The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis

Abstract The emergence of multi and extensively drug resistant tuberculosis (MDRTB and XDRTB) has increased the concern of public health authorities around the world. The World Health Organization has defined MDRTB as tuberculosis (TB) caused by organisms resistant to at least isoniazid and rifampicin, the main first-line drugs used in TB therapy, whereas XDRTB refers to TB resistant not only to isoniazid and rifampicin, but also to a fluoroquinolone and to at least one of the three injectable second-line drugs, kanamycin, amikacin and capreomycin. Resistance in Mycobacterium tuberculosis is mainly due to the occurrence of spontaneous mutations and followed by selection of mutants by subsequent treatment. However, some resistant clinical isolates do not present mutations in any genes associated with resistance to a given antibiotic, which suggests that other mechanism(s) are involved in the development of drug resistance, namely the presence of efflux pump systems that extrude the drug to the exterior of the cell, preventing access to its target. Increased efflux activity can occur in response to prolonged exposure to subinhibitory concentrations of anti-TB drugs, a situation that may result from inadequate TB therapy. The inhibition of efflux activity with a non-antibiotic inhibitor may restore activity of an antibiotic subject to efflux and thus provide a way to enhance the activity of current anti-TB drugs. The work described in this thesis foccus on the study of efflux mechanisms in the development of multidrug resistance in M. tuberculosis and how phenotypic resistance, mediated by efflux pumps, correlates with genetic resistance. In order to accomplish this goal, several experimental protocols were developed using biological models such as Escherichia coli, the fast growing mycobacteria Mycobacterium smegmatis, and Mycobacterium avium, before their application to M. tuberculosis. This approach allowed the study of the mechanisms that result in the physiological adaptation of E. coli to subinhibitory concentrations of tetracycline (Chapter II), the development of a fluorometric method that allows the detection and quantification of efflux of ethidium bromide (Chapter III), the characterization of the ethidium bromide transport in M. smegmatis (Chapter IV) and the contribution of efflux activity to macrolide resistance in Mycobacterium avium complex (Chapter V). Finally, the methods developed allowed the study of the role of efflux pumps in M. tuberculosis strains induced to isoniazid resistance (Chapter VI). By this manner, in Chapter II it was possible to observe that the physiological adaptation of E. coli to tetracycline results from an interplay between events at the genetic level and protein folding that decrease permeability of the cell envelope and increase efflux pump activity. Furthermore, Chapter III describes the development of a semi-automated fluorometric method that allowed the correlation of this efflux activity with the transport kinetics of ethidium bromide (a known efflux pump substrate) in E. coli and the identification of efflux inhibitors. Concerning M. smegmatis, we have compared the wild-type M. smegmatis mc2155 with knockout mutants for LfrA and MspA for their ability to transport ethidium bromide. The results presented in Chapter IV showed that MspA, the major porin in M. smegmatis, plays an important role in the entrance of ethidium bromide and antibiotics into the cell and that efflux via the LfrA pump is involved in low-level resistance to these compounds in M. smegmatis. Chapter V describes the study of the contribution of efflux pumps to macrolide resistance in clinical M. avium complex isolates. It was demonstrated that resistance to clarithromycin was significantly reduced in the presence of efflux inhibitors such as thioridazine, chlorpromazine and verapamil. These same inhibitors decreased efflux of ethidium bromide and increased the retention of [14C]-erythromycin in these isolates. Finaly, the methods developed with the experimental models mentioned above allowed the study of the role of efflux pumps on M. tuberculosis strains induced to isoniazid resistance. This is described in Chapter VI of this Thesis, where it is demonstrated that induced resistance to isoniazid does not involve mutations in any of the genes known to be associated with isoniazid resistance, but an efflux system that is sensitive to efflux inhibitors. These inhibitors decreased the efflux of ethidium bromide and also reduced the minimum inhibitory concentration of isoniazid in these strains. Moreover, expression analysis showed overexpression of genes that code for efflux pumps in the induced strains relatively to the non-induced parental strains. In conclusion, the work described in this thesis demonstrates that efflux pumps play an important role in the development of drug resistance, namely in mycobacteria. A strategy to overcome efflux-mediated resistance may consist on the use of compounds that inhibit efflux activity, restoring the activity of antimicrobials that are efflux pump substrates, a useful approach particularly in TB where the most effective treatment regimens are becoming uneffective due to the increase of MDRTB/XDRTB.

Au-nanossondas aplica����o na detec����o de Mycobacterium tuberculosis e plasmodium berghei

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para a obten����o do grau de Mestre em Biotecnologia

Caracteriza����o de estirpes Mycobacterium Tuberculosis da fam��lia Lisboa: an��lise de regi��es de diferen��a e single nucleotide polymorphisms

Disserta����o apresentada para a obten����o do Grau de Mestre em Gen��tica Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ci��ncias e Tecnologia

Gold nanoprobes for the detection of mutations associated with antibiotic resistance in Mycobacterium tuberculosis complex

Disserta����o para obten����o do Grau de Mestre em Gen��tica Molecular e Biomedicina

Novas estrat��gias no combate �� tuberculose: a NADH-MENAQUINONA desidrogenase do tipo II de Mycobacterium tuberculosis

Disserta����o para obten����o do Grau de Mestre em Microbiologia M��dica

Exploring motor neuron degeneration in ALS - prevention by glycoursodeoxycholic acid and signaling to microglia

Disserta����o para obten����o do Grau de Mestre em Gen��tica Molecular e Biomedicina

Implementation of a pilot project for the treatment and prevention of substance abuse in children and adolescents : strengthening families for parents and youths 12-16

RESUMO: Antecendentes: Uma avalia����o dos servi��os de abuso de subst��ncias em Barbados identificou a necessidade de programas e servi��os que s��o projetados especificamente para crian��as e adolescentes. Objetivo: Realizar programa com base em evid��ncias para reduzir a incid��ncia de abuso de drogas entre crian��as e adolescentes por meio do fortalecimento da unidade familiar atrav��s de parentalidade positiva, de maior funcionamento familiar e de resist��ncia dos jovens. M��todo: Dois projetos-piloto foram realizadas com base no programa "Fortalecer as Fam��lias para Pais e Jovens de 12 a 16 anos (SFPY). O programa de nove semanas foi empregado como uma interven����o para criar la��os familiares mais fortes, aumentar a resist��ncia dos jovens e reduzir o abuso de drogas entre crian��as e adolescentes de idades de 11 a 16 anos. A decis��o foi tomada para incluir participantes de 11 anos desde que as crian��as possam estar no primeiro ano da escola secund��ria nessa idade. IMPLEMENTATION OF SUBSTANCE ABUSE PILOT PROJECT FOR CHILDREN AND ADOLESCENTS 5 Resultados: Quinze fam��lias participaram em dois projetos-piloto e a avalia����o final mostrou que os jovens ap��s o programa, geralmente tornaram-se mais positivos sobre o seu lugar na unidade familiar e sentiram que sua participa����o no programa foi ben��fica. Os pais, da mesma forma, relataram que eles conquistaram, com o programa uma rela����o mais positiva, uma melhor compreens��o das necessidades, e consci��ncia das mudan��as de desenvolvimento de seus jovens. Desta forma, considera-se que o programa atingiu o resultado desejado de criar unidades familiares mais fortes. Conclus��o: O Projeto Piloto ���SFPY��� foi bem sucedido em fazer pais e jovens mais conscientes de suas necessidades individuais e de responsabilidades dentro da unidade familiar. Como resultado, o relacionamentos das respectivas fam��lias melhorou. Estudos baseados em evid��ncias t��m demonstrado que um rela����o familiar mais forte diminui a incid��ncia de uso e abuso de drogas na popula����o adolescente, aumentando os fatores de prote����o e diminuindo os fatores de risco. A implementa����o do programa, que foi desenvolvido e testado no ambiente norte-americano, demonstrou que era transfer��vel para a sociedade de Barbados. No entanto, seu impacto total s�� pode ser determinado atrav��s de um estudo comparativo envolvendo um grupo de controle e / ou uma interven����o alternativa ao abuso de subst��ncias. Portanto, �� recomend��vel que um estudo comparativo da interven����o SFPY deve envolver uma amostra representativa de adolescentes que est��o em est��gio de desenvolvimento anterior mais cedo. Evid��ncias j�� demonstram que o programa �� mais eficaz, com impacto mais longo sobre os jovens que participam em uma idade maisABSTRACT:Background: An evaluation of substance abuse services in Barbados has identified the need for programmes and services that are specifically designed for children and adolescents. Aim: To conduct an evidence-based programme to reduce the incidence of substance abuse among children and adolescents by strengthening the family unit through positive parenting, enhanced family functioning and youth resilience. Method: Two pilot projects were conducted based on the ���Strengthening Families for Parents and Youths 12��� 16��� (SFPY) programme. The nine-week programme was employed as an intervention to create stronger family connections, increase youth resiliency and reduce drug abuse among children and adolescents between the ages of 11 to 16. The decision was made to include participants from age 11 since children may be in the first year of secondary school at this age. IMPLEMENTATION OF SUBSTANCE ABUSE PILOT PROJECT FOR CHILDREN AND ADOLESCENTS 3 Results: Fifteen families participated in two pilot projects and an evaluation conducted at the conclusion showed that the youth were generally more positive about their perceived place in the family unit and felt that the being in the programme was generally beneficial. The parents similarly reported they had a more positive relationship with their youths and also had a better understanding of their needs, and an awareness of their developmental changes. This affirmed that the programme had achieved its desired outcome to create stronger family units. Conclusion: The SFPY Pilot Project was successful in making parents and youths more aware of their individual needs and responsibilities within the family unit. As a result relationships within their respective families were strengthened. Evidence-based studies have shown that enhanced family functioning decreases the incidence of substance use and abuse in the adolescent population by increasing protective factors and decreasing risk factors. The implementation of the programme, which was developed and tested in the North American environment, demonstrated that it was transferable to the Barbadian society. However, its full impact can only be determined through a comparative study involving a control group and/or an alternative substance abuse intervention. It is therefore recommended that a comparative study of the SFPY intervention should be delivered to a representative sample of adolescents who are at an earlier developmental stage. Evidence has shown that the programme is more effective, with longer impact on youths who participate at a younger age.

Lab on paper: biossensores colorim��tricos de papel para a detec����o de membros do complexo Mycobacterium tuberculosis

Este trabalho foi realizado no ��mbito do projecto Lab on Paper, desenvolvido no Centro de Investiga����o de Materiais (CENIMAT) da Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa (FCT - UNL)

Infe����o por Mycobacterium Tuberculosis em profissionais de sa��de : metodologia de avalia����o do risco e sua aplica����o num hospital central

RESUMO - A Tuberculose surge, de acordo com o ��ltimo relat��rio da Organiza����o Mundial da Sa��de, como a segunda principal causa de morte em todo o mundo, de entre as doen��as infeciosas. Em 2012, 1.3 milh��es de pessoas morreram devido a esta patologia e surgiram 8.6 milh��es de novos casos. De entre os grupos de risco de infe����o, surgem os profissionais de sa��de. A dificuldade no diagn��stico da Tuberculose, o contacto pr��ximo com os pacientes, as medidas de controlo de infe����o por vezes inadequadas s��o algumas das raz��es que explicam o risco mais elevado de contrair Tuberculose no local de trabalho. Esta Disserta����o de Mestrado pretende estabelecer uma nova classifica����o de risco de infe����o por M. tuberculosis em estabelecimentos de sa��de, com vista a promover a sa��de destes profissionais, inovadora nos crit��rios de avalia����o das medidas de controlo de infe����o e de an��lise dos casos de exposi����o n��o protegida a Tuberculose ativa. Esta metodologia de avalia����o foi o resultado de uma revis��o bibliogr��fica sobre a tem��tica, tendo sido aplicada num hospital para verificar a sua adequabilidade e mais-valia.

National suicide prevention strategy for Afghanistan (NSPS)

RESUMO: Do suic��dio no Afeganist��o �� uma prioridade de sa��de p��blica. O Afeganist��o �� um pa��s de baixo rendimento, emergindo de tr��s d��cadas de conflitos. H�� uma alta preval��ncia de sofrimento psicol��gico, perturba����es mentais e abuso de subst��ncias. Existem v��rias quest��es sociais, tais como, desequil��brio/viol��ncia de g��nero, pobreza, atitudes e costumes obsoletos, r��pidas mudan��as s��cio-culturais, viola����o dos direitos humanos e especialmente dos direitos das mulheres e das crian��as. Estes fatores de risco contribuem para o aumento da vulnerabilidade da popula����o em rela����o ao suic��dio. A relativa alta taxa de suic��dio no Afeganist��o �� especialmente significativa comparada com as taxas baixas em todos os pa��ses isl��micos. Os estudos mostraram predomin��ncia de suic��dio nas mulheres (95%) e em pessoas jovens. Existe, por isso, uma necessidade urgente do pa��s ter uma estrat��gia de preven����o do suic��dio. A estrat��gia foi desenvolvida pela cria����o de um grupo t��cnico/ de assessoria multi-sectorial de diferentes intervenientes tais como governo, ONGs, ag��ncias doadoras, as fam��lias das v��timas e outraas partes interessadas. A estrat��gia baseia-se os seguintes valores chave:, respeito pelas diversidades; sensibilidade para as quest��es s��cio-cultura-religiosa e de g��nero; promo����o da dignidade da sociedade; respeito pelos direitos humanoss.. Os 'seis pontos estrat��gicos' s��o: envolvimento das principais partes interessadas e cria����o de colabora����o intersectorial coordenada; fornecimento de cuidados ��s pessoas que fazem tentativas de suic��dio e ��s suas fam��lias; melhoria dos servi��os para pessoas com doen��a mental e problemas psicossociais; promover uma comunica����o e imagem adequada dos comportamentos suicidas, pelos meios de comunica����o; reduzir o acesso aos meios de suic��dio e coligir informa����o sobre as taxas de suic��dio, os fatores de risco, os fatores protetores e as interven����es eficazes. A estrat��gia nacional de preven����o do suic��dio ser�� inicialmente implementada por 5 anos, com uma avalia����o anual do plano de ac����o para entender os seus pontos fortes e limita����es. Recomenda����es e sugest��es ser��o incorporadas nos pr��xima planos anuais para uma interven����o eficaz. Um sistema de monitoriza����o ir�� medir o progresso na implementa����o da estrat��gia.-----------------------------ABSTRACT: Suicide in Afghanistan is a public health priority. Afghanistan is a low-income country, emerging from three decades of conflicts. There is high prevalence of mental distress, mental disorders and substance abuse. There are multiple social issues, such as gender imbalance/violence, poverty, obsolete attitudes and customs, rapid social-cultural changes, human right violations, and especially women and children rights. These risk factors contribute to increase the vulnerability of the population for suicide. The relative high rate of suicide in Afghanistan is especially significant as the rates are low in all Islamic countries. Research studies have shown predominance of suicide in women (95%) and in young age people. There is an urgent need for the country to have a suicide prevention strategy. The strategy has been developed by establishing a multi-sectoral technical/advisory group of different stakeholders from government, NGOs, donor agencies, victim���s families, and interested parties. The strategy is based on the following key values, namely, respect for diversities; sensitiveness to socio-culture-religious and gender issues; promotion of the society dignity and respect for the human rights of people. The six ���Strategic directions��� are: involving key stakeholders and creating coordinated inter-sectoral collaboration; providing after care for people making a suicide attempt and their families; improving services for people with mental disorders and psycho-social problems; promoting the safe reporting and image of suicidal behaviour by media; reducing access to the means of suicide and gathering information about suicide rates, risk factor, protective factors and effective interventions. The National Suicide Prevention Strategy will be initially implemented for 5 years, with an annual evaluation of the action plan to understand the strengths and limitations. Recommendations and suggestions will be incorporated into the next annual plans for effective intervention. A monitoring framework will measure progress in implementing the strategy.