Use of short-term culture for identification of Mycobacterium avium subsp paratuberculosis in tissue from Crohn's disease patients

Objective To investigate the role of Mycobacterium avium subp. paratubercul osis (MAP) in Crohn's disease (CD), using short-term mycobacterial culture media. Methods Sixty-three tissue specimens from 27 CD patients and 36 controls we re processed and inoculated into a modified 7H9 broth base medium and incub ated at 37 degrees C and 5% CO2 for up to 1 year Acid-fast staining, determ ination of mycobactin dependency, PCR analysis using two IS900-derived olig onucleotides and hybridization with an internal probe were performed. Results MAP was present in six of seven (86%) surgically resected tissue sa mples and in four of 20 (20%) biopsies, with an overall 37% from CD patient s, as compared to two of 36 (5.6%) of control specimens. The presence of MA P in Mycobacterial Growth Indicator Tube (MGIT) cultures was detected withi n 10-12 weeks for surgically resected tissue and after 40 weeks for biopsy specimens, with no MAP growth detected in 22B* Bactec cultures. Conclusions Because MAP was present in 86% of resected tissue compared to 2 0% of biopsy specimens from CD patients, we speculate that MAP resides in t he submucosal layer closer to the active part of the ulcer rather than on t he surface of the mucosal cells. Thus, surgically resected tissue cultured in MGIT medium is a favorable protocol for rapid cultivation of MAP and for investigating its role in CD pathogenesis. The data support the mycobacter ial role in CD pathogenesis.