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 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/.The Bacillus genus is an extensive heterogeneous group encompassing 83 validly described species to date (http://www .bacterio.cict.fr/b/bacillus.html). Many species in this taxon are of major clinical importance, such as the B. cereus group (comprised of B. cereus, B. anthracis, B. thuringiensis, B. mycoides, and B. weihenstephanensis), but unfortunately, members of this group share a great deal of morphological and biochemical similarities (3,8,16). In contrast, the environmental and nonpathogenic species of this genus exhibit a wide range of physiology, DNA base content, and nutritional requirements (2,4,15). Since the biochemical approach for species identification can be tedious, expensive, and inaccurate, a rapid, definitive method is greatly needed. Molecular procedures are increasingly being used for rapid species identification. However, some methods used for this genus such as restriction digests of a target gene (i.e., 16S rRNA gene) (11) or randomly amplified polymorphic DNA analysis (22) are limiting in discriminating between a large group of species (6). Sequencing of the 16S rRNA gene and select housekeeping genes has shown to be particularly useful, generating large public sequence databases due to the tangible, exact nature of sequence data. With the increasing use of these methods and decreased expense of running sequencing reactions after the initial equipment investment, more laboratories are relying on sequence data for species identification (21).A previous study using the 16S rRNA gene for rapid identification of the Bacillus genus was undertaken by Goto et al. (6). At this time, the validity of using a hypervariable region (nucleotides [nt] 70 to 344) of the gene was proven adequate to discrimina...

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Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n ‫؍‬ 8), SIT92 (n ‫؍‬ 3), SIT70 (n ‫؍‬ 2), and SIT3038 (n ‫؍‬ 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.