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Subspecific Differentiation of Mycobacterium avium Complex Strains by Automated Sequencing of a Region of the Gene (hsp65) Encoding a 65-Kilodalton Heat Shock Protein

DOUGLAS S. SWANSON^1,2, VIVEK KAPUR^2,, KATHRYN STOCKBAUER², XI PAN², RICHARD FROTHINGHAM^3,4 and JAMES M. MUSSER^2,5,*

¹ Section of Infectious Diseases, Department of Pediatrics, Houston, Texas 77030
² Section of Molecular Pathobiology, Department of Pathology, Houston, Texas 77030
⁵ Baylor College of Medicine, and Clinical Microbiology Laboratory and Molecular Diagnostics Laboratory; The Methodist Hospital, Houston, Texas 77030
³ Infectious Disease Section, Durham Veterans Affairs Medical Center, Durham, North Carolina 27705
⁴ Division of Infectious Diseases and International Health, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

^* Corresponding author. Mailing address: Department of Pathology, Baylor College of Medicien, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-4198. Fax: (713) 798-4595. E-mail: jmusser{at}path.bcm.tmc.edu.

ABSTRACT

To develop a strategy for rapid species assignment and strain differentiation of Mycobacterium avium complex (MAC) organisms, the sequence of a 360-bp region of the gene (hsp65) encoding a 65-kDa heat shock protein was determined for 56 isolates, including 21 patient isolates and 35 reference strains. Eleven hsp65 alleles were identified, and there was no sharing of alleles between strains classified as M. avium and Mycobacterium intracellulare based on serovar and species-specific DNA hybridization probes. Phylogenetic analysis showed that 30 strains had one of two hsp65 alleles which were found in known M. avium organisms, 23 strains had one of six alleles allied with known M. intracellulare organisms, and three MAC isolates had one of three hsp65 alleles that differed substantially from the consensus M. avium and M. intracellulare hsp65 sequences. Estimates of strain relationships based on the sequences of hsp65 and the 16S-23S ribosomal DNA internal transcribed spacer were similar. Automated DNA sequencing of a 360-bp region of the hsp65 gene from MAC organisms provides a rapid and unambiguous marker system for strain differentiation and permits specific assignment of these acid-fast organisms for diagnostic purposes.

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