RAPID MYCOBACTERIUM SPECIES ASSIGNMENT AND UNAMBIGUOUS IDENTIFICATIONOF MUTATIONS ASSOCIATED WITH ANTIMICROBIAL RESISTANCE IN MYCOBACTERIUM-TUBERCULOSIS BY AUTOMATED DNA-SEQUENCING

Objective.-To develop and demonstrate the utility of automated DNA seq uencing strategies for rapid and unambiguous identification of Mycobac terium species and mutations associated with antimicrobial resistance in Mycobacterium tuberculosis. Design and Specimens.-A 360-base pair s egment of the gene (hsp65) encoding a 65-kd heat shock protein was cha racterized from 91 isolates assigned to 24 Mycobacterium species by tr aditional biochemical techniques. Areas of seven genes recently shown to contain mutations associated with antimicrobial resistance in M tub erculosis strains were also sequenced in a sample of 128 resistant org anisms. Early positive BACTEC 460 cultures and acid-fast, bacterium-po sitive sputum specimens from patients with tuberculosis were also stud ied. Results.-Automated DNA sequencing identified species-specific pol ymorphism in the target segment of hsp65, successfully identified orga nisms to the species level in smear-positive sputum samples, and unamb iguously characterized seven genes associated with antimicrobial resis tance in M tuberculosis. Conclusions.-Rapid identification of M tuberc ulosis and other Mycobacterium species is possible by automated DNA se quencing of a portion of hsp65. The technique is also feasible for ana lysis of some smear-positive sputum specimens. Unambiguous characteriz ation of target segments of genes harboring mutations associated with antimicrobial resistance in M tuberculosis is possible from primary pa tient specimens. Taken together, the data demonstrate the feasibility of mycobacterial species identification and potential to identify muta tions associated with antimicrobial resistance in less than 48 hours.