Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment

pH Modulation of Efflux Pump Activity of Multi-Drug Resistant Escherichia coli: Protection During Its Passage and Eventual Colonization of the Colon

BACKGROUND:Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB). METHODOLOGY/PRINCIPAL FINDINGS:E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated. CONCLUSIONS/SIGNIFICANCE:The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host

Project 8 Phase III Design Concept

We present a working concept for Phase III of the Project 8 experiment, aiming to achieve a neutrino mass sensitivity of $2~\mathrm{eV}$ ($90~\%$ C.L.) using a large volume of molecular tritium and a phased antenna array. The detection system is discussed in detail.Comment: 3 pages, 3 figures, Proceedings of Neutrino 2016, XXVII International Conference on Neutrino Physics and Astrophysics, 4-9 July 2016, London, U

Results from the Project 8 phase-1 cyclotron radiation emission spectroscopy detector

The Project 8 collaboration seeks to measure the absolute neutrino mass scale by means of precision spectroscopy of the beta decay of tritium. Our technique, cyclotron radiation emission spectroscopy, measures the frequency of the radiation emitted by electrons produced by decays in an ambient magnetic field. Because the cyclotron frequency is inversely proportional to the electron's Lorentz factor, this is also a measurement of the electron's energy. In order to demonstrate the viability of this technique, we have assembled and successfully operated a prototype system, which uses a rectangular waveguide to collect the cyclotron radiation from internal conversion electrons emitted from a gaseous $^{83m}$Kr source. Here we present the main design aspects of the first phase prototype, which was operated during parts of 2014 and 2015. We will also discuss the procedures used to analyze these data, along with the features which have been observed and the performance achieved to date.Comment: 3 pages; 2 figures; Proceedings of Neutrino 2016, XXVII International Conference on Neutrino Physics and Astrophysics, 4-9 July 2016, London, U

Laboratory services contribution to supporting public health and health services in Portuguese-speaking countries

Objetivo: promover o debate sobre a importância do laboratório no apoio à saúde pública e aos serviços de saúde nos países de língua portuguesa, numa perspetiva global e de uma só saúde. Métodos: revisão de literatura, análise dos documentos preparatórios das comunicações do 5º Congresso Nacional de Medicina Tropical, apresentações realizadas pelos oradores de Cabo Verde, Angola e Portugal e o debate que se seguiu. Resultados: identificaram-se os desafios que deverão ser ultrapassados de forma a criar uma rede de laboratórios de diagnóstico que possa estimular o acesso universal das populações aos cuidados de saúde, tendo-se proposto medidas e estratégias com base na experiência dos sistemas de saúde dos diferentes países de língua portuguesa. Reviu-se o contributo dos Laboratórios do IHMT/NOVA para esta estratégia. Conclusão: não há acesso universal a cuidados de saúde de qualidade sem serviços de diagnóstico laboratorial de qualidade. Assim, deve-se promover uma correta, eficaz e sustentável inter-relação entre laboratórios de referência nacionais, hospitalares e privados e ainda laboratórios de investigação, todos cooperando em sintonia com os serviços de saúde de modo a garantir os melhores cuidados de saúde. Objective: To promote the debate on the importance of the laboratory services in supporting public health and health services in Portuguese-speaking countries, from a global and One Health perspective. Methods: literature review, analysis of the preparatory papers for the 5th Tropical Medicine National Congress, presentations by speakers from Cape Verde, Angola and Portugal and the ensuing debate. Results: we identified the challenges that must be overcome in order to create a network of diagnostic laboratories that can stimulate universal access to health care for the populations. Measures and strategies based on the experience of health systems in different Portuguese-speaking countries are proposed. The contribution of the IHMT/NOVA Laboratories to this strategy was reviewed. Conclusion: There is no universal access to quality health care with-out quality laboratory diagnostic services and without promoting a correct, effective and sustainable interrelationship between national reference laboratories, hospital and private, and research laboratories, all cooperating in line with health services to ensure the best quality health care.publishersversionpublishe