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      Glyburide Reduces Bacterial Dissemination in a Mouse Model of Melioidosis

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          Burkholderia pseudomallei infection (melioidosis) is an important cause of community-acquired Gram-negative sepsis in Northeast Thailand, where it is associated with a ∼40% mortality rate despite antimicrobial chemotherapy. We showed in a previous cohort study that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality and attenuated inflammatory responses compared to patients not taking glyburide. We sought to define the mechanism underlying this observation in a murine model of melioidosis.


          Mice (C57BL/6) with streptozocin-induced diabetes were inoculated with ∼6×10 2 cfu B. pseudomallei intranasally, then treated with therapeutic ceftazidime (600 mg/kg intraperitoneally twice daily starting 24 h after inoculation) in order to mimic the clinical scenario. Glyburide (50 mg/kg) or vehicle was started 7 d before inoculation and continued until sacrifice. The minimum inhibitory concentration of glyburide for B. pseudomallei was determined by broth microdilution. We also examined the effect of glyburide on interleukin (IL) 1β by bone-marrow-derived macrophages (BMDM).


          Diabetic mice had increased susceptibility to melioidosis, with increased bacterial dissemination but no effect was seen of diabetes on inflammation compared to non-diabetic controls. Glyburide treatment did not affect glucose levels but was associated with reduced pulmonary cellular influx, reduced bacterial dissemination to both liver and spleen and reduced IL1β production when compared to untreated controls. Other cytokines were not different in glyburide-treated animals. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Glyburide directly reduced the secretion of IL1β by BMDMs in a dose-dependent fashion.


          Diabetes increases the susceptibility to melioidosis. We further show, for the first time in any model of sepsis, that glyburide acts as an anti-inflammatory agent by reducing IL1β secretion accompanied by diminished cellular influx and reduced bacterial dissemination to distant organs. We found no evidence for a direct effect of glyburide on the bacterium.

          Author Summary

          Burkholderia pseudomallei infection (also called melioidosis) is a common cause of bacterial infection in Northeast Thailand, where the mortality rate is 43% despite appropriate antibiotic treatment. We showed previously that patients taking glyburide ( = glibenclamide) prior to admission have lower mortality rates and lower levels of inflammation in the blood. In this study, we used a mouse model to better understand the mechanism underlying this observation. In this study, we used a mouse model of diabetes and infected the mice with B. pseudomallei. Half the mice were treated with glyburide and half were not. We also performed in vitro experiments to find the minimum concentration of glyburide that would inhibit the growth of B. pseudomallei. We found that glyburide treatment was associated with reduced inflammation (as measured by the flow of cells into the lungs and by interleukin-1β production) and reduced spread of the bacterium to liver and spleen when compared to untreated controls. There was no direct effect of glyburide on B. pseudomallei growth in vitro or in vivo. Because the effect of glyburide is on the host and not on the bacterium, it is possible that this effect will be seen in other causes of sepsis, not just melioidosis.

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          Most cited references 42

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          Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei.

          Burkholderia pseudomallei is a potential bioterror agent and the causative agent of melioidosis, a severe disease that is endemic in areas of Southeast Asia and Northern Australia. Infection is often associated with bacterial dissemination to distant sites, and there are many possible disease manifestations, with melioidosis septic shock being the most severe. Eradication of the organism following infection is difficult, with a slow fever-clearance time, the need for prolonged antibiotic therapy and a high rate of relapse if therapy is not completed. Mortality from melioidosis septic shock remains high despite appropriate antimicrobial therapy. Prevention of disease and a reduction in mortality and the rate of relapse are priority areas for future research efforts. Studying how the disease is acquired and the host-pathogen interactions involved will underpin these efforts; this review presents an overview of current knowledge in these areas, highlighting key topics for evaluation.
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            A genetic and physiological study of impaired glucose homeostasis control in C57BL/6J mice.

            C57BL/6J mice exhibit impaired glucose tolerance. The aims of this study were to map the genetic loci underlying this phenotype, to further characterise the physiological defects and to identify candidate genes. Glucose tolerance was measured in an intraperitoneal glucose tolerance test and genetic determinants mapped in an F2 intercross. Insulin sensitivity was measured by injecting insulin and following glucose disposal from the plasma. To measure beta cell function, insulin secretion and electrophysiological studies were carried out on isolated islets. Candidate genes were investigated by sequencing and quantitative RNA analysis. C57BL/6J mice showed normal insulin sensitivity and impaired insulin secretion. In beta cells, glucose did not stimulate a rise in intracellular calcium and its ability to close KATP channels was impaired. We identified three genetic loci responsible for the impaired glucose tolerance. Nicotinamide nucleotide transhydrogenase (Nnt) lies within one locus and is a nuclear-encoded mitochondrial proton pump. Expression of Nnt is more than sevenfold and fivefold lower respectively in C57BL/6J liver and islets. There is a missense mutation in exon 1 and a multi-exon deletion in the C57BL/6J gene. Glucokinase lies within the Gluchos2 locus and shows reduced enzyme activity in liver. The C57BL/6J mouse strain exhibits plasma glucose intolerance reminiscent of human type 2 diabetes. Our data suggest a defect in beta cell glucose metabolism that results in reduced electrical activity and insulin secretion. We have identified three loci that are responsible for the inherited impaired plasma glucose tolerance and identified a novel candidate gene for contribution to glucose intolerance through reduced beta cell activity.
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              Deletion of nicotinamide nucleotide transhydrogenase: a new quantitive trait locus accounting for glucose intolerance in C57BL/6J mice.

              The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to nicotinamide nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.

                Author and article information

                Role: Editor
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                October 2013
                17 October 2013
                : 7
                : 10
                [1 ]Center for Experimental and Molecular Medicine, Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
                [2 ]Warwick Medical School, University of Warwick, Coventry, United Kingdom
                [3 ]Department of Infection and Tropical Medicine, Heartlands Hospital, Birmingham, United Kingdom
                [4 ]Department of Medicine, University of Cambridge, Cambridge, United Kingdom
                [5 ]Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand
                [6 ]Friedrich Loeffler Institute of Medical Microbiology, University Medicine Greifswald, Greifswald, Germany
                Swiss Tropical and Public Health Institute, Switzerland
                Author notes

                I have read the journal's policy and have the following conflicts to declare: SJP. has received consulting fees from Pfizer. The other authors have no conflicts to report. This does not alter our adherence to all PLOS policies on sharing data and materials.

                Conceived and designed the experiments: GCKWK KB LMK AB SJP TvdP IS WJW. Performed the experiments: GCKWK TAW KK HKdJ LMK AJH. Analyzed the data: GCKWK TAW KB AJH AB TvdP IS WJW. Contributed reagents/materials/analysis tools: KB KK AJH TvdP IS WJW. Wrote the paper: GCKWK TAW TvdP WJW.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 10
                GCKWK received unrestricted funding for this project from the Wellcome Trust of Great Britain (086532/Z/08/Z, www.wellcome.ac.uk). WJW and TvdP received funding from The Netherlands Organisation for Scientific Research (NOW, www.nwo.nl) and The Netherlands Organisation for Health Research and Development (ZonMw, www.zonmw.nl). TvdP also received funding from the AMC Research Council, BEGETU, the European Union, de Landsteiner Stichting voor Bloedtransfusie Research and de Nederlands Astma Fonds. LMK received funding from the Netherlands Organisation for Health Research and Development (ZonMw grant number 92003504, www.zonmw.nl) and the Stichting BeGeTu. SJP received funding from the National Institutes of Health Cambridge Biomedical Research Center, Health Protection Agency (UK), and the UK Clinical Research Collaboration. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article

                Infectious disease & Microbiology


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