Identification of Clinical isolates of <i>Mycobacterium</i> spp. by sequence analysis of the 16S ribosomal RNA gene

Holberg-Petersen, Mona; Steinbakk, Martin; Figenschau, K. J.; Jantzen, Erik; Eng, Jan; Melby, K

doi:10.1111/j.1699-0463.1999.tb01549.x

Cited by 28 publications

(15 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our findings are consistent with other reports which compared phenotypic identification to molecular sequencing (12,18,26). Due to reduced repeat rates, we found the MicroSeq 500 system to be faster than our conventional identification scheme and also more accurate when the RIDOM database was consulted.…”

Section: Discussionsupporting

confidence: 83%

Identification of Mycobacterium spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries

et al. 2002

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 83%

Identification of Mycobacterium spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries

et al. 2002

View full text Add to dashboard Cite

show abstract

“…Cost-effective methodologies are without purpose in the face of inaccu- rate results. The accuracy of 16S rDNA sequencing in the identification of NTM to the species level is well supported in the literature (4,9,12,15,17,20). Multiple studies have confirmed the speed and accuracy of 16S rDNA sequencing in the identification of mycobacteria by directly comparing traditional phenotypic methodology with 16S rDNA sequencing in the species determination of difficult to identify mycobacteria.…”

Section: Discussionmentioning

confidence: 54%

Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis

Cook¹,

Turenne

Wolfe

et al. 2003

J Clin Microbiol

View full text Add to dashboard Cite

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.

show abstract

“…To minimize turnaround time, 16S rRNA sequencing is performed on all isolates received and has become our primary tool for identification. Studies using 16S ribosomal DNA (rDNA) sequence-based identification methods for the identification of mycobacteria show that this technique is more rapid (hours versus weeks) and more accurate than conventional methods (18,23,28,39). It also provides information on the taxonomic relatedness of new species, which may not be possible with other technologies.…”

mentioning

confidence: 99%

Necessity of Quality-Controlled 16S rRNA Gene Sequence Databases: Identifying Nontuberculous Mycobacterium Species

et al. 2001

View full text Add to dashboard Cite

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing ϳ440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1-to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.The notion that sequence-based methodologies will take their place in routine clinical laboratories is an increasing reality. The initial cost of equipment, i.e., automated sequencers, can quickly be recovered with savings in personnel, time, and ultimately in health care costs. Laboratories are beginning to rely more on genotypic characterization, which is more specific and easier to standardize than conventional tests (14).Leaders in the field of diagnostic mycobacteriology acknowledge the need for advanced methods for more accurate identification of mycobacteria to the species level (36,39,44). The number of members within the genus is highly underestimated, and of the 92 currently established species, as listed in J. P. Euzéby's: "List of bacterial names with standing in nomenclature" (http://www.bacterio.cict.fr/m/mycobacterium.html), many are considered potentially pathogenic. Biochemical characteristics have been well established for the most well-known Mycobacterium species, but with a rapid increase of newly described species, this becomes more and more complex, difficult, and perhaps obsolete. Also, many species that are biochemically inert or extremely slow growing contribute further to these problems.The 16S rRNA gene is the most widely accepte...

show abstract

Identification of Clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene

Cited by 28 publications

References 30 publications

Identification of Mycobacterium spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries

Identification of Mycobacterium spp. by Using a Commercial 16S Ribosomal DNA Sequencing Kit and Additional Sequencing Libraries

Conventional Methods versus 16S Ribosomal DNA Sequencing for Identification of Nontuberculous Mycobacteria: Cost Analysis

Necessity of Quality-Controlled 16S rRNA Gene Sequence Databases: Identifying Nontuberculous Mycobacterium Species

Contact Info

Product

Resources

About