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Proceedings of the National Academy of... Jul 2023BpeB and BpeF are multidrug efflux transporters from that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å...
BpeB and BpeF are multidrug efflux transporters from that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from , where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.
Topics: Burkholderia pseudomallei; Membrane Transport Proteins; Biological Transport; Drug Resistance, Multiple; Binding Sites; Anti-Bacterial Agents
PubMed: 37428905
DOI: 10.1073/pnas.2215072120 -
Scientific Reports Aug 2017Little is known about the evolution, adaptation and pathogenesis of Burkholderia pseudomallei within host during acute melioidosis infection. Melioidosis is a potential...
Little is known about the evolution, adaptation and pathogenesis of Burkholderia pseudomallei within host during acute melioidosis infection. Melioidosis is a potential life threatening disease contracted through inhalation, ingestion, inoculation or direct entry of the organism into the blood stream via wounds or skin abrasions from contaminated soil and water. Environmental B. pseudomallei strain (Bp ), isolated during a melioidosis outbreak in Pahang, Malaysia was injected intra-peritoneally into a mouse and passaged strain was recovered from spleen (Bp). A gel-based comparative proteomics profiling approach was used, to map and identify differentially expressed proteins (fold-change ≥ 2; p-value ≤ 0.05) between the strains. A total of 730 and 685 spots were visualised in the Bp and Bp strains, respectively. Of the 730 spots (Bp as reference gel), 87 spots were differentially regulated (44 up- and 43 down-regulated). The identified proteins were classified as proteins related to metabolism, stress response, virulence, signal transduction, or adhesion. In comparison, it was found that those proteins related to adhesins, virulence factors and stress- response were up-regulated and could possibly explain the adaptation of the bacteria in the host. Investigating the differentially expressed proteins may provide better perspective of bacterial factors which aid survivability of B. pseudomallei in host.
Topics: Adaptation, Physiological; Animals; Bacterial Proteins; Burkholderia pseudomallei; Disease Models, Animal; Electrophoresis, Gel, Two-Dimensional; Melioidosis; Mice, Inbred BALB C; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Virulence; Virulence Factors
PubMed: 28827633
DOI: 10.1038/s41598-017-09373-0 -
Scientific Reports Jul 2019Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to...
Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B. pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B. pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.
Topics: Animals; Bacterial Proteins; Burkholderia pseudomallei; Cell Membrane; Cell Shape; Computational Biology; Melioidosis; Mice; Mice, Inbred BALB C; Peptidoglycan Glycosyltransferase; Virulence
PubMed: 31363151
DOI: 10.1038/s41598-019-47483-z -
Microbial Drug Resistance (Larchmont,... Sep 2021Current antimicrobial treatment recommendations for melioidosis, the disease caused by , are largely based on studies of strains isolated from the Eastern Hemisphere...
Current antimicrobial treatment recommendations for melioidosis, the disease caused by , are largely based on studies of strains isolated from the Eastern Hemisphere (EH), where most human cases are identified and reported. In this study, we evaluated the antimicrobial susceptibility of 26 strains in the CDC (Centers for Diseases Control and Prevention) collection from the Western Hemisphere (WH) isolated from 1960 to 2015. Minimal inhibitory concentration (MIC) values were measured by standard broth microdilution for 16 antimicrobials following Clinical and Laboratory Standards Institute (CLSI) guidelines. Twenty-four of the 26 WH strains were susceptible to the six antimicrobials with CLSI-defined MIC susceptibility interpretive criteria for : amoxicillin/clavulanate, ceftazidime, imipenem, doxycycline, tetracycline, and trimethoprim/sulfamethoxazole. One WH strain demonstrated intermediate amoxicillin/clavulanate resistance and another strain had intermediate resistance to tetracycline. For all antimicrobials tested, the susceptibility profiles of WH isolates were comparable with previously reported MIC results of EH strains. The overall similarities suggest that the same antimicrobials are useful for melioidosis treatment in both the WH and EH. Using analyses of WH genomes, we identified a novel amino acid substitution P258S in the beta-lactamase PenA, which may contribute to decreased susceptibility to amoxicillin/clavulanate in .
Topics: Anti-Bacterial Agents; Burkholderia pseudomallei; Drug Resistance, Multiple, Bacterial; Genes, Bacterial; Genomics; Humans; Microbial Sensitivity Tests; Phenotype; beta-Lactamases
PubMed: 33570476
DOI: 10.1089/mdr.2020.0362 -
Medicine Mar 2019Burkholderia pseudomallei is the causative agent of meliodosis, and the cases in China are gradually increasing. The present retrospective study aimed to surveil the... (Observational Study)
Observational Study
Burkholderia pseudomallei is the causative agent of meliodosis, and the cases in China are gradually increasing. The present retrospective study aimed to surveil the molecular epidemiological characteristics and antibiotic resistance of B pseudomallei isolates. B pseudomallei strains were isolated and verified from meliodosis patients with relevant epidemiological information from 2004 to 2016 in Hainan, China. Pulsed-field gel electrophoresis based on Spe I digestion was carried out, and antimicrobial resistance of B pseudomallei strains was observed against 9 frequently-used antimicrobials. A total of 164 B pseudomallei isolates were successfully divided into 60 pulsed-field gel electrophoresis (PFGE) patterns, including 33 clusters and 27 single types, at an 85% similarity level. The isolates also exhibited a high level of ceftazidime resistance rate (12.8%, 21/164). B pseudomallei strains were mainly heterogenous with no predominant type, but there were some clonal populations, dominate clusters prevalent and the resistance rates of cephems antimicrobial increased significantly between 2004 and 2016 along with the number of melioidosis cases collected in Hainan (cefoperazone-sulbactam [SCF], rs = 0.96, P = .04; ceftazidime [CAZ], rs = 0.98, P = .01). In conclusion, this study will help to enhance our understanding of molecular characteristics and antibiotic resistance of B pseudomallei.
Topics: Anti-Bacterial Agents; Burkholderia pseudomallei; Cefoperazone; Ceftazidime; China; Cluster Analysis; Drug Resistance, Bacterial; Electrophoresis, Gel, Pulsed-Field; Female; Humans; Male; Melioidosis; Prevalence; Retrospective Studies; Sulbactam
PubMed: 30817562
DOI: 10.1097/MD.0000000000014461 -
Journal of Proteome Research May 2011Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses...
Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation.
Topics: Bacterial Outer Membrane Proteins; Burkholderia mallei; Burkholderia pseudomallei; Chromatography, Liquid; Computational Biology; Proteome; Tandem Mass Spectrometry; Trypsin
PubMed: 21391724
DOI: 10.1021/pr1012398 -
PLoS Neglected Tropical Diseases Sep 2019Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood,...
BACKGROUND
Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated.
METHODOLOGY/PRINCIPAL FINDINGS
We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp.
CONCLUSIONS/SIGNIFICANCE
B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.
Topics: Burkholderia; Burkholderia pseudomallei; Genomic Islands; Melioidosis; Phylogeny; Polymerase Chain Reaction; Puerto Rico; Sequence Analysis, DNA; Soil Microbiology; Water Microbiology
PubMed: 31487287
DOI: 10.1371/journal.pntd.0007727 -
MicrobiologyOpen Feb 2018The ability of Burkholderia pseudomallei to persist and survive in the environment is a health problem worldwide. Therefore, the antibacterial activities of chitosan...
The ability of Burkholderia pseudomallei to persist and survive in the environment is a health problem worldwide. Therefore, the antibacterial activities of chitosan against four environmental isolates of B. pseudomallei from soil in Khon Kaen, Thailand, were investigated. Antibacterial activities were assessed by a plate count technique after treatment with 0.2, 0.5, 1, 2 or 5 mg ml chitosan for 0, 24 and 48 hr. Chitosan at 5 mg ml completely killed all four B. pseudomallei isolates within 24 hr, whilst 2 mg ml chitosan lowered the viability of B. pseudomallei by 20% within the same time span. Chitosan may act by disruption of the cell membrane, releasing intracellular components that can be detected spectrophotometrically at 260 and 280 nm. Transmission electron microscopy inspection of chitosan-treated B. pseudomallei revealed damage to the bacterial membranes. This study demonstrated the effective antibacterial activity by chitosan against B. pseudomallei. Chitosan causes disruption of the bacterial cell membrane, release of intracellular constituents and cell death. This study revealed the inhibitory potential of chitosan for mitigating B. pseudomallei occurrences.
Topics: Anti-Bacterial Agents; Burkholderia pseudomallei; Cell Membrane; Chitosan; Colony Count, Microbial; Microbial Sensitivity Tests; Microbial Viability; Microscopy, Electron, Transmission; Soil Microbiology; Spectrophotometry; Thailand
PubMed: 29178614
DOI: 10.1002/mbo3.534 -
Infection, Genetics and Evolution :... Dec 2023Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to...
Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.
Topics: Burkholderia; Burkholderia pseudomallei; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thailand; Soil Microbiology; Water Microbiology
PubMed: 37995885
DOI: 10.1016/j.meegid.2023.105532 -
Applied and Environmental Microbiology Apr 2017is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of in the...
is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 ( = 153) or BPSS0745 ( = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; < 0.001) and 9.0 (95% CI, 3.1 to 26.4; < 0.001), respectively. High genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for The worldwide environmental distribution of the soil bacterium remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a -specific qPCR approach can detect significantly higher numbers of -positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent -specific qPCR targets further increased the detection rate of compared with that from single targets. Samples with a high molecular load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of infections in different parts of the world.
Topics: Bacteriological Techniques; Burkholderia pseudomallei; Environment; Humans; Melioidosis; Open Reading Frames; Real-Time Polymerase Chain Reaction; Soil Microbiology; Thailand; Type III Secretion Systems
PubMed: 28188208
DOI: 10.1128/AEM.03212-16