Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.

During the implementation of a PCR-based assay to differentiate between Mycobacterium abscessus and M. chelonae using the 16S to 23S internal transcribed spacer region (1), a sequence variant of M. abscessus was discovered. The sequence variant did not bind the M. abscessus specific probe, resulting in no or low fluorescence detected. The base change was found at position 39 of the amplicon (GenBank accession no. DQ177308). The base change was confirmed by sequencing with four additional patient isolates. A total of 6 isolates out of 129 tested from 3 March 2005 to 21 July 2005 had low fluorescence detected for a frequency of 4.65%.The PCR assay has since been improved by redesigning the M. abscessus probe (Nanogen, Inc., Bothell, WA) to incorporate proprietary modified bases for more-efficient hybridization characteristics. The probe modification included incorporation of a super G (proprietary modified base G) at the site of the polymorphism as well as a super A (proprietary modified base A) further downstream ( Table 1). The improved assay allowed for a detectable level of fluorescence for all isolates. The fluorescence detected in the FAM (6-carboxyfluorescein) channel on the SmartCycler II (Cepheid, Inc., Sunnyvale, CA) increased from an average of 41.7 Ϯ 36 (range of 0 to 100) fluorescence units to 180 Ϯ 22 (range of 150 to 200) fluorescence units.The assay with the modified probe performed with 100% sensitivity after testing 22 M. abscessus isolates, 6 of which were sequence variants, and 100% specificity after testing 10 M. chelonae isolates. All samples tested were proven by 16S rRNA gene sequencing to be among the M. chelonae-M. abscessus complex, as previously reported (1).