Routine rapid Mycobacterium species assignment based on species-specific allelic variation in the 65-kilodalton heat shock protein gene (hsp65).
OBJECTIVE
To assess the utility of automated DNA sequencing strategies for Mycobacterium species assignment and surrogate rifampin susceptibility testing of Mycobacterium tuberculosis complex isolates in a hospital-based clinical microbiology laboratory.


METHODS
Consecutive patient specimens (n = 161) cultured in BACTEC 12B medium (growth index of 50 or greater) or on solid media (Löwenstein-Jensen) were analyzed. A 360-bp segment of a gene (hsp65) encoding a 65-kd heat shock protein was sequenced to identify species-specific allelic polymorphism. Identification of sequence variation in the rpoB gene encoding the beta subunit of RNA polymerase was used as a surrogate method to assess rifampin susceptibility in M tuberculosis complex isolates.


RESULTS
The automated DNA sequencing strategies rapidly identified virtually all mycobacteria (158 [98%] of 161) to the species level and unambiguously characterized the region of rpoB that contains mutations responsible for rifampin resistance in M tuberculosis strains. With few exceptions, DNA sequence-based species assignment data agreed with diagnostic information obtained by conventional methods. All discrepancies were due to ambiguous biochemical test data or interpretation. The rifampin susceptibility phenotype was correctly predicted for all strains by rpoB sequencing.


CONCLUSIONS
Rapid mycobacterial species assignment based on hsp65 sequencing can be routinely performed in a hospital diagnostic microbiology laboratory setting. The method is especially useful for identification of fastidious organisms, such as Mycobacterium genavense. Sequencing of the rifampin-resistance-determining region of rpoB provides a convenient surrogate strategy for predicting rifampin susceptibility in M tuberculosis complex isolates.
Pai
S
S
Clinical Microbiology Laboratory, Methodist Hospital Houston, Tex, USA.
Esen
N
N
Pan
X
X
Musser
J M
JM
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D018834:Chaperonin 60; D018833:Chaperonins / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D009161:Mycobacterium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology; D017422:Sequence Analysis, DNA / Q000379:methods
D001426:Bacterial Proteins; D018834:Chaperonin 60; D017931:DNA Primers; D004269:DNA, Bacterial; C074119:heat-shock protein 65, Mycobacterium; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D018833:Chaperonins; D012293:Rifampin