Joyce Wolfe

Necessity of quality-controlled 16S rRNA gene sequence databases : Identifying nontuberculous Mycobacterium species

ABSTRACT 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 ∼440 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.

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

ABSTRACT 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:

Application of Mycobacterial Interspersed Repetitive Unit Typing to Manitoba Tuberculosis Cases: Can Restriction Fragment Length Polymorphism Be Forgotten?

80.93 for slowly growing, biochemically active NTM;

Evaluation of 24 locus MIRU-VNTR genotyping of Mycobacterium tuberculosis isolates in Canada

173.23 for slowly growing, biochemically inert NTM; and

Identification of Mycobacterium Species by Multiple-Fluorescence PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene

129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was

A High Proportion of Novel Mycobacteria Species Identified by 16S rDNA Analysis Among Slowly Growing AccuProbe-Negative Strains in a Clinical Setting

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.

Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry

ABSTRACT Since 1993, all Mycobacterium tuberculosis isolates recovered in the province of Manitoba, Canada, have been genotyped by the standard IS6110-restriction fragment length polymorphism (RFLP) method for routine surveillance, prevention, and control purposes. To date, our laboratory has collected 1,290 isolates, from which we have identified approximately 390 unique fingerprint patterns or “types.” Although the standard method is well known for being a lengthy and labor-intensive procedure, a more efficient alternative for typing tuberculosis isolates, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method, has recently gained acceptance. Consequently, all isolates acquired in 2003 (n = 126) were typed by both methods in order to determine the utility of replacing the RFLP method with MIRU typing for all future isolates. Application of Hunters discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (DMIRU = 0.831 to 0.984 versus DRFLP = 0.821 to 0.997). Clustering of isolates by using MIRU data correlated with RFLP-derived clustering, lending useful information for either an investigation or confirmation of an incidence of recent transmission. In addition, it was determined that each predominant RFLP type in Manitoba had a corresponding, recognizable MIRU type. It is conceivable that in the future RFLP typing can be replaced with MIRU for real-time, ongoing tuberculosis surveillance in the province.

Phenotypic and Molecular Characterization of Clinical Isolates of Mycobacterium elephantis from Human Specimens

The current gold standard for Mycobacterium tuberculosis complex (MTBC) genotyping is insertion sequence (IS) 6110 restriction fragment length polymorphism (RFLP) as it provides the highest discriminatory power of all available MTBC genotyping methods. However, RFLP is labour intensive and the interpretation of data from this method can be susceptible to errors. In 2001 a rapid, reproducible variable number of tandem repeat (VNTR) based typing method using 12 mycobacterial interspersed repetitive units (MIRU) was developed. Despite this advancement, this method lacked the discriminatory power of IS6110-RFLP. More recently a set of 24 MIRU-VNTR loci was reported to have greater discriminatory power than the original 12 locus system and may exceed that of RFLP when combined with spoligotyping. We compared the 24 locus method to the 12 locus method in order to improve surveillance of tuberculosis in Canada. A random sample of 650 MTBC isolates from British Columbia, Saskatchewan, Manitoba and Quebec Canada was genotyped using the 24 MIRU loci. Comparison of the data for the 12 and 24 MIRU loci showed an increase of the Hunter-Gaston discriminatory index (HGDI) from 0.895 (12 loci) to 0.920 (24 loci). The implementation of the 24 locus MIRU-VNTR methods offers improvement in discriminatory power over the traditional 12 locus method. For long-term surveillance of MTBC within Canada, the use of 24 MIRU-VNTR loci will provide rapid, highly discriminatory molecular epidemiology information.

Polyphasic Characterization Reveals that the Human Pathogen Mycobacterium peregrinum Type II Belongs to the Bovine Pathogen Species Mycobacterium senegalense

ABSTRACT Identification of mycobacteria to the species level by growth-based methodologies is a process that has been fraught with difficulties due to the long generation times of mycobacteria. There is an increasing incidence of unusual nontuberculous mycobacterial infections, especially in patients with concomitant immunocompromised states, which has led to the discovery of new mycobacterial species and the recognition of the pathogenicity of organisms that were once considered nonpathogens. Therefore, there is a need for rapid and sensitive techniques that can accurately identify all mycobacterial species. Multiple-fluorescence-based PCR and subsequent single-strand conformation polymorphism (SSCP) analysis (MF-PCR-SSCP) of four variable regions of the 16S rRNA gene were used to identify species-specific patterns for 30 of the most common mycobacterial human pathogens and environmental isolates. The species-specific SSCP patterns generated were then entered into a database by using BioNumerics, version 1.5, software with a pattern-recognition capability, among its multiple uses. Patient specimens previously identified by 16S rRNA gene sequencing were subsequently tested by this method and were identified by comparing their patterns with those in the reference database. Fourteen species whose SSCP patterns were included in the database were correctly identified. Five other test organisms were correctly identified as unique species or were identified by their closest relative, as they were not in the database. We propose that MF-PCR-SSCP offers a rapid, specific, and relatively inexpensive identification tool for the differentiation of mycobacterial species.

Multidrug and Extensively Drug-resistant Tuberculosis in Canada 1997–2008: Demographic and Disease Characteristics

Sequencing of the 16S ribosomal DNA (rDNA) for identification of nontuberculous mycobacteria (NTM) has contributed to the establishment of more than 35 new species during the last decade. Increasingly, NTM are accepted as potential or proven pathogens. We identified, by 16S rDNA sequence analysis, slowly growing NTM isolates negative by AccuProbe (GenProbe, San Diego, CA) that previously were identified by using conventional biochemical techniques, to determine the accuracy of reporting AccuProbe-negative NTM prior to sequence-based identification. Of 82 strains, 30 were deemed novel. An attempt was made to determine the clinical importance of previously misidentified novel species. Clinical cases are described for a number of strains previously identified as Mycobacterium terrae complex, Mycobacterium scrofulaceum, and Mycobacterium avium complex. As sequence-based identification methods become more commonplace in clinical microbiology laboratories, there is a need to understand the significance of previously undescribed species, which often mimic and subsequently are identified as well-established species.