Characterization of Mycobacterium fortuitum isolates from animals and their environment by PCR-RFLP analysis (PRA) of hsp65 and rpoB genes

Mycobacterium fortuitum strains isolated from soil and water samples from animal dwellings and animal tissue specimens were characterized by biochemical tests and PRA ofhsp65 and rpoB genes. Specimens (161) comprising 81 soil samples, 20 water samples and 60 animal tissues yielded 30 (85.7%) M.fortuitum isolates from soil. 3 (8.6%) from water and 2 (5.7%) from animal tissues. In PCR ofhsp65 gene all the isolates generated an amplification product of439 bp product. RE digestion ofthe product with BstEll and analysis ofdigests by 3% agarose gel electrophoresis revealed identical RFLP patterns of3 bands (245 bp, 120 bp and 80 bp) in all the isolates including reference strain. However, with HaeIII the isolates were differentiated into 2 groups. First group (22 isolates including reference strain) showing 2 fragments of 155 bp and 135 bp size was identified as M.fortuitum I whereas the other group (10 isolates) producing fragments of 150 bp and 135 bp size was identified as M. fortuitumll. In PCR of rpoB all the M. fortuitum isolates showed an amplification product of360 bp. RE digestion of the product with HaeIII revealed identical RFLP patterns in all the isolates however MspI digestion separated the isolates into 2 groups. The discrimination of M. fortuitum isolates that was observed in PRA ofhsp65 gene was also achieved by PRA ofrpoB gene. Both the PRA methods were useful in identification ofM.jortuitum isolates to species level and their further differentiation to the level ofsubspecies.