multi-locus sequence typing, SNP-profiling, and whole genome sequence typing. Each technique has different molecular epidemiologic advantages, all of which provided evidence that the infecting strain was most similar to those found in Southeast Asia, possibly originating in, or around, Malaysia. Advancements in new typing technologies provide genotyping resolution not previously available to public health investigators, allowing for more accurate source identification

BurkDiff: A Real-Time PCR Allelic Discrimination Assay for Burkholderia Pseudomallei and B. mallei

A real-time PCR assay, BurkDiff, was designed to target a unique conserved region in the B. pseudomallei and B. mallei genomes containing a SNP that differentiates the two species. Sensitivity and specificity were assessed by screening BurkDiff across 469 isolates of B. pseudomallei, 49 isolates of B. mallei, and 390 isolates of clinically relevant non-target species. Concordance of results with traditional speciation methods and no cross-reactivity to non-target species show BurkDiff is a robust, highly validated assay for the detection and differentiation of B. pseudomallei and B. mallei

Diversity of 16S-23S rDNA Internal Transcribed Spacer (ITS) Reveals Phylogenetic Relationships in Burkholderia pseudomallei and Its Near-Neighbors

Length polymorphisms within the 16S-23S ribosomal DNA internal transcribed spacer (ITS) have been described as stable genetic markers for studying bacterial phylogenetics. In this study, we used these genetic markers to investigate phylogenetic relationships in Burkholderia pseudomallei and its near-relative species. B. pseudomallei is known as one of the most genetically recombined bacterial species. In silico analysis of multiple B. pseudomallei genomes revealed approximately four homologous rRNA operons and ITS length polymorphisms therein. We characterized ITS distribution using PCR and analyzed via a high-throughput capillary electrophoresis in 1,191 B. pseudomallei strains. Three major ITS types were identified, two of which were commonly found in most B. pseudomallei strains from the endemic areas, whereas the third one was significantly correlated with worldwide sporadic strains. Interestingly, mixtures of the two common ITS types were observed within the same strains, and at a greater incidence in Thailand than Australia suggesting that genetic recombination causes the ITS variation within species, with greater recombination frequency in Thailand. In addition, the B. mallei ITS type was common to B. pseudomallei, providing further support that B. mallei is a clone of B. pseudomallei. Other B. pseudomallei near-neighbors possessed unique and monomorphic ITS types. Our data shed light on evolutionary patterns of B. pseudomallei and its near relative species

Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA

<p>Abstract</p> <p>Background</p> <p>Brucellosis is a worldwide anthropozoonotic disease caused by an in vivo intracellular pathogen belonging to genus <it>Brucella</it>. The characterization of brucelae transcriptome's during host-pathogen interaction has been limited due to the difficulty of obtaining an adequate quantity of good quality eukaryotic RNA-free pathogen RNA for downstream applications.</p> <p>Findings</p> <p>Here, we describe a combined protocol to prepare RNA from intracellular <it>B. melitensis </it>in a quantity and quality suitable for pathogen gene expression analysis. Initially, <it>B. melitensis </it>total RNA was enriched from a host:pathogen mixed RNA sample by reducing the eukaryotic RNA..Then, to increase the <it>Brucella </it>RNA concentration and simultaneously minimize the contaminated host RNA in the mixed sample, a specific primer set designed to anneal to all <it>B. melitensis </it>ORF allows the selective linear amplification of sense-strand prokaryotic transcripts in a previously enriched RNA sample.</p> <p>Conclusion</p> <p>The novelty of the method we present here allows analysis of the gene expression profile of <it>B. melitensis </it>when limited amounts of pathogen RNA are present, and is potentially applicable to both <it>in vivo </it>and <it>in vitro </it>models of infection, even at early infection time points.</p

Characterization of Ceftazidime Resistance Mechanisms in Clinical Isolates of Burkholderia pseudomallei from Australia

Burkholderia pseudomallei is a Gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen

Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

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

Within-Host Evolution of Burkholderia pseudomallei in Four Cases of Acute Melioidosis

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum

The Early Stage of Bacterial Genome-Reductive Evolution in the Host

The equine-associated obligate pathogen Burkholderia mallei was developed by reductive evolution involving a substantial portion of the genome from Burkholderia pseudomallei, a free-living opportunistic pathogen. With its short history of divergence (∼3.5 myr), B. mallei provides an excellent resource to study the early steps in bacterial genome reductive evolution in the host. By examining 20 genomes of B. mallei and B. pseudomallei, we found that stepwise massive expansion of IS (insertion sequence) elements ISBma1, ISBma2, and IS407A occurred during the evolution of B. mallei. Each element proliferated through the sites where its target selection preference was met. Then, ISBma1 and ISBma2 contributed to the further spread of IS407A by providing secondary insertion sites. This spread increased genomic deletions and rearrangements, which were predominantly mediated by IS407A. There were also nucleotide-level disruptions in a large number of genes. However, no significant signs of erosion were yet noted in these genes. Intriguingly, all these genomic modifications did not seriously alter the gene expression patterns inherited from B. pseudomallei. This efficient and elaborate genomic transition was enabled largely through the formation of the highly flexible IS-blended genome and the guidance by selective forces in the host. The detailed IS intervention, unveiled for the first time in this study, may represent the key component of a general mechanism for early bacterial evolution in the host