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Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host., Competing Interests: The authors have declared that no competing interests exist.

Burkholderia pseudomallei is the environmental bacillus that causes melioidosis; a disease clinically significant in Australia and Southeast Asia but emerging in tropical and sub-tropical regions around the globe. Previous studies have placed the ancestral population of the organism in Australia with a single lineage disseminated to Southeast Asia. We have previously characterized B. pseudomallei isolates from New Guinea and the Torres Strait archipelago; remote regions that share paleogeographic ties with Australia. These studies identified regional biogeographical boundaries. In this study, we utilize whole-genome sequencing to reconstruct ancient evolutionary relationships and ascertain correlations between paleogeography and present-day distribution of this bacterium in Australasia. Our results indicate that B. pseudomallei from New Guinea fall into a single clade within the Australian population. Furthermore, clades from New Guinea are region-specific; an observation possibly linked to limited recent anthropogenic influence in comparison to mainland Australia and Southeast Asia. Isolates from the Torres Strait archipelago were distinct yet scattered among those from mainland Australia. These results provide evidence that the New Guinean and Torres Strait lineages may be remnants of an ancient portion of the Australian population. Rising sea levels isolated New Guinea and the Torres Strait Islands from each other and the Australian mainland, and may have allowed long-term isolated evolution of these lineages, providing support for a theory of microbial biogeography congruent with that of macro flora and fauna. Moreover, these findings indicate that contemporary microbial biogeography theories should consider recent and ongoing impacts of globalisation and human activity., Competing Interests: The authors have declared that no competing interests exist.

Twelve Burkholderia pseudomallei isolates collected over a 32-month period from a patient with chronic melioidosis demonstrated identical multilocus sequence types (STs). However, whole-genome sequencing suggests a polyclonal infection. This study is the first to report a mixed infection with the same ST., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Melioidosis is an emerging infectious disease caused by the soil bacterium Burkholderia pseudomallei. In diagnostic and forensic settings, molecular detection assays need not only high sensitivity with low limits of detection but also high specificity. In a direct comparison of published and newly developed TaqMan PCR assays, we found the TTS1-orf2 assay to be superior in detecting B. pseudomallei directly from clinical specimens. The YLF/BTFC multiplex assay (targeting the Yersinia-like fimbrial/Burkholderia thailandensis-like flagellum and chemotaxis region) also showed high diagnostic sensitivity and provides additional information on possible geographic origin.

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.

Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.

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.

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.

Endemic melioidosis is caused by genetically diverse Burkholderia pseudomallei strains. However, clonal outbreaks (multiple cases caused by 1 strain) have occurred, such as from contaminated potable water. B. pseudomallei is designated a group B bioterrorism agent, which necessitates rapidly recognizing point-source outbreaks. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) can identify genetically related isolates, but results take several days to obtain. We developed a simplified 4-locus multilocus variable number tandem repeat analysis (MLVA-4) for rapid typing and compared results with PFGE and MLST for a large number of well-characterized B. pseudomallei isolates. MLVA-4 compared favorably with MLST and PFGE for the same isolates; it discriminated between 65 multilocus sequence types and showed relatedness between epidemiologically linked isolates from outbreak clusters and between isolates from individual patients. MLVA-4 can establish or refute that a clonal outbreak of melioidosis has occurred within 8 hours of receipt of bacterial strains.

Molecular typing methods for Burkholderia pseudomallei have been successful at assigning isolates into epidemiologically related groups, but have not been able to detect differences and define evolutionary patterns within groups. Our variable number tandem repeat (VNTR) analysis of a set of 121 Australian B. pseudomallei isolates, 104 of which were associated with nine epidemiological groups, provides fine scale differentiation even among very closely related isolates. We used a Bayesian model based upon mutation accumulation patterns to define the close phylogenetic relationships within these epidemiological groups. Our results reveal that genetic diversity can exist within a very small geographic area, and that low levels of diversity can exist even within a single infection. These methods provide the ability to generate robust evolutionary hypotheses that enable tracking of B. pseudomallei in forensic and epidemiological outbreaks at fine phylogenetic scales.

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 highlevel 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. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identied. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is a bacterium endemic to Southeast Asia and northern Australia, but it has not been found to occur endemically in the United States. We report an ostensibly autochthonous case of melioidosis in the United States. Despite an extensive investigation, the source of exposure was not identified. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]