Correction: Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

[This corrects the article DOI: 10.1371/journal.pbio.3001052.].

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

An investigation of the seasonal relationships between meteorological factors, water quality, and sporadic cases of Legionnaires' disease in Washington, DC.

Since the discovery of Legionnaires' disease (LD), limited progress has been made in understanding the epidemiology of sporadic cases of LD. Outbreaks have confirmed that air conditioning and potable water systems can be sources of community-acquired LD. However, studying the association between water quality and LD incidence has been challenging due to the heterogeneity of water systems across large geographic areas. Furthermore, although seasonal trends in incidence have been linked to increased rainfall and temperatures, the large geographic units have posed similar difficulties. To address this issue, a retrospective ecological study was conducted in Washington, DC, from 2001 to 2019. The study identified aseasonal pattern of LD incidence, with the majority of cases occurring between June and December, peaking in August, October, and November. Increased temperature was found to be associated with LD incidence. In surface water, higher concentrations of manganese, iron, and strontium were positively associated with LD, while aluminum and orthophosphate showed a negative association. Intreatment plant water, higher concentrations of total organic carbon, aluminum, barium, and chlorine were positively associated with LD, while strontium, zinc, and orthophosphate showed a negative association. The results for orthophosphates and turbidity were inconclusive, indicating the need for further research.

Sigma Receptor Ligands Prevent COVID Mortality In Vivo: Implications for Future Therapeutics.

The emergence of lethal coronaviruses follows a periodic pattern which suggests a recurring cycle of outbreaks. It remains uncertain as to when the next lethal coronavirus will emerge, though its eventual emergence appears to be inevitable. New mutations in evolving SARS-CoV-2 variants have provided resistance to current antiviral drugs, monoclonal antibodies, and vaccines, reducing their therapeutic efficacy. This underscores the urgent need to investigate alternative therapeutic approaches. Sigma receptors have been unexpectedly linked to the SARS-CoV-2 life cycle due to the direct antiviral effect of their ligands. Coronavirus-induced cell stress facilitates the formation of an ER-derived complex conducive to its replication. Sigma receptor ligands are believed to prevent the formation of this complex. Repurposing FDA-approved drugs for COVID-19 offers a timely and cost-efficient strategy to find treatments with established safety profiles. Notably, diphenhydramine, a sigma receptor ligand, is thought to counteract the virus by inhibiting the creation of ER-derived replication vesicles. Furthermore, lactoferrin, a well-characterized immunomodulatory protein, has shown antiviral efficacy against SARS-CoV-2 both in laboratory settings and in living organisms. In the present study, we aimed to explore the impact of sigma receptor ligands on SARS-CoV-2-induced mortality in ACE2-transgenic mice. We assessed the effects of an investigational antiviral drug combination comprising a sigma receptor ligand and an immunomodulatory protein. Mice treated with sigma-2 receptor ligands or diphenhydramine and lactoferrin exhibited improved survival rates and rapid rebound in mass following the SARS-CoV-2 challenge compared to mock-treated animals. Clinical translation of these findings may support the discovery of new treatment and research strategies for SARS-CoV-2.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened ∼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.

Identification of antiviral antihistamines for COVID-19 repurposing.

There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. Although repurposed drugs with favorable safety profiles could have significant benefit, widely available prevention or treatment options for COVID-19 have yet to be identified. Efforts to identify approved drugs with in vitro activity against SARS-CoV-2 resulted in identification of antiviral sigma-1 receptor ligands, including antihistamines in the histamine-1 receptor binding class. We identified antihistamine candidates for repurposing by mining electronic health records of usage in population of more than 219,000 subjects tested for SARS-CoV-2. Usage of diphenhydramine, hydroxyzine and azelastine was associated with reduced incidence of SARS-CoV-2 positivity in subjects greater than age 61. We found diphenhydramine, hydroxyzine and azelastine to exhibit direct antiviral activity against SARS-CoV-2 in vitro. Although mechanisms by which specific antihistamines exert antiviral effects is not clear, hydroxyzine, and possibly azelastine, bind Angiotensin Converting Enzyme-2 (ACE2) and the sigma-1 receptor as off-targets. Clinical studies are needed to measure the effectiveness of diphenhydramine, hydroxyzine and azelastine for disease prevention, for early intervention, or as adjuvant therapy for severe COVID-19.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose.

BACKGROUND: The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. RESULTS: In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant-) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. CONCLUSIONS: Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.

Spatial transcriptomes within the Pseudomonas aeruginosa biofilm architecture.

Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.

An avirulent Burkholderia pseudomallei ∆purM strain with atypical type B LPS: expansion of the toolkit for biosafe studies of melioidosis.

BACKGROUND: The work was undertaken to expand the tools available for researching Burkholderia pseudomallei (Bp), the etiological agent of the tropical disease melioidosis. Melioidosis has the potential to pose a severe threat to public health and safety. In the United States, Bp is listed as a Tier-1 select agent by the Centers for Disease Control and Prevention (CDC), thus requiring high levels of regulation and biosafety level 3 (BSL3) facilities for experimental manipulation of live organisms. An avirulent ∆purM derivative of strain 1026b (Bp82) has proven to be a valuable tool for biosafe research as a select-agent excluded strain, but the high level of genetic diversity between Bp strains necessitates an expansion of the biosafe toolset. RESULTS: The ∆purM mutation was recapitulated in the Bp 576a strain, a serotype B background. An important difference between strains 1026b and 576a is the lipopolysaccharide (LPS), a major virulence factor and protective antigen. Polyclonal sera from 1026b-challenged non-human primates showed no cross reactivity with strain 576a LPS and low reactivity with whole cell lysate. Strain 576a replicates to higher levels in mouse organs and induces more TNF-α in the lungs of BALB/c mice compared to 1026b. The newly created Bp 576a ∆purM strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was abrogated with single-copy complementation. Bp 576mn was unable to replicate in human cells and was avirulent in BALB/c mice following high-dose intranasal inoculation, similar to Bp82. Organ loads indicated a significant reduction in bacterial replication. CONCLUSIONS: In this work, the new biosafe strain 576mn with atypical type B LPS was generated. This strain should prove a valuable addition to the toolkit for biosafe studies of Bp and development of therapeutic and preventative strategies aimed at combatting melioidosis. Strain 576mn is an ideal candidate for select-agent exclusion.

Transcript amplification from single bacterium for transcriptome analysis.

Total transcript amplification (TTA) from single eukaryotic cells for transcriptome analysis is established, but TTA from a single prokaryotic cell presents additional challenges with much less starting material, the lack of poly(A)-tails, and the fact that the messages can be polycistronic. Here, we describe a novel method for single-bacterium TTA using a model organism, Burkholderia thailandensis, exposed to a subinhibitory concentration of the antibacterial agent, glyphosate. Utilizing a B. thailandensis microarray to assess the TTA method showed low fold-change bias (less than twofold difference and Pearson correlation coefficient R ≈ 0.87-0.89) and drop-outs (4%-6% of 2842 detectable genes), compared with data obtained from the larger-scale nonamplified RNA samples. Further analysis of the microarray data suggests that B. thailandensis, when exposed to the aromatic amino acid biosynthesis inhibitor glyphosate, induces (or represses) genes to possibly recuperate and balance the intracellular amino acid pool. We validated our single-cell microarray data at the multi-cell and single-cell levels with lacZ and gfp reporter-gene fusions, respectively. Sanger sequencing of 192 clones generated from the TTA product of a single cell, with and without enrichment by elimination of rRNA and tRNA, detected only B. thailandensis sequences with no contamination. These data indicate that RNA-seq of TTA from a single cell is possible using this novel method.

Genomic and Phylogenetic Analysis of Bacillus cereus Biovar anthracis Isolated from Archival Bone Samples Reveals Earlier Natural History of the Pathogen.

(1) Background: Bacillus cereus biovar anthracis (Bcbva) was the causative agent of an anthrax-like fatal disease among wild chimpanzees in 2001 in Côte d'Ivoire. Before this, there had not been any description of an anthrax-like disease caused by typically avirulent Bacillus cereus. Genetic analysis found that B. cereus had acquired two anthrax-like plasmids, one a pXO1-like toxin producing plasmid and the other a pXO2-like plasmid encoding capsule. Bcbva caused animal fatalities in Cameroon, Democratic Republic of Congo, and the Central African Republic between 2004 and 2012. (2) Methods: The pathogen had acquired plasmids in the wild and that was discovered as the cause of widespread animal fatalities in the early 2000s. Primate bones had been shipped out of the endemic zone for anthropological studies prior to the realized danger of contamination with Bcbva. Spores were isolated from the bone fragments and positively identified as Bcbva. Strains were characterized by classical microbiological methods and qPCR. Four new Bcbva isolates were whole-genome sequenced. Chromosomal and plasmid phylogenomic analysis was performed to provide temporal and spatial context to these new strains and previously sequenced Bcbva. Tau and principal component analyses were utilized to identify genetic and spatial case patterns in the Taï National Park anthrax zone. (3) Results: Preliminary studies positively identified Bcbva presence in several archival bone fragments. The animals in question died between 1994 and 2010. Previously, the earliest archival strains of Bcbva were identified in 1996. Though the pathogen has a homogeneous genome, spatial analyses of a subset of mappable isolates from Taï National Park revealed strains found closer together were generally more similar, with strains from chimpanzees and duikers having the widest distribution. Ancestral strains were located mostly in the west of the park and had lower spatial clustering compared to more recent isolates, indicating a local increase in genetic diversity of Bcbva in the park over space and time. Global clustering analysis indicates patterns of genetic diversity and distance are shared between the ancestral and more recently isolated type strains. (4) Conclusions: Our strains have the potential to unveil historical genomic information not available elsewhere. This information sheds light on the evolution and emergence of a dangerous anthrax-causing pathogen.

Beyond the spore, the exosporium sugar anthrose impacts vegetative Bacillus anthracis gene regulation in cis and trans.

The Bacillus anthracis exosporium nap is the outermost portion of spore that interacts with the environment and host systems. Changes to this layer have the potential to impact wide-ranging physiological and immunological processes. The unique sugar, anthrose, normally coats the exosporium nap at its most distal points. We previously identified additional mechanisms rendering B. anthracis anthrose negative. In this work, several new ant - B. anthracis strains are identified and the impact of anthrose negativity on spore physiology is investigated. We demonstrate that live-attenuated Sterne vaccines as well as culture filtrate anthrax vaccines generate antibodies targeting non-protein components of the spore. The role of anthrose as a vegetative B. anthracis Sterne signaling molecule is implicated by luminescent expression strain assays, RNA-seq experiments, and toxin secretion analysis by western blot. Pure anthrose and the sporulation-inducing nucleoside analogue decoyinine had similar effects on toxin expression. Co-culture experiments demonstrated gene expression changes in B. anthracis depend on intracellular anthrose status (cis) in addition to anthrose status of extracellular interactions (trans). These findings provide a mechanism for how a unique spore-specific sugar residue affects physiology, expression and genetics of vegetative B. anthracis with impacts on the ecology, pathogenesis, and vaccinology of anthrax.

Spatial and phylogenetic patterns reveal hidden infection sources of Bacillus anthracis in an anthrax outbreak in Son La province, Vietnam.

Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

Characterization of Bacillus anthracis replication and persistence on environmental substrates associated with wildlife anthrax outbreaks.

Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 105 spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.

Genome-scale CRISPR screens identify host factors that promote human coronavirus infection.

BACKGROUND: The COVID-19 pandemic has resulted in 275 million infections and 5.4 million deaths as of December 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as antigenically novel SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. METHODS: To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. Gene knockdown, CRISPR knockout, and small molecule testing in Vero, HEK293, and human small airway epithelial cells were used to verify our findings. RESULTS: While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. The website https://sarscrisprscreens.epi.ufl.edu/ was created to allow visualization and comparison of SARS-CoV2 CRISPR screens in a uniformly analyzed way. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. The role of EDC4 and XRN1 in coronavirus replication in human small airway epithelial cells was verified. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. CONCLUSIONS: Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.

An Investigation of Burkholderia pseudomallei Seroprevalence in Market Pigs Slaughtered at Selected Pig Abattoirs in Uganda.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a disease of humans and animals. It is primarily transmitted through direct contact with contaminated soil and surface water. The epidemiology of this pathogen in Africa, including Uganda, is largely unknown. The objectives of this study were to estimate the seroprevalence of B. pseudomallei in pigs slaughtered in central Uganda and to identify potential hotspots for this pathogen in the country. A total of 1035 pig sera were analyzed for serological responses to B. pseudomallei with type A and type B LPS using OPS type A and OPS type B ELISAs. Of the 1035 samples, 75 (7.25%, 95% CI: 5.8-9%) were seropositive to the OPS-A ELISA using a two standard deviations (SD) cutoff and 19 (1.84%, 95% CI: 1.2-2.9%) at 3 SD. For the OPS-B ELISA, 93/1035 (8.99%, 95% CI: 7.4-10.9%) were seropositive at the 2 SD cutoff, and 28/1035 (2.71%, 95% CI: 1.9-3.9%) at the 3 SD cutoff. Pigs slaughtered in central Uganda were exposed to B. pseudomallei, and there is a higher seroprevalence in the rainy months. Public health awareness campaigns about melioidosis may be needed.

The Burkholderia pseudomallei Δasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice.

Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b Δasd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei Δasd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented Δasd mutant. RAW264.7 cells infected by the Δasd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The Δasd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei Δasd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

Identification of a PadR-type regulator essential for intracellular pathogenesis of Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease endemic to the tropics. Melioidosis manifests in various ways ranging from acute skin lesions to pneumonia and, in rare cases, infection of the central nervous system. Bp is a facultative intracellular pathogen and it can infect various cell types. The Bp intracellular lifecycle has been partially elucidated and is highly complex. Herein, we have identified a transcriptional regulator, BP1026B_II1198, that is differentially expressed as Bp transits through host cells. A deletion mutant of BP1026B_II1198 was attenuated in RAW264.7 cell and BALB/c mouse infection. To further characterize the function of this transcriptional regulator, we endeavored to determine the regulon of BP1026B_II1198. RNA-seq analysis showed the global picture of genes regulated while ChIP-seq analysis identified two specific BP1026B_II1198 binding regions on chromosome II. We investigated the transposon mutants of these genes controlled by BP1026B_II1198 and confirmed that these genes contribute to pathogenesis in RAW264.7 murine macrophage cells. Taken together, the data presented here shed light on the regulon of BP1026B_II1198 and its role during intracellular infection and highlights an integral portion of the highly complex regulation network of Bp during host infection.

The Burkholderia pseudomallei intracellular 'TRANSITome'.

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.