Using Gene Overexpression as a Potential Method to Elucidate Antibiotic Resistance Mechanisms in Mycobacteria
Bacalao, Maria Alexandra
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Mycobacterium Tuberculosis remains a major public health threat, with 9 million new cases and 1.5 million deaths in 2013. Also concerning is the rise of antbiotic resistance, leading to the development of Multiple Drug Resistant (MDR) and Extensively Drug Resistance (XDR) Mycobacterium tuberculosis strains. The development of these strains is multifactorial involving both extrinsic factors and intrinsic factors. Extrinsic factors include treatment noncompliance and a delay in diagnosis with existing drug sensitivity methods. Intrinsic factors include heterogeneity in the bacterial population and localization in cavitations and other areas where antibiotic penetration is difficult. Our study analyzed the potential role of a potential mechanism of antibiotic resistance, overexpression of existing wild type M. tuberculosis genes involved in mycobacterial antibiotic defense. We cloned seven M. tuberculosis wild type genes using PCR and the subcloned those constructs into E. Coli. Further subcloning steps placed these genes into shuttle vectors that allowed for transformation into M tuberculosis and M. smegmatis. The M. Smegmatis mutant overexpressing the GyrA gene was selected for dose-response studies to establish whether GyrA overexpression led to increased fluoroquinolone resistance. Two M. smegmatis GyrA mutant and one wild type strain were incubated with increasing concentration of Moxifloxacin. Initial dose-response studies did not show yield an adequate dose-response curves under the study conditions. However, the MIC was higher for the GyrA mutant strains than for wild type M. smegmatis, showing that these GyrA mutants were likely more resistant than wild type and that the role of gene overexpression in M. tuberculosis antibiotic resistance merits further study.