Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn's disease.

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 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.

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.