Basic Research
Copyright ©2008 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. May 7, 2008; 14(17): 2723-2730
Published online May 7, 2008. doi: 10.3748/wjg.14.2723
Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn’s disease
Daniel R Beckler, Sammer Elwasila, George Ghobrial, John F Valentine, Saleh A Naser
Daniel R Beckler, Sammer Elwasila, George Ghobrial, Saleh A Naser, Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, Center for Biomolecular Sciences, College of Medicine, University of Central Florida, Florida, FL 32816, United States
John F Valentine, Department of Medicine, University of Florida, Gainesville, Florida, FL 32810, United States
Author contributions: Beckler DR, Elwasila S, Ghobrial G, Valentine JF, Naser SA contributed equally to the work; Beckler DR, Elwasila S, and Ghobrial G participated in the experiments design and the data collection; Valentine JF provided the clinical samples and assisted in data interpretation; Naser SA participated in research design, supervising the daily experiments, interpretation of the data and editing the manuscript.
Correspondence to: Saleh A Naser, Professor, Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 4000 Central Florida Blvd, Florida, FL 32816, United States.
Telephone: +1-407-823-0955
Fax: +1-407-823-0956
Received: December 20, 2007
Revised: March 14, 2008
Published online: May 7, 2008

AIM: To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in clinical MAP strains with susceptibility to RIF and RFB.

METHODS: 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 11 MAP isolates in attempt to seek correlation with rpoB sequences.

RESULTS: We determined that MAP strain 18 had an MIC of > 30 mg/L and ≤ 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of ≤ 1.0 mg/L 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. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected 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 mg/L and > 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r.

CONCLUSION: The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. 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.

Keywords: Mycobacterium paratuberculosis, Crohn’s disease, Rifabutin, Rifampicin, rpoB, Minimum inhibitory concentration