Emerging rise in microbial drug resistance and the slow-growing characteristic of some intracellular pathogens such as MAP (Mycobacterium avium subspecies paratuberculosis) strongly urges the need for an effective approach for unconventional drug susceptibility testing. We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 10 MAP isolates in attempt to seek correlation with rpoB sequences. We determined that MAP strain 18 had an MIC ≥ 30 ug/ml and ≥ 5 ug/ml for RIF and RFB respectively, and a significant rpoB mutation C1367T, compared to an MIC of ≤ 1.0 ug/ml for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site; the beta subunit of RNA polymerase. Our in vitro induced mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC ≥ 30 ug/ml and ≥ 10 ug/ml for RIF and RFB respectively. The T1442C mutation resulted in a Leu481 to Pro481 amino acid change, consequently altering the beta subunit sequence. Sequencing the entire 3.5 kb rpoB in strains 18 and UCF5-RIF16r revealed no additional expressed nucleotide mutation. Of the 10 MAP strains analyzed, an additional one strain (UCF4) exhibited a slight increase in the MIC against RIF and RFB compared to the wild-type. Nucleotide sequencing of the rpoB gene revealed an A2284C mutation in strain UCF4 that occurred further downstream of the expected probable rpoB region and resulted in an amino acid alteration Asn762 to His762. The location of this mutation outside the binding site and its correlation with the minor increase in MIC suggests a possible secondary interaction between the drug and the beta subunit. We have provided three dimensional images through the utilization of PyMol Molecular-based Graphics to display a clear comparison of the mutations observed in the beta subunit for MAP strains 18, UCF5-RIF16r, and UCF4. We propose that these alterations may have caused a less stable interaction between RIF and the beta subunit, resulting in the observed increased in MIC. Furthermore, the change in amino acid sequence did not affect the viability for our RIF resistant strains. The data clearly illustrates that clinical and in vitro-induced MAP mutants with rpoB mutations result in resistance to RIF and RFB. Consequently, unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd-4079 |
| Date | 01 January 2007 |
| Creators | Beckler, Daniel |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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