K S Blackwood

Publications of K S Blackwood

• Application of mycobacterial interspersed repetitive unit typing to Manitoba tuberculosis cases: can restriction fragment length polymorphism be forgotten?

  Authors: K S Blackwood, J N Wolfe, A M Kabani

  Journal of clinical microbiology. 12/2004; 42(11):5001-6.

  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) methodSince 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 Hunter's discriminatory index to the available study population showed that the MIRU method was close in discriminatory power (D) to the RFLP method (D(MIRU) = 0.831 to 0.984 versus D(RFLP) = 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.

• Reassessment of sequence-based targets for identification of bacillus species.

  Authors: K S Blackwood, C Y Turenne, D Harmsen, A M Kabani

  Journal of clinical microbiology. 05/2004; 42(4):1626-30.

  The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification andThe Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/.

• Evaluation of recA sequences for identification of Mycobacterium species.

  Authors: K S Blackwood, C He, J Gunton, C Y Turenne, J Wolfe, A M Kabani

  Journal of clinical microbiology. 08/2000; 38(8):2846-52.

  16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium. Previous studies have shown that Mycobacterium16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium. Previous studies have shown that Mycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recA gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between M. gastri and M. kansasii to 75.7% between M. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose that recA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the Mycobacterium species.