Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn's disease.
OBJECTIVE
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 <or= 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of <or= 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; the beta subunit of RNA polymerase. 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.


CONCLUSIONS
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.
Beckler
Daniel R
DR
Department of Molecular Biology and Microbiology, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Florida, FL 32816, United States.
Elwasila
Sammer
S
Ghobrial
George
G
Valentine
John F
JF
Naser
Saleh A
SA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000627:therapeutic use*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003424:Crohn Disease / Q000188:drug therapy*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D016927:Mycobacterium avium subsp. paratuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D018579:Patient Selection; D016133:Polymerase Chain Reaction; D017828:Rifabutin / Q000627:therapeutic use*; D012293:Rifampin / Q000627:therapeutic use*; D017422:Sequence Analysis, DNA; D017211:Treatment Failure
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017828:Rifabutin; D012293:Rifampin