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PLoS Neglected Tropical Diseases Aug 2022Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the...
Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.
Topics: Burkholderia pseudomallei; CRISPR-Cas Systems; DNA; Genomics; Humans; Melioidosis; Sensitivity and Specificity
PubMed: 36037185
DOI: 10.1371/journal.pntd.0010659 -
Scientific Reports Jul 2022Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a life-threatening disease. The interaction of B. pseudomallei with its...
Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a life-threatening disease. The interaction of B. pseudomallei with its host is complicated, and cellular response to B. pseudomallei infection is still largely unknown. In this study, we aimed to determine host-cell responses to B. pseudomallei at the proteomics level. We performed proteomic profiling of B. pseudomallei HNBP001-infected mouse macrophage RAW264.7 cells to characterize the cellular response dynamics during infection. Western blot analysis was utilized for the validation of changes in protein expression. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the clusterProfiler R package. Compared with the negative control (NC) group, 811 common proteins varied over time, with a cut-off level of two fold change and an adjusted P-value less than 0.05. The bioinformatics analysis revealed that the proteins significantly changed in the B. pseudomallei HNBP001 infection group (Bp group) were enriched in glycometabolism pathways, including glycolysis, fructose and mannose metabolism, pentose phosphate pathway, galactose metabolism, and carbon metabolism. Western blot analysis verified three selected proteins involved in glycometabolism pathways, namely PGM1, PKM, and PGK1 were increase over time post the infection. Furthermore, in vitro functional analysis revealed an increased glucose uptake and decreased ATP production and O-GlcNAcylation in the Bp group compared with control group, suggesting that B. pseudomallei HNBP001 infection induces changes in glycometabolism in RAW264.7 cells. These results indicate that glycometabolism pathways change in RAW264.7 cells post B. pseudomallei HNBP001 infection, providing important insights into the intimate interaction between B. pseudomallei and macrophages.
Topics: Animals; Burkholderia pseudomallei; Cell Line; Melioidosis; Mice; Proteomics; RAW 264.7 Cells
PubMed: 35869254
DOI: 10.1038/s41598-022-16716-z -
BMC Infectious Diseases Feb 2019Melioidosis is a life-threatening infectious disease that is caused by gram negative bacteria Burkholderia pseudomallei. This bacteria occurs as an environmental...
BACKGROUND
Melioidosis is a life-threatening infectious disease that is caused by gram negative bacteria Burkholderia pseudomallei. This bacteria occurs as an environmental saprophyte typically in endemic regions of south-east Asia and northern Australia. Therefore, patients with melioidosis are at high risk of being misdiagnosed and/or under-diagnosed in South Asia.
CASE PRESENTATION
Here, we report two cases of melioidosis from Nepal. Both of them were diabetic male who presented themselves with fever, multiple abscesses and developed sepsis. They were treated with multiple antimicrobial agents including antitubercular drugs before being correctly diagnosed as melioidosis. Consistent with this, both patients were farmer by occupation and also reported travelling to Malaysia in the past. The diagnosis was made consequent to the isolation of B. pseudomallei from pus samples. Accordingly, they were managed with intravenous meropenem followed by oral doxycycline and cotrimoxazole.
CONCLUSION
The case reports raise serious concern over the existing unawareness of melioidosis in Nepal. Both of the cases were left undiagnosed for a long time. Therefore, clinicians need to keep a high index of suspicion while encountering similar cases. Especially diabetic-farmers who present with fever and sepsis and do not respond to antibiotics easily may turn out to be yet another case of melioidosis. Ascertaining the travel history and occupational history is of utmost significance. In addition, the microbiologist should be trained to correctly identify B. pseudomallei as it is often confused for other Burkholderia species. The organism responds only to specific antibiotics; therefore, correct and timely diagnosis becomes crucial for better outcomes.
Topics: Abscess; Adult; Anti-Bacterial Agents; Burkholderia pseudomallei; Diabetes Mellitus; Diagnostic Errors; Doxycycline; Fever; Humans; Malaysia; Male; Melioidosis; Meropenem; Middle Aged; Nepal; Travel; Trimethoprim, Sulfamethoxazole Drug Combination
PubMed: 30782129
DOI: 10.1186/s12879-019-3793-x -
BMC Microbiology May 2021Burkholderia pseudomallei, a facultative intracellular bacterium, is the aetiological agent of melioidosis that is responsible for up to 40% sepsis-related mortality in...
BACKGROUND
Burkholderia pseudomallei, a facultative intracellular bacterium, is the aetiological agent of melioidosis that is responsible for up to 40% sepsis-related mortality in epidemic areas. However, no effective vaccine is available currently, and the drug resistance is also a major problem in the treatment of melioidosis. Therefore, finding new clinical treatment strategies in melioidosis is extremely urgent.
RESULTS
We demonstrated that tauroursodeoxycholic acid (TUDCA), a clinically available endoplasmic reticulum (ER) stress inhibitor, can promote B. pseudomallei clearance both in vivo and in vitro. In this study, we investigated the effects of TUDCA on the survival of melioidosis mice, and found that treatment with TUDCA significantly decreased intracellular survival of B. pseudomallei. Mechanistically, we found that B. pseudomallei induced apoptosis and activated IRE1 and PERK signaling ways of ER stress in RAW264.7 macrophages. TUDCA treatment could reduce B. pseudomallei-induced ER stress in vitro, and TUDCA is protective in vivo.
CONCLUSION
Taken together, our study has demonstrated that B. pseudomallei infection results in ER stress-induced apoptosis, and TUDCA enhances the clearance of B. pseudomallei by inhibiting ER stress-induced apoptosis both in vivo and in vitro, suggesting that TUDCA could be used as a potentially alternative treatment for melioidosis.
Topics: Animals; Apoptosis; Burkholderia pseudomallei; Cell Line; Endoplasmic Reticulum Stress; Melioidosis; Mice; Signal Transduction; Survival Analysis; Taurochenodeoxycholic Acid
PubMed: 33947331
DOI: 10.1186/s12866-021-02199-x -
BMC Microbiology Mar 2022Burkholderia pseudomallei (B. pseudomallei), as a highly pathogenic organism, causes melioidosis, which is a disease of public health importance in many tropical...
BACKGROUND
Burkholderia pseudomallei (B. pseudomallei), as a highly pathogenic organism, causes melioidosis, which is a disease of public health importance in many tropical developing countries. Here, we present and validate a novel detection technique, termed multiple cross displacement amplification combined with nanoparticles-based lateral flow biosensor (MCDA-NB), for identifying B. pseudomallei and diagnosing melioidosis.
RESULTS
B. pseudomallei-MCDA targets the TTS1 (Type III secretion system gene cluster 1) to specifically design ten MCDA primers. The nanoparticles-based biosensor (NB) can be combined with B. pseudomallei-MCDA for visually, objective, simply and rapidly reporting reaction results. The optimal amplification conditions of B. pseudomallei-MCDA were 66 °C for 30 min. Assay's sensitivity was 100 fg of genomic DNA in the pure cultures, and the analytical specificity was 100% by the examination of 257 strains, including 228 B. pseudomallei and 29 non-B. pseudomallei. As a result, the whole detection procedure was completed within 50 min, including 15 min for genomic DNA preparation, 30 min for l MCDA reaction, and 2 min for the interpretation of the results visually by biosensor.
CONCLUSIONS
B. pseudomallei-MCDA assay is a rapid, sensitive and specific method for the detection of B. pseudomallei, and can be used as a potential tool for melioidosis diagnose in basic, field and clinical laboratories.
Topics: Biosensing Techniques; Burkholderia pseudomallei; Humans; Melioidosis; Nucleic Acid Amplification Techniques; Sensitivity and Specificity
PubMed: 35272632
DOI: 10.1186/s12866-022-02485-2 -
PloS One 2014Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via...
Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.
Topics: Acute Disease; Animals; Bacterial Infections; Burkholderia mallei; Burkholderia pseudomallei; Disease Models, Animal; Female; Host-Pathogen Interactions; Humans; Immunity, Innate; Mice; Mice, Inbred BALB C; Post-Exposure Prophylaxis
PubMed: 25503969
DOI: 10.1371/journal.pone.0115066 -
BMC Microbiology Mar 2015Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a...
BACKGROUND
Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together.
RESULTS
We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase.
CONCLUSION
Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation.
Topics: Agar; Antibiosis; Bacterial Proteins; Biological Factors; Burkholderia; Burkholderia pseudomallei; Culture Media; Culture Media, Conditioned; Flagella; Hydrogen-Ion Concentration; Movement; Protein Stability; Proteolysis; Soil Microbiology; Thailand
PubMed: 25879538
DOI: 10.1186/s12866-015-0395-7 -
PLoS Neglected Tropical Diseases May 2021Burkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first...
BACKGROUND
Burkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first described in 1911 in Myanmar. Melioidosis is a common cause of sepsis and death in South and South-east Asia, but it is rarely diagnosed in Myanmar. We conducted a nationwide soil study to identify areas where B. pseudomallei is present.
METHODOLOGY/PRINCIPAL FINDINGS
We collected soil samples from 387 locations in all 15 states and regions of Myanmar between September 2017 and June 2019. At each site, three samples were taken at each of three different depths (30, 60 and 90 cm) and were cultured for B. pseudomallei separately, along with a pooled sample from each site (i.e. 10 cultures per site). We used a negative binomial regression model to assess associations between isolation of B. pseudomallei and environmental factors (season, soil depth, soil type, land use and climate zones). B. pseudomallei was isolated in 7 of 15 states and regions. Of the 387 sites, 31 (8%) had one or more positive samples and of the 3,870 samples cultured, 103 (2.7%) tested positive for B. pseudomallei. B. pseudomallei was isolated more frequently during the monsoon season [RR-2.28 (95% CI: 0.70-7.38)] and less in the hot dry season [RR-0.70 (95% CI: 0.19-2.56)] compared to the cool dry season, and in the tropical monsoon climate zone [RR-2.26; 95% CI (0.21-6.21)] compared to the tropical dry winter climate zone. However, these associations were not statistically significant. B. pseudomallei was detected at all three depths and from various soil types (clay, silt and sand). Isolation was higher in agricultural land (2.2%), pasture land (8.5%) and disused land (5.8%) than in residential land (0.4%), but these differences were also not significant.
CONCLUSION/SIGNIFICANCE
This study confirms a widespread distribution of B. pseudomallei in Myanmar. Clinical studies should follow to obtain a better picture of the burden of melioidosis in Myanmar.
Topics: Burkholderia pseudomallei; Geography; Humans; Melioidosis; Myanmar; Soil Microbiology
PubMed: 34029325
DOI: 10.1371/journal.pntd.0009372 -
PloS One 2020Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two...
Identification of bacterial virulence factors is critical for understanding disease pathogenesis, drug discovery and vaccine development. In this study we used two approaches to predict virulence factors of Burkholderia pseudomallei, the Gram-negative bacterium that causes melioidosis. B. pseudomallei is naturally antibiotic resistant and there are no clinically available melioidosis vaccines. To identify B. pseudomallei protein targets for drug discovery and vaccine development, we chose to search for substrates of the B. pseudomallei periplasmic disulfide bond forming protein A (DsbA). DsbA introduces disulfide bonds into extra-cytoplasmic proteins and is essential for virulence in many Gram-negative organism, including B. pseudomallei. The first approach to identify B. pseudomallei DsbA virulence factor substrates was a large-scale genomic analysis of 511 unique B. pseudomallei disease-associated strains. This yielded 4,496 core gene products, of which we hypothesise 263 are DsbA substrates. Manual curation and database screening of the 263 mature proteins yielded 81 associated with disease pathogenesis or virulence. These were screened for structural homologues to predict potential B-cell epitopes. In the second approach, we searched the B. pseudomallei genome for homologues of the more than 90 known DsbA substrates in other bacteria. Using this approach, we identified 15 putative B. pseudomallei DsbA virulence factor substrates, with two of these previously identified in the genomic approach, bringing the total number of putative DsbA virulence factor substrates to 94. The two putative B. pseudomallei virulence factors identified by both methods are homologues of PenI family β-lactamase and a molecular chaperone. These two proteins could serve as high priority targets for future B. pseudomallei virulence factor characterization.
Topics: Amino Acid Sequence; Bacterial Proteins; Bacterial Vaccines; Burkholderia pseudomallei; Cysteine; Epitopes, B-Lymphocyte; Gene Ontology; Genome, Bacterial; Models, Molecular; Sequence Homology, Amino Acid; Substrate Specificity; Virulence Factors
PubMed: 33216758
DOI: 10.1371/journal.pone.0241306 -
Emerging Infectious Diseases Feb 2021We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during...
We report an analysis of the genomic diversity of isolates of Burkholderia pseudomallei, the cause of melioidosis, recovered in Colombia from routine surveillance during 2016-2017. B. pseudomallei appears genetically diverse, suggesting it is well established and has spread across the region.
Topics: Burkholderia pseudomallei; Colombia; Genomics; Humans; Melioidosis; Multilocus Sequence Typing
PubMed: 33496648
DOI: 10.3201/eid2702.202824