<p>Normal healthy people are not
susceptible to tuberculosis (TB) but immunocompromised and HIV positive
patients are at high risk of TB. The treatment regimen (rifampin, isoniazid and
amikacin) for TB patients is 6-9 months for normal patients but if <i>Mycobacterium tuberculosis</i> (Mtb) becomes
multidrug resistant, it takes 20-30 months to treat. According to the World Health Organization in
2018, there were about half a million new cases among which 78% were multidrug
resistant TB. This antibiotic resistance is due in part to its ability to
survive in the macrophage in our body by entering a non-replicating persistent
state. Mtb also contains efflux pumps that increase antibiotic tolerance by
pumping out the drugs. Therefore, if the efflux pump activity can be blocked by
using efflux pump inhibitors, then it might increase antibiotic susceptibility
of the pathogen. In our study, we used <i>Mycobacterium
smegmatis</i> (Msm) as a model organism for Mtb and subjected it to a
combination of three stresses (low oxygen, low pH and low nutrients) that mimic
the physiological stresses in the human body and report that these conditions
produced a non-replicating state in Msm. This is the first report of the use of
this combination of stresses to produce a non-replicating state in Msm. Our
results show that non-replicating Msm became completely tolerant to isoniazid
and displayed increased tolerance to rifampin and clarithromycin by nearly
2-fold when compared to log-phase cells. Moreover, the efflux pump inhibitor
verapamil decreased the antibiotic tolerance of the nonreplicating Msm to the
antibiotics by 6-10 fold and the efflux pump inhibitor piperine decreased
tolerance to the antibiotics by 2-4 fold. Also, in this study we attempted to
construct a gene knockout mutant lacking two potential ATP-binding cassette
transporters to study their functions as drug exporters. However, we were
unable to obtain homologous recombination mutants. Further studies on efflux
pump inhibitors could potentially enable greater understanding of antibiotic
tolerance mechanisms in non-replicating, drug tolerant Mtb and enable the
development of novel therapies that shorten treatment time for tuberculosis.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/12226886 |
| Date | 01 May 2020 |
| Creators | Sushanta Ratna (8787791) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/INVESTIGATIONS_ON_THE_ROLES_OF_EFFLUX_PUMP_INHIBITORS_ON_THE_ANTIBIOTIC_TOLERANCE_OF_NON-REPLICATING_MYCOBACTERIUM_SMEGMATIS/12226886 |
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