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

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 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 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 codefor 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.

O aumento da tuberculose multirresistente e extensivamente resistente (TBMR e TBXDR) gerou um agravamento das preocupações por parte das autoridades de Saúde Pública em todo o mundo. A Organização Mundial de Saúde (OMS) define TBMR como tuberculose (TB) resistente pelo menos à isoniazida e à rifampicina, os principais fármacos de primeira-linha utilizados no tratamento da TB, enquanto que a TBXDR refere-se a casos de TB resistente não só à isoniazida e à rifampicina, mas também a uma fluoroquinolona e a pelo menos a um dos três fármacos de segunda-linha injectáveis canamicina, amicacina e capreomicina. A resistência em Mycobacterium tuberculosis deve-se principalmente à ocorrência de mutações espontâneas, à qual se segue a selecção de mutantes resistentes durante o tratamento. No entanto, algumas estirpes clínicas de M. tuberculosis resistentes não apresentam mutação em qualquer um dos genes que se sabe estarem associados à aquisição de resistência a um determinado fármaco, o que sugere que outro(s) mecanismo(s) deverão estar envolvidos no desenvolvimento de resistência, nomeadamente a presença de sistemas de bombas de efluxo que efectuam a extrusão do composto para o exterior da célula, evitando que o mesmo atinja o seu alvo. Um aumento da actividade de efluxo pode ocorrer devido a uma exposição prolongada a concentrações subinibitórias dos antibacilares, uma situação que pode resultar de uma terapia inadequada. A inibição da actividade de efluxo com um inibidor que não seja um antibiótico poderá restaurar a actividade de um antibiótico que seja substrato de bombas de efluxo e desta forma consistir uma forma de aumentar a actividade dos actuais fármacos utilizados no tratamento da TB. O trabalho descrito nesta dissertação tem como objectivo o estudo dos mecanismos de efluxo no desenvolvimento de multirresistência em M. tuberculosis e de como a resistência fenotípica mediada por bombas de efluxo se correlaciona com a resistência genética. De forma a alcançar este objectivo, foram desenvolvidos vários protocolos experimentais utilizando modelos biológicos, tais como Escherichia coli, Mycobacterium smegmatis uma micobactéria de crescimento rápido e Mycobacterium avium, antes da sua aplicação ao estudo de M. tuberculosis. Esta abordagem permitiu o estudo dos mecanismos que resultam na adaptação fisiológica de E. coli à tetraciclina por exposição a concentrações subinibitórias deste antibiótico (Capítulo II), o desenvolvimento de um método fluorimétrico que permite a detecção e quantificação da actividade de efluxo (Capítulo III), a caracterização do transporte de brometo de etídeo em M. smegmatis (Capítulo IV) e a contribuição da actividade de efluxo para a resistência aos macrólidos no complexo Mycobacterium avium (Capítulo V). Por fim, os métodos desenvolvidos permitiram o estudo do papel das bombas de efluxo em estirpes de M. tuberculosis induzidas a resistência à isoniazida. Assim, como descrito no Capítulo II, foi possível observar que a adaptação fisiológica de E. coli à presença de tetraciclina resulta de uma interacção entre mecanismos a nível genético e modificações pós-traducionais a nível da conformação de proteínas que diminui a permeabilidade da parede celular e aumenta a actividade das bombas de efluxo. Para além disso, o Capítulo III descreve o desenvolvimento de um método fluorimétrico semi-automático que permitiu correlacionar esta actividade de fluxo com a cinética de transporte do brometo de etídeo (um conhecido substrato de bombas de efluxo) em E. coli e também a identificação de inibidores do efluxo. Relativamente a M. smegmatis, comparou-se a estirpe selvagem M. smegmatismc2155 com mutantes "knockout" para LfrA e MspA, no que respeita à sua capacidade de transportar brometo de etídeo. Os resultados apresentados no Capítulo IV demonstraram que MspA, a principal porina de M. smegmatis, desempenha um papel importante na entrada de brometo de etídeo e antibióticos na célula e que o efluxo através da bomba LfrA está envolvido na resistência de baixo nível a estes compostos em M. smegmatis. O Capítulo V descreve o estudo da contribuição de bombas de efluxo na resistência aos macrólidos em estirpes clínicas do complexo M. avium. Demonstrou-se que a resistência à claritromicina sofreu uma redução significativa na presença dos inibidores de efluxo tioridazina, clorpromazina e verapamil. Estes inibidores também diminuíram o efluxo de brometo de etideo e aumentaram a retenção de eritromicina marcada com 14C nestas estirpes. Por fim, os métodos desenvolvidos com os modelos experimentais referidos acima permitiram o estudo do papel das bombas de efluxo em estirpes de M. tuberculosisinduzidas à resistência à isoniazida. Este trabalho encontra-se descrito no Capítulo VI desta dissertação, onde se demonstra que a indução de resistência à isoniazida não resultou da ocorrência de mutações em qualquer um dos genes associados com a resistência a este fármaco, mas de um sistema de efluxo que é sensível a inibidores do efluxo. Estes inibidores provocaram a diminuição do efluxo de brometo de etídeo e também a redução da concentração mínima inibitória da isoniazida nestas estirpes. Para além disso, a análise de expressão genética demonstrou a sobre-expressão de genes que codificam para bombas de efluxo nas estirpes induzidas comparativamente com as estirpes originais não induzidas. Concluindo, o trabalho descrito nesta dissertação demonstra que as bombas de efluxo desempenham um papel importante no desenvolvimento de resistência, em particular nas micobactérias. Uma estratégia para ultrapassar a resistência mediada por mecanismos de efluxo poderia passar pela utilização de compostos que inibem a actividade de efluxo, restaurando a actividade de antimicrobianos que são substratos de bombas de efluxo, uma abordagem útil particularmente em TB em que os regimes de tratamento mais eficazes se estão a tornar ineficazes face ao aumento da TBMR/TBXR.