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|Title: ||The role of the efflux mechanisms in multidrug resistance in Mycobacterium tuberculosis|
|Authors: ||Rodrigues, Liliana|
|Advisor: ||Viveiros, Miguel|
|Issue Date: ||2010|
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.|
|Appears in Collections:||IHMT: MM - Teses de Doutoramento|
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