Genomic analyses of Mycobacterium species and clinical Mycobacterium tuberculosis isolates from Utah

Resistance of Mycobacterium tuberculosis to antitubercular drugs is a global problem, which is on the rise; and due to the slow growth of M. tuberculosis, it takes several weeks to identify the species and determine drug susceptibility of the infecting strain. We investigated whether amplified fragment length polymorphism (AFLP) would be an effective way to quickly detect drug resistance and to type mycobacteria. Over one hundred clinical isolates of M. tuberculosis and numberous nontuberculosis isolates were examined by AFLP. Two pairs of enzymes were used in this analysis: EcoRI - MseI and ApaI - TaqI. The restriction digest was then ligated to specific linkers and the fragments were then amplified by PCR with nonselective primers. Selective FAM-labeled primers were used to amplify fragments. These fragments were analyzed using an ABI 377 DNA sequencer under denaturing conditions and GeneScan software was used to analyze the fragments generated. The banding patterns were compared and trees generated by neighbor joining using PAUP*4.0. AFLP easily distinguished different species of Mycobacterium. Mycobacteria other than M. tuberculosis generated very different profiles from those of M. tuberculosis. Specific cluster groups of similar M. tuberculosis isolates were also resolved. Although AFLP patterns were not associated with specific drug resistance, there was significant clustering of drug-resistant strains in general. The possible genetic commonality for this clustering warrants further study.