scholarly journals Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248119
Author(s):  
Sam Barker ◽  
Sarah V. Harding ◽  
David Gray ◽  
Mark I. Richards ◽  
Helen S. Atkins ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Bacterial Species ◽  
Initial Study ◽  
Treatment Burden ◽  
Surrogate Optimization ◽  
Middle Income ◽  
Intravenous Treatment ◽  
Culturable Cell ◽  
The Tropics ◽  
High Throughput Assay

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z’ factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six “best in class” compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

scholarly journals Drug screening to identify compounds to act as co-therapies for the treatment of pathogenic Burkholderia

2019 ◽  
Author(s):  
Sam Barker ◽  
Sarah V. Harding ◽  
David Gray ◽  
Mark I. Richards ◽  
Helen S. Atkins ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Bacterial Species ◽  
Treatment Burden ◽  
Surrogate Optimization ◽  
Middle Income ◽  
Intravenous Treatment ◽  
Efficacious Treatment ◽  
Culturable Cell ◽  
The Tropics ◽  
High Throughput Assay

AbstractBurkholderia pseudomallei is a soil-dwelling organism present throughout the tropics, and is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to most currently available antibiotics. The most efficacious treatment for melioidosis requires at least two weeks of intravenous treatment with ceftazidime or meropenem. This places a large treatment burden on the predominantly middle income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z’ factor of 0.68. We screened a library of 61,250 compounds, and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six “best in class” compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, preliminary data also demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy against these bacteria.

scholarly journals Investigation of Melioidosis Outbreak in Pig Farms in Southern Thailand

2020 ◽  
Vol 7 (1) ◽  
pp. 9 ◽  
Author(s):  
Wiyada Kwanhian ◽  
Treenate Jiranantasak ◽  
Aleeza T. Kessler ◽  
Bryn E. Tolchinsky ◽  
Sarah Parker ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Contaminated Water ◽  
Environmental Investigation ◽  
Source Of Infection ◽  
Local Sequence ◽  
Southern Thailand ◽  
Life Threatening ◽  
The Tropics ◽  
Privately Owned ◽  
Sequence Types

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a potentially life-threatening infection that can affect humans and a wide variety of animals in the tropics. In December 2017, a swine melioidosis case was discovered during a meat inspection at a privately-owned slaughterhouse in Nakhon Si Thammarat Province in southern Thailand. The infection, which continued for several months, caused a dispute about where the disease began. An environmental investigation into two farms—both involved in raising the first infected pig—ensued. Through genetic analysis, the investigation revealed that a contaminated water supply at one farm was the probable source of infection. The three local sequence types identified in the investigation were types 51, 298 and 392.

scholarly journals 728. Genomic Characterization of Burkholderia pseudomallei Isolates in Colombia

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S463-S463
Author(s):  
Catalina Espitia-Acero ◽  
Rafael Rios ◽  
Monica Gabriela huertas ◽  
Sandra Vargas ◽  
Carolina Duarte Valderrama ◽  
...  
Keyword(s):  
Population Structure ◽  
Rural Areas ◽  
Burkholderia Pseudomallei ◽  
Virulence Determinants ◽  
Secretion Systems ◽  
Research Support ◽  
Middle Income ◽  
Resistance Determinants ◽  
Low Middle Income Countries ◽  
Genomic Characterization

Abstract Background Melioidosis is a serious infection caused by Burkholderia pseudomallei (Bps), an opportunistic organism, highly adaptable and with a wide array of intrinsic virulence factors and antimicrobial resistance determinants. Bps is underdiagnosed due to its slow growth on routine laboratory media and the lack of robust diagnostic infrastructure in rural areas of low/middle income countries. Recent data indicates that Bps infections are increasing in Colombia (COL). However, the understanding of the genomic epidemiology and population structure of the emerging Bps isolates in COL is unknown. Here we characterize the genomic features of Bps isolates from infected patients in COL. Methods We identified 13 Bps clinical isolates recovered in 5 Colombian cities between 2018 and 2020. We performed WGS and phylogenomic analyses using Bayesian methods. For comparisons, we included 82 publicly available genomes from Bps recovered worldwide (including 10 additional isolates from COL). Additionally, we characterized the resistome, virulome and MLST of all isolates. Results 12 out of the 13 isolates were confirmed as Bps and 1 belonged to the B. cepacia complex. The Bps population structure was divided in two main clades: clade 1 with isolates from Asia and Australia, and clade 2 with isolates from Africa, America, and the Caribbean (Figure 1). We found two groups of Colombian isolates, the first was related to ST518 and the second, highly diverse including 11 different STs (1742, 1748, 92, among others). Genomic characterization showed the presence of β-lactamases PenA (n=11) and OXA-57 (n=1). We also identified a T584A substitution in PBP3 (n=11). All genomes contained virulence determinants of motility (BimA), invasion (Flagella), signaling (CdpA) and adherence (Type IV pili). Type III and VI secretion systems, were also found in all isolates resembling Bps from other parts of the world. Figure 1. Maximum clade credibility tree of 82 genomes of Bps. The inner ring shows the ST for each genome, while the outer ring shows the geographical region associated with them. Groups highlighted in red show the location of the Colombian genomes and those related to them. Conclusion Bps is an emerging pathogen in COL and its population structure seems highly diverse, predominantly of the American lineage and absence of Australasians strains. A high prevalence ( >90%) of resistance determinants, particularly related to β-lactams, suggest that active surveillance of these emergent pathogens is needed in countries like COL. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

scholarly journals Patients With Common Variable Immunodeficiency (CVID) Show Higher Gut Bacterial Diversity and Levels of Low-Abundance Genes Than the Healthy Housemates

2021 ◽  
Vol 12 ◽  
Author(s):  
Juraj Bosák ◽  
Matej Lexa ◽  
Kristýna Fiedorová ◽  
Darshak C. Gadara ◽  
Lenka Micenková ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Common Variable Immunodeficiency ◽  
Metabolic Pathways ◽  
Bacterial Species ◽  
Initial Study ◽  
Fecal Microbiota ◽  
Fecal Microbiome ◽  
Length Of Treatment ◽  
Low Levels ◽  
Common Variable

Common variable immunodeficiency (CVID) is a clinically and genetically heterogeneous disorder with inadequate antibody responses and low levels of immunoglobulins including IgA that is involved in the maintenance of the intestinal homeostasis. In this study, we analyzed the taxonomical and functional metagenome of the fecal microbiota and stool metabolome in a cohort of six CVID patients without gastroenterological symptomatology and their healthy housemates. The fecal microbiome of CVID patients contained higher numbers of bacterial species and altered abundance of thirty-four species. Hungatella hathewayi was frequent in CVID microbiome and absent in controls. Moreover, the CVID metagenome was enriched for low-abundance genes likely encoding nonessential functions, such as bacterial motility and metabolism of aromatic compounds. Metabolomics revealed dysregulation in several metabolic pathways, mostly associated with decreased levels of adenosine in CVID patients. Identified features have been consistently associated with CVID diagnosis across the patients with various immunological characteristics, length of treatment, and age. Taken together, this initial study revealed expansion of bacterial diversity in the host immunodeficient conditions and suggested several bacterial species and metabolites, which have potential to be diagnostic and/or prognostic CVID markers in the future.

Morphometric Analysis of Portal Hepatocytes from Rats Treated with Compound 48/80

1982 ◽  
Vol 40 ◽  
pp. 64-65
Author(s):  
R.V.W. Dimlich ◽  
R.R. Cardell
Keyword(s):  
Mast Cell ◽  
Smooth Endoplasmic Reticulum ◽  
Initial Study ◽  
Hepatic Glycogen ◽  
Electron Microscopic ◽  
Intravenous Treatment ◽  
Apparent Decrease ◽  
Hepatic Portal ◽  
Unpublished Observation ◽  
Mast Cell Degranulating

Compound 48/80, a mast cell degranulating agent, produces marked hypotension in the rat. Thirty minutes after intravenous treatment with compound 48/80 there is a significant increase in blood-glucose with a concommitant decrease in hepatic glycogen similar to that observed in shock-like states. Electron microscopic observations of hepatocytes from rats treated with 48/80 have shown an apparent decrease in hepatic glycogen and increase in smooth endoplasmic reticulum (SER). Mitochondria in these cells seemed to contain fewer matrix granules. The present analysis was performed to quantify these observations. Since light microscopic observations of tissue from these rats revealed an apparent greater depletion of glycogen in the portal areas (unpublished observation), hepatic portal cells were analyzed in this initial study.

scholarly journals Groundwater Seeps Facilitate Exposure to Burkholderia pseudomallei

2011 ◽  
Vol 77 (20) ◽  
pp. 7243-7246 ◽  
Author(s):  
Anthony Baker ◽  
Donald Tahani ◽  
Christopher Gardiner ◽  
Keith L. Bristow ◽  
Andrew R. Greenhill ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Extreme Weather Events ◽  
Clinical Samples ◽  
Residential Areas ◽  
Content Type ◽  
Weather Events ◽  
Potential Reservoir ◽  
Site Access ◽  
The Tropics ◽  
Pcr Targeting

ABSTRACTBurkholderia pseudomalleiis a saprophytic bacterium which is the causative agent of melioidosis, a common cause of fatal bacterial pneumonia and sepsis in the tropics. The incidence of melioidosis is clustered spatially and temporally and is heavily linked to rainfall and extreme weather events. Clinical case clustering has recently been reported in Townsville, Australia, and has implicated Castle Hill, a granite monolith in the city center, as a potential reservoir of infection. Topsoil and water from seasonal groundwater seeps were collected around the base of Castle Hill and analyzed by quantitative real-time PCR targeting the type III secretion system genes for the presence ofB. pseudomallei. The organism was identified in 65% (95% confidence interval [CI], 49.5 to 80.4) of soil samples (n= 40) and 92.5% (95% CI, 83.9 to 100) of seasonal groundwater samples (n= 40). Further sampling of water collected from roads and gutters in nearby residential areas after an intense rainfall event found that 88.2% (95% CI, 72.9 to 100) of samples (n= 16) contained viableB. pseudomalleiat concentrations up to 113 CFU/ml. Comparison of isolates using multilocus sequence typing demonstrated clinical matches and close associations between environmental isolates and isolates derived from clinical samples from patients in Townsville. This study demonstrated that waterborneB. pseudomalleifrom groundwater seeps around Castle Hill may facilitate exposure toB. pseudomalleiand contribute to the clinical clustering at this site. Access to this type of information will advise the development and implementation of public health measures to reduce the incidence of melioidosis.

Burkholderia pseudomallei pathogenesis and survival in different niches

2020 ◽  
Vol 48 (2) ◽  
pp. 569-579
Author(s):  
Chee-Hoo Yip ◽  
Ahmad-Kamal Ghazali ◽  
Sheila Nathan
Keyword(s):  
Burkholderia Pseudomallei ◽  
Salt Water ◽  
Mortality Rates ◽  
Current Treatment ◽  
Water Bodies ◽  
Intracellular Pathogen ◽  
Stagnant Water ◽  
Wide Range ◽  
The Tropics ◽  
Highly Virulent

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease of the tropics with high clinical mortality rates. To date, no vaccines are approved for melioidosis and current treatment relies on antibiotics. Conversely, common misdiagnosis and high pathogenicity of Bp hamper efforts to fight melioidosis. This bacterium can be isolated from a wide range of niches such as waterlogged fields, stagnant water bodies, salt water bodies and from human and animal clinical specimens. Although extensive studies have been undertaken to elucidate pathogenesis mechanisms of Bp, little is known about how a harmless soil bacterium adapts to different environmental conditions, in particular, the shift to a human host to become a highly virulent pathogen. The bacterium has a large genome encoding an armory of factors that assist the pathogen in surviving under stressful conditions and assuming its role as a deadly intracellular pathogen. This review presents an overview of what is currently known about how the pathogen adapts to different environments. With in-depth understanding of Bp adaptation and survival, more effective therapies for melioidosis can be developed by targeting related genes or proteins that play a major role in the bacteria's survival.

scholarly journals Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity

2012 ◽  
Vol 80 (9) ◽  
pp. 3247-3255 ◽  
Author(s):  
Claudia M. Müller ◽  
Laura Conejero ◽  
Natasha Spink ◽  
Matthew E. Wand ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Galleria Mellonella ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Virulence Gene Expression ◽  
Control And Prevention

ABSTRACTBurkholderia pseudomalleiis a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created arelA spoTdouble mutant inB. pseudomalleistrain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotypein vitroandin vivo. TheB. pseudomalleiΔrelAΔspoTmutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in theGalleria mellonellainsect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelAΔspoTmutant resulted in partial protection against infection with wild-typeB. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation inB. pseudomallei, and the ΔrelAΔspoTmutant may be a promising live-attenuated vaccine candidate.

scholarly journals Dictyostelium discoideum as a Model System for Identification of Burkholderia pseudomallei Virulence Factors

2011 ◽  
Vol 79 (5) ◽  
pp. 2079-2088 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Maharsh K. Patel ◽  
Mark A. Schell
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Burkholderia Pseudomallei ◽  
Bacterial Pathogen ◽  
Model System ◽  
Virulence Determinants ◽  
Content Type ◽  
Throughput Model ◽  
Human Infections ◽  
High Throughput Assay

ABSTRACTBurkholderia pseudomalleiis an emerging bacterial pathogen and category B biothreat. Human infections withB. pseudomallei(called melioidosis) present as a range of manifestations, including acute septicemia and pneumonia. Although melioidosis can be fatal, little is known about the molecular basis ofB. pseudomalleipathogenicity, in part because of the lack of simple, genetically tractable eukaryotic models to facilitateen masseidentification of virulence determinants or explore host-pathogen interactions. Two assays, one high-throughput and one quantitative, were developed to monitor levels of resistance ofB. pseudomalleiand the closely related nearly avirulent speciesBurkholderia thailandensisto predation by the phagocytic amoebaDictyostelium discoideum. The quantitative assay showed that levels of resistance to, and survival within, amoeba by these bacteria and their known virulence mutants correlate well with their published levels of virulence in animals. Using the high-throughput assay, we screened a 1,500-memberB. thailandensistransposon mutant library and identified 13 genes involved in resistance to predation byD. discoideum. Orthologs of these genes were disrupted inB. pseudomallei, and nearly all mutants had similarly decreased resistance to predation byD. discoideum. For some mutants, decreased resistance also correlated with reduced survival in and cytotoxicity toward macrophages, as well as attenuated virulence in mice. These observations suggest that some factors required byB. pseudomalleifor resistance to environmental phagocytes also aid in resistance to phagocytic immune cells and contribute to disease in animals. Thus,D. discoideumprovides a novel, high-throughput model system for facilitating inquiry intoB. pseudomalleivirulence.

scholarly journals Identification of Spacer and Protospacer Sequence Requirements in the Vibrio cholerae Type I-E CRISPR/Cas System

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Jacob Bourgeois ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Immune System ◽  
Vibrio Cholerae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Adaptive Immune System ◽  
Initial Study ◽  
Type I ◽  
Content Type ◽  
Protospacer Adjacent Motif ◽  
Sequence Elements

ABSTRACT The prokaryotic adaptive immune system CRISPR/Cas serves as a defense against bacteriophage and invasive nucleic acids. A type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5′-TT-3′ protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5′-TT-3′ PAM do not interfere as expected, indicating that further investigation of this system is necessary. In this study, we identified additional conserved sequences, including a pyrimidine in the 5′ position of the spacer and a purine in the complementary position of the protospacer using 873 unique spacers and 2,267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5′-RTT-3′ PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally, by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay, that a 5′-RTT-3′ PAM is necessary for CRISPR interference, and we discover functional consequences for spacer efficacy related to the identity of the 5′ spacer pyrimidine. IMPORTANCE Bacterial CRISPR/Cas systems provide immunity by defending against phage and other invading elements. A thorough comprehension of the molecular mechanisms employed by these diverse systems will improve our understanding of bacteriophage-bacterium interactions and bacterial adaptation to foreign DNA. The Vibrio cholerae type I-E system was previously identified in an extinct classical biotype and was partially characterized for its function. Here, using both bioinformatic and functional assays, we extend that initial study. We have found that the type I-E system still exists in modern strains of V. cholerae. Furthermore, we defined additional sequence elements both in the CRISPR array and in target DNA that are required for immunity. CRISPR/Cas systems are now commonly used as precise and powerful genetic engineering tools. Knowledge of the sequences required for CRISPR/Cas immunity is a prerequisite for the effective design and experimental use of these systems. Our results greatly facilitate the effective use of one such system. Furthermore, we provide a publicly available software program that assists in the detection and validation of CRISPR/Cas immunity requirements when such a system exists in a bacterial species.

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