Comparative phylogenomics and evolution of the Brucellae reveal a path to virulence.

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as "stealth pathogens" that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing "atypical" strains and a highly conserved "classical" core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.

When outgroups fail; phylogenomics of rooting the emerging pathogen, Coxiella burnetii.

Rooting phylogenies is critical for understanding evolution, yet the importance, intricacies and difficulties of rooting are often overlooked. For rooting, polymorphic characters among the group of interest (ingroup) must be compared to those of a relative (outgroup) that diverged before the last common ancestor (LCA) of the ingroup. Problems arise if an outgroup does not exist, is unknown, or is so distant that few characters are shared, in which case duplicated genes originating before the LCA can be used as proxy outgroups to root diverse phylogenies. Here, we describe a genome-wide expansion of this technique that can be used to solve problems at the other end of the evolutionary scale: where ingroup individuals are all very closely related to each other, but the next closest relative is very distant. We used shared orthologous single nucleotide polymorphisms (SNPs) from 10 whole genome sequences of Coxiella burnetii, the causative agent of Q fever in humans, to create a robust, but unrooted phylogeny. To maximize the number of characters informative about the rooting, we searched entire genomes for polymorphic duplicated regions where orthologs of each paralog could be identified so that the paralogs could be used to root the tree. Recent radiations, such as those of emerging pathogens, often pose rooting challenges due to a lack of ingroup variation and large genomic differences with known outgroups. Using a phylogenomic approach, we created a robust, rooted phylogeny for C. burnetii. [Coxiella burnetii; paralog SNPs; pathogen evolution; phylogeny; recent radiation; root; rooting using duplicated genes.].

Phylogeography of Francisella tularensis subsp. holarctica, Europe.

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002-00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups.

Molecular epidemiologic investigation of an anthrax outbreak among heroin users, Europe.

In December 2009, two unusual cases of anthrax were diagnosed in heroin users in Scotland. A subsequent anthrax outbreak in heroin users emerged throughout Scotland and expanded into England and Germany, sparking concern of nefarious introduction of anthrax spores into the heroin supply. To better understand the outbreak origin, we used established genetic signatures that provided insights about strain origin. Next, we sequenced the whole genome of a representative Bacillus anthracis strain from a heroin user (Ba4599), developed Ba4599-specific single-nucleotide polymorphism assays, and genotyped all available material from other heroin users with anthrax. Of 34 case-patients with B. anthracis-positive PCR results, all shared the Ba4599 single-nucleotide polymorphism genotype. Phylogeographic analysis demonstrated that Ba4599 was closely related to strains from Turkey and not to previously identified isolates from Scotland or Afghanistan, the presumed origin of the heroin. Our results suggest accidental contamination along the drug trafficking route through a cutting agent or animal hides used to smuggle heroin into Europe.

Genotyping of Brucella species using clade specific SNPs.

BACKGROUND: Brucellosis is a worldwide disease of mammals caused by Alphaproteobacteria in the genus Brucella. The genus is genetically monomorphic, requiring extensive genotyping to differentiate isolates. We utilized two different genotyping strategies to characterize isolates. First, we developed a microarray-based assay based on 1000 single nucleotide polymorphisms (SNPs) that were identified from whole genome comparisons of two B. abortus isolates , one B. melitensis, and one B. suis. We then genotyped a diverse collection of 85 Brucella strains at these SNP loci and generated a phylogenetic tree of relationships. Second, we developed a selective primer-extension assay system using capillary electrophoresis that targeted 17 high value SNPs across 8 major branches of the phylogeny and determined their genotypes in a large collection ( n = 340) of diverse isolates. RESULTS: Our 1000 SNP microarray readily distinguished B. abortus, B. melitensis, and B. suis, differentiating B. melitensis and B. suis into two clades each. Brucella abortus was divided into four major clades. Our capillary-based SNP genotyping confirmed all major branches from the microarray assay and assigned all samples to defined lineages. Isolates from these lineages and closely related isolates, among the most commonly encountered lineages worldwide, can now be quickly and easily identified and genetically characterized. CONCLUSIONS: We have identified clade-specific SNPs in Brucella that can be used for rapid assignment into major groups below the species level in the three main Brucella species. Our assays represent SNP genotyping approaches that can reliably determine the evolutionary relationships of bacterial isolates without the need for whole genome sequencing of all isolates.

Thyroid hormone-dependent development in Xenopus laevis: a sensitive screen of thyroid hormone signaling disruption by municipal wastewater treatment plant effluent.

Because thyroid hormones (THs) are conserved modulators of development and physiology, identification of compounds adversely affecting TH signaling is critical to human and wildlife health. Anurans are an established model for studying disruption of TH signaling because metamorphosis is dependent upon the thyroid system. In order to strengthen this model and identify new gene transcript biomarkers for TH disruption, we performed DNA microarray analysis of Xenopus laevis tadpole tail transcriptomes following treatment with triiodothyronine (T(3)). Comparison of these results with previous studies in frogs and mammals identified 36 gene transcripts that were TH-sensitive across clades. We then tested molecular biomarkers for sensitivity to disruption by exposure to wastewater effluent (WWE). X. laevis tadpoles, exposed to WWE from embryo through metamorphosis, exhibited an increased developmental rate compared to controls. Cultured tadpole tails showed dramatic increases in levels of four TH-sensitive gene transcripts (thyroid hormone receptor ß (TRß), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T(3) and WWE extracts. TRß, DIO2, and CRHBP were identified as TH sensitive in other studies, while FAPα mRNA transcripts were highly TH sensitive in our array. The results validate the array and demonstrate TH-disrupting activity by WWE. Our findings demonstrate the usefulness of cross-clade analysis for identification of gene transcripts that provide sensitivity to endocrine disruption. Further, the results suggest that development is disrupted by exposure to complex mixes of compounds found in WWE possibly through interference with TH signaling.

An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics.

BACKGROUND: An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990. RESULTS: We demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media. CONCLUSIONS: We propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.

Next-generation sequencing of Coccidioides immitis isolated during cluster investigation.

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. RESULTS: We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. CONCLUSIONS: We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Development of a rapid, cost-effective TaqMan Real-Time PCR Assay for identification and differentiation of Coccidioides immitis and Coccidioides posadasii.

Coccidioidomycosis is an infection caused by Coccidioides immitis or C. posadasii. We developed a TaqMan real-time PCR assay that rapidly and accurately differentiates the species. This assay can be used as a tool to improve disease surveillance, increase understanding of the natural history of the infection, and assist in clinical differentiation studies.

Whole-genome-based phylogeny and divergence of the genus Brucella.

Brucellae are worldwide bacterial pathogens of livestock and wildlife, but phylogenetic reconstructions have been challenging due to limited genetic diversity. We assessed the taxonomic and evolutionary relationships of five Brucella species-Brucella abortus, B. melitensis, B. suis, B. canis, and B. ovis-using whole-genome comparisons. We developed a phylogeny using single nucleotide polymorphisms (SNPs) from 13 genomes and rooted the tree using the closely related soil bacterium and opportunistic human pathogen, Ochrobactrum anthropi. Whole-genome sequencing and a SNP-based approach provided the requisite level of genetic detail to resolve species in the highly conserved brucellae. Comparisons among the Brucella genomes revealed 20,154 orthologous SNPs that were shared in all genomes. Rooting with Ochrobactrum anthropi reveals that the B. ovis lineage is basal to the rest of the Brucella lineage. We found that B. suis is a highly divergent clade with extensive intraspecific genetic diversity. Furthermore, B. suis was determined to be paraphyletic in our analyses, only forming a monophyletic clade when the B. canis genome was included. Using a molecular clock with these data suggests that most Brucella species diverged from their common B. ovis ancestor in the past 86,000 to 296,000 years, which precedes the domestication of their livestock hosts. Detailed knowledge of the Brucella phylogeny will lead to an improved understanding of the ecology, evolutionary history, and host relationships for this genus and can be used for determining appropriate genotyping approaches for rapid detection and diagnostic assays for molecular epidemiological and clinical studies.

Phylogeography of Francisella tularensis: global expansion of a highly fit clone.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

Genomic islands from five strains of Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species. RESULTS: We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described. CONCLUSION: Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.

Novel human alpha1a-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function.

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the alpha(1a)-adrenoceptor (alpha(1a)AR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type alpha(1a)AR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose-response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for alpha(1a)AR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor-G protein coupling. In conclusion, four naturally-occurring human alpha(1a)AR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide alpha(1a)AR SNP choice for future clinical studies.

Incongruence between multi-locus sequence analysis (MLSA) and whole-genome-based phylogenies: Pseudomonas syringae pathovar pisi as a cautionary tale.

Previous phylogenies, built using a subset of genomic loci, split Pseudomonas syringae pv. pisi into two well-supported clades and implied convergence in host range for these lineages. The analysis of phenotypic and genotypic data within the context of this phylogenetic relationship implied further convergence at the level of virulence gene loss and acquisition. We generate draft genome assemblies for two additional P. syringae strains, isolated from diseased pea plants, and demonstrate incongruence between phylogenies created from a subset of the data compared with the whole genomes. Our whole-genome analysis demonstrates that strains classified as pv. pisi actually form a coherent monophyletic clade, so that apparent convergence is actually the product of shared ancestry. We use this example to urge caution when making evolutionary inferences across closely related strains of P. syringae.

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

BACKGROUND: The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. RESULTS: Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. CONCLUSION: The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

Phylogeography of Bacillus anthracis in the country of Georgia shows evidence of population structuring and is dissimilar to other regional genotypes.

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.

A decade of plague in Mahajanga, Madagascar: insights into the global maritime spread of pandemic plague.

UNLABELLED: A cluster of human plague cases occurred in the seaport city of Mahajanga, Madagascar, from 1991 to 1999 following 62 years with no evidence of plague, which offered insights into plague pathogen dynamics in an urban environment. We analyzed a set of 44 Mahajanga isolates from this 9-year outbreak, as well as an additional 218 Malagasy isolates from the highland foci. We sequenced the genomes of four Mahajanga strains, performed whole-genome sequence single-nucleotide polymorphism (SNP) discovery on those strains, screened the discovered SNPs, and performed a high-resolution 43-locus multilocus variable-number tandem-repeat analysis of the isolate panel. Twenty-two new SNPs were identified and defined a new phylogenetic lineage among the Malagasy isolates. Phylogeographic analysis suggests that the Mahajanga lineage likely originated in the Ambositra district in the highlands, spread throughout the northern central highlands, and was then introduced into and became transiently established in Mahajanga. Although multiple transfers between the central highlands and Mahajanga occurred, there was a locally differentiating and dominant subpopulation that was primarily responsible for the 1991-to-1999 Mahajanga outbreaks. Phylotemporal analysis of this Mahajanga subpopulation revealed a cycling pattern of diversity generation and loss that occurred during and after each outbreak. This pattern is consistent with severe interseasonal genetic bottlenecks along with large seasonal population expansions. The ultimate extinction of plague pathogens in Mahajanga suggests that, in this environment, the plague pathogen niche is tenuous at best. However, the temporary large pathogen population expansion provides the means for plague pathogens to disperse and become ecologically established in more suitable nonurban environments. IMPORTANCE: Maritime spread of plague led to the global dissemination of this disease and affected the course of human history. Multiple historical plague waves resulted in massive human mortalities in three classical plague pandemics: Justinian (6th and 7th centuries), Middle Ages (14th to 17th centuries), and third (mid-1800s to the present). Key to these events was the pathogen's entry into new lands by "plague ships" via seaport cities. Although initial disease outbreaks in ports were common, they were almost never sustained for long and plague pathogens survived only if they could become established in ecologically suitable habitats. Although plague pathogens' ability to invade port cities has been essential for intercontinental spread, these regions have not proven to be a suitable long-term niche. The disease dynamics in port cities such as Mahajanga are thus critical to plague pathogen amplification and dispersal into new suitable ecological niches for the observed global long-term maintenance of plague pathogens.

Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection.

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications.

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.