Sequencing by Binding rivals error-corrected Sequencing by Synthesis technology for accurate detection and quantification of minor (<0.1%) subpopulation variants.

Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications including detection of drug resistant pathogens and somatic variant detection in oncology. To enable these applications, wet lab enhancements and bioinformatic error correction methods have been developed for 'sequencing by synthesis' technology to reduce its inherent sequencing error rate. A recently available sequencing approach termed 'sequencing by binding' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using 'sequencing by binding' for the detection of ultra-rare subpopulations down to 0.001%.

Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study.

BACKGROUND: Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment. METHODS: We did a retrospective longitudinal cohort study of adults (aged ≥18 years) with culture-positive pulmonary tuberculosis who received at least 4 months of a bedaquiline-containing regimen from 12 drug-resistant tuberculosis treatment facilities in Cape Town, South Africa, between Jan 20, 2016, and Nov 20, 2017. Sputum was programmatically collected at baseline (ie, before bedaquiline initiation) and each month to monitor treatment response per the national algorithm. The last available isolate from the sputum collected at or after 4 months of bedaquiline was designated the follow-up isolate. Phenotypic drug susceptibility testing for bedaquiline was done on baseline and follow-up isolates in MGIT960 media (WHO-recommended critical concentration of 1 µg/mL). Targeted deep sequencing for Rv0678, atpE, and pepQ, as well as whole-genome sequencing were also done. FINDINGS: In total, 40 (31%) of 129 patients from an estimated pool were eligible for this study. Overall, three (8%) of 38 patients assessable by phenotypic drug susceptibility testing for bedaquiline had primary resistance, 18 (47%) gained resistance (acquired or reinfection), and 17 (45%) were susceptible at both baseline and follow-up. Several Rv0678 and pepQ single-nucleotide polymorphisms and indels were associated with resistance. Although variants occurred in Rv0676c and Rv1979c, these variants were not associated with resistance. Targeted deep sequencing detected low-level variants undetected by whole-genome sequencing; however, none were in genes without variants already detected by whole-genome sequencing. Patients with baseline fluoroquinolone resistance, clofazimine exposure, and four or less effective drugs were more likely to have bedaquiline-resistant gain. Resistance gain was primarily due to acquisition; however, some reinfection by resistant strains occurred. INTERPRETATION: Bedaquiline-resistance gain, for which we identified risk factors, was common in these programmatically treated patients with sustained culture positivity. Our study highlights risks associated with implementing life-saving new drugs and shows evidence of bedaquiline-resistance transmission. Routine drug susceptibility testing should urgently accompany scale-up of new drugs; however, rapid drug susceptibility testing for bedaquiline remains challenging given the diversity of variants observed. FUNDING: Doris Duke Charitable Foundation, US National Institute of Allergy and Infectious Diseases, South African Medical Research Council, National Research Foundation, Research Foundation Flanders, Stellenbosch University Faculty of Medicine Health Sciences, South African National Research Foundation, Swiss National Science Foundation, and Wellcome Trust.

NHP BurkPx: A multiplex serodiagnostic bead assay to monitor Burkholderia pseudomallei exposures in non-human primates.

BACKGROUND: Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, infecting humans and non-human primates (NHP) through contaminated soil or water. World-wide there are an estimated 165,000 human melioidosis cases each year, but recordings of NHP cases are sporadic. Clinical detection of melioidosis in humans is primarily by culturing B. pseudomallei, and there are no standardized detection protocols for NHP. NHP are an important animal model for melioidosis research including clinical trials and development of biodefense countermeasures. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the diagnostic potential of the multiple antigen serological assay, BurkPx, in NHP using two sera sets: (i) 115 B. pseudomallei-challenged serum samples from 80 NHP collected each week post-exposure (n = 52) and at euthanasia (n = 47), and (ii) 126 B. pseudomallei-naïve/negative serum samples. We observed early IgM antibody responses to carbohydrate antigens followed by IgG antibody recognition to multiple B. pseudomallei protein antigens during the second week of infection. B. pseudomallei negative serum samples had low to intermediate antibody cross reactivity to the antigens in this assay. Infection time was predicted as the determining factor in the variation of antibody responses, with 77.67% of variation explained by the first component of the principal component analysis. A multiple antigen model generated a binary prediction metric ([Formula: see text]), which when applied to all data resulted in 100% specificity and 63.48% sensitivity. Removal of week 1 B. pseudomallei challenged serum samples increased the sensitivity of the model to 95%. CONCLUSION/SIGNIFICANCE: We employed a previously standardized assay for humans, the BurkPx assay, and assessed its diagnostic potential for detection of B. pseudomallei exposure in NHP. The assay is expected to be useful for surveillance in NHP colonies, in investigations of suspected accidental releases or exposures, and for identifying vaccine correlates of protection.

Phylogeography and transmission of M. tuberculosis in Moldova: A prospective genomic analysis.

BACKGROUND: The incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries. METHODS AND FINDINGS: We conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited. CONCLUSIONS: The MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis.

Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin-resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of pncA fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.

The Role of Growth Directors in Controlling the Morphology of Hematite Nanorods.

The control of the growth of hematite nanoparticles from iron chloride solutions under hydrothermal conditions in the presence of two different structure promoters has been studied using a range of both structural and spectroscopic techniques including the first report of photo induced force microscopy (PiFM) to map the topographic distribution of the structure-directing agents on the developing nanoparticles. We show that the shape of the nanoparticles can be controlled using the concentration of phosphate ions up to a limit determined to be ~6 × 10-3 mol. Akaganéite (ß-FeOOH) is a major component of the nanoparticles formed in the absence of structure directors but only present in the very early stages (< 8 h) of particle growth when phosphate is present. The PiFM data suggest a correlation between the areas in which phosphate ions are adsorbed and areas where akaganéite persists on the surface. In contrast, goethite (α-FeOOH) is a directly observed precursor of the hematite nanorods when 1,2-diamino propane is present. The PiFM data shows goethite in the center of the developing particles consistent with a mechanism in which the iron hydroxide re-dissolves and precipitates at the nanorod ends as hematite.

Utility of Targeted, Amplicon-Based Deep Sequencing To Detect Resistance to First-Line Tuberculosis Drugs in Botswana.

Multidrug-resistant tuberculosis (TB) is an alarming threat, and targeted deep sequencing (DS) may be an effective method for rapid identification of drug-resistant profiles, including detection of heteroresistance. We evaluated the sensitivity and specificity of targeted DS versus phenotypic drug susceptibility testing (pDST) among patients starting first-line anti-TB therapy in Botswana. Overall, we found high concordance between DS and pDST. Lower sensitivity of DS, which targets established high-confidence resistance variants, was observed for detecting isoniazid resistance among HIV-infected patients.

Mixed Mycobacterium tuberculosis-Strain Infections Are Associated With Poor Treatment Outcomes Among Patients With Newly Diagnosed Tuberculosis, Independent of Pretreatment Heteroresistance.

Background: Heteroresistant Mycobacterium tuberculosis infections (defined as concomitant infection with drug-resistant and drug-susceptible strains) may explain the higher risk of poor tuberculosis treatment outcomes observed among patients with mixed-strain M. tuberculosis infections. We investigated the clinical effect of mixed-strain infections while controlling for pretreatment heteroresistance in a population-based sample of patients with tuberculosis starting first-line tuberculosis therapy in Botswana. Methods: We performed 24-locus mycobacterial interspersed repetitive unit-variable number tandem-repeat analysis and targeted deep sequencing on baseline primary cultured isolates to detect mixed infections and heteroresistance, respectively. Drug-sensitive, micro-heteroresistant, macro-heteroresistant, and fixed-resistant infections were defined as infections in which the frequency of resistance was <0.1%, 0.1%-4%, 5%-94%, and ≥95%, respectively, in resistance-conferring domains of the inhA promoter, the katG gene, and the rpoB gene. Results: Of the 260 patients with tuberculosis included in the study, 25 (9.6%) had mixed infections and 30 (11.5%) had poor treatment outcomes. Micro-heteroresistance, macro-heteroresistance, and fixed resistance were found among 11 (4.2%), 2 (0.8%), and 11 (4.2%), respectively, for isoniazid and 21 (8.1%), 0 (0%), and 10 (3.8%), respectively, for rifampicin. In multivariable analysis, mixed infections but not heteroresistant infections independently predicted poor treatment outcomes. Conclusions: Among patients starting first-line tuberculosis therapy in Botswana, mixed infections were associated with poor tuberculosis treatment outcomes, independent of heteroresistance.

Mycobacterium tuberculosis Subculture Results in Loss of Potentially Clinically Relevant Heteroresistance.

Multidrug-resistant tuberculosis (TB) presents a major public health dilemma. Heteroresistance, the coexistence of drug-resistant and drug-susceptible strains or of multiple drug-resistant strains with discrete haplotypes, may affect accurate diagnosis and the institution of effective treatment. Subculture, or passage of cells onto fresh growth medium, is utilized to preserve Mycobacterium tuberculosis cell lines and is universally employed in TB diagnostics. The impact of such passages, typically performed in the absence of drug, on drug-resistant subpopulations is hypothesized to vary according to the competitive costs of genotypic resistance-associated variants. We applied ultradeep next-generation sequencing to 61 phenotypically rifampin-monoresistant (n = 17) and preextensively (n = 41) and extensively (n = 3) drug-resistant isolates with presumptive heteroresistance at two time points in serial subculture. We found significant dynamic loss of minor-variant resistant subpopulations across all analyzed resistance-determining regions, including eight isolates (13%) whose antibiogram data would have transitioned from resistant to susceptible for at least one drug through subculture. Surprisingly, some resistance-associated variants appeared to be selected for in subculture.

Cryptic Microheteroresistance Explains Mycobacterium tuberculosis Phenotypic Resistance.

RATIONALE: Minority drug-resistant Mycobacterium tuberculosis subpopulations can be associated with phenotypic resistance but are poorly detected by Sanger sequencing or commercial molecular diagnostic assays. OBJECTIVES: To determine the role of targeted next-generation sequencing in resolving these minor variant subpopulations. METHODS: We used single molecule overlapping reads (SMOR), a targeted next-generation sequencing approach that dramatically reduces sequencing error, to analyze primary cultured isolates phenotypically resistant to rifampin, fluoroquinolones, or aminoglycosides, but for which Sanger sequencing found no resistance-associated variants (RAVs) within respective resistance-determining regions (study group). Isolates also underwent single-colony selection on antibiotic-containing agar, blinded to sequencing results. As a positive control, isolates with multiple colocalizing chromatogram peaks were also analyzed (control group). MEASUREMENTS AND MAIN RESULTS: Among 61 primary culture isolates (25 study group and 36 control group), SMOR described 66 (49%) and 45 (33%) of 135 total heteroresistant RAVs at frequencies less than 5% and less than 1% of the total mycobacterial population, respectively. In the study group, SMOR detected minor resistant variant subpopulations in 80% (n = 20/25) of isolates with no Sanger-identified RAVs (median subpopulation size, 1.0%; interquartile range, 0.2-3.9%). Single-colony selection on drug-containing media corroborated SMOR results for 90% (n = 18/20) of RAV-containing specimens, and the absence of RAVs in 60% (n = 3/5) of isolates. Overall, Sanger sequencing was concordant with SMOR for 77% (n = 53/69) of macroheteroresistant (5-95% total population), but only 5% of microheteroresistant (<5%) subpopulations (n = 3/66) across both groups. CONCLUSIONS: Cryptic minor variant mycobacterial subpopulations exist below the resolving capability of current drug susceptibility testing methodologies, and may explain an important proportion of false-negative resistance determinations.

Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.

During routine screening for Burkholderia pseudomallei from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43T, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were coisolated with B. pseudomallei on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analyses demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole-genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the B. pseudomallei complex. Molecular analyses clearly demonstrated that strains MSMB43T, MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed, with the type strain MSMB43T (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382).IMPORTANCEBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species, with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov. is phylogenetically distinct from B. pseudomallei and other members of the B. pseudomallei complex, making it the fifth member of this important group of bacteria.

Development of Immunoassays for Burkholderia pseudomallei Typical and Atypical Lipopolysaccharide Strain Typing.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used by B. pseudomallei Isolates of B. pseudomallei have been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS-specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide-specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains of Burkholderia and non-Burkholderia species showed that the assays are reactive to B. pseudomallei and Burkholderia mallei strains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable for B. pseudomallei LPS typing.

Bilayer Networks within a Hydrogel Shell: A Robust Chassis for Artificial Cells and a Platform for Membrane Studies.

The ability to make artificial lipid bilayers compatible with a wide range of environments, and with sufficient structural rigidity for manual handling, would open up a wealth of opportunities for their more routine use in real-world applications. Although droplet interface bilayers (DIBs) have been demonstrated in a host of laboratory applications, from chemical logic to biosynthesis reaction vessels, their wider use is hampered by a lack of mechanical stability and the largely manual methods employed in their production. Multiphase microfluidics has enabled us to construct hierarchical triple emulsions with a semipermeable shell, in order to form robust, bilayer-bound, droplet networks capable of communication with their external surroundings. These constructs are stable in air, water, and oil environments and overcome a critical obstacle of achieving structural rigidity without compromising environmental interaction. This paves the way for practical application of artificial membranes or droplet networks in diverse areas such as medical applications, drug testing, biophysical studies and their use as synthetic cells.

The Effects of Signal Erosion and Core Genome Reduction on the Identification of Diagnostic Markers.

UNLABELLED: Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives. IMPORTANCE: A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei/mallei were identified. By using only public genomes, a larger number of markers were identified, due to undersampling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined.

Diverse Burkholderia Species Isolated from Soils in the Southern United States with No Evidence of B. pseudomallei.

The global distribution of the soil-dwelling bacterium Burkholderia pseudomallei, causative agent of melioidosis, is poorly understood. We used established culturing methods developed for B. pseudomallei to isolate Burkholderia species from soil collected at 18 sampling sites in three states in the southern United States (Arizona (n = 4), Florida (n = 7), and Louisiana (n = 7)). Using multi-locus sequence typing (MLST) of seven genes, we identified 35 Burkholderia isolates from these soil samples. All species belonged to the B. cepacia complex (Bcc), including B. cenocepacia, B. cepacia, B. contaminans, B. diffusa, B. metallica, B. seminalis, B. vietnamiensis and two unnamed members of the Bcc. The MLST analysis provided a high level of resolution among and within these species. Despite previous clinical cases within the U.S. involving B. pseudomallei and its close phylogenetic relatives, we did not isolate any of these taxa. The Bcc contains a number of opportunistic pathogens that cause infections in cystic fibrosis patients. Interestingly, we found that B. vietnamiensis was present in soil from all three states, suggesting it may be a common component in southern U.S. soils. Most of the Burkholderia isolates collected in this study were from Florida (30/35; 86%), which may be due to the combination of relatively moist, sandy, and acidic soils found there compared to the other two states. We also investigated one MLST gene, recA, for its ability to identify species within Burkholderia. A 365bp fragment of recA recovered nearly the same species-level identification as MLST, thus demonstrating its cost effective utility when conducting environmental surveys for Burkholderia. Although we did not find B. pseudomallei, our findings document that other diverse Burkholderia species are present in soils in the southern United States.

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

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

Genomic characterization of Burkholderia pseudomallei isolates selected for medical countermeasures testing: comparative genomics associated with differential virulence.

Burkholderia pseudomallei is the causative agent of melioidosis and a potential bioterrorism agent. In the development of medical countermeasures against B. pseudomallei infection, the US Food and Drug Administration (FDA) animal Rule recommends using well-characterized strains in animal challenge studies. In this study, whole genome sequence data were generated for 6 B. pseudomallei isolates previously identified as candidates for animal challenge studies; an additional 5 isolates were sequenced that were associated with human inhalational melioidosis. A core genome single nucleotide polymorphism (SNP) phylogeny inferred from a concatenated SNP alignment from the 11 isolates sequenced in this study and a diverse global collection of isolates demonstrated the diversity of the proposed Animal Rule isolates. To understand the genomic composition of each isolate, a large-scale blast score ratio (LS-BSR) analysis was performed on the entire pan-genome; this demonstrated the variable composition of genes across the panel and also helped to identify genes unique to individual isolates. In addition, a set of ~550 genes associated with pathogenesis in B. pseudomallei were screened against the 11 sequenced genomes with LS-BSR. Differential gene distribution for 54 virulence-associated genes was observed between genomes and three of these genes were correlated with differential virulence observed in animal challenge studies using BALB/c mice. Differentially conserved genes and SNPs associated with disease severity were identified and could be the basis for future studies investigating the pathogenesis of B. pseudomallei. Overall, the genetic characterization of the 11 proposed Animal Rule isolates provides context for future studies involving B. pseudomallei pathogenesis, differential virulence, and efficacy to therapeutics.

Burkholderia pseudomallei type G in Western Hemisphere.

Burkholderia pseudomallei isolates from the Western Hemisphere are difficult to differentiate from those from regions in which melioidosis is traditionally endemic. We used internal transcribed spacer typing to determine that B. pseudomallei isolates from the Western Hemisphere are consistently type G. Knowledge of this relationship might be useful for epidemiologic investigations.

Detection of Burkholderia pseudomallei O-antigen serotypes in near-neighbor species.

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