Computational protocol: Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

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Protocol publication

[…] Bacterial strains - Twenty-nine strains including B. cereus or B. thuringiensis and B. mycoides were included in this study (). All these strains were originally present in Coleção de Culturas do Gênero Bacillus e Gêneros Correlatos - Bacillus Collection/Oswaldo Cruz Institute/Oswaldo Cruz Foundation. Details of each strain can be found in Zahner et al. (2005). Molecular methods - Extraction and purification of genomic DNA has been described previously (Zahner et al. 2005). The presence the gene be, encoding emetic toxin, was examined by PCR as described by Toh et al. (2004) and be-PCR amplification products were sequenced. The cytK sequences were detected according to the protocol of Fagerlund et al. (2004), with the primers CytkF 5'-CCAACCCAGTTACCAGTTCC-3' and CytkR 5'-AACAGATATCGGTCAAAATGC-3' and the inA gene was amplified according to the methods of Gutmann and Ellar (2000). Nested-PCR for amplification of the structural genes of the entire complement of B. anthracis toxins found on pXO1 (pag, lef, cya) and the capsular antigen gene of pXO2 (cap) was carried out as described by Jackson et al. (1998). Details of primers used are provided in . Strains showing a negative result for the be sequence by PCR assay were submitted to Southern blotting and hybridization with a be probe as previously reported (Zahner et al. 1998). Each PCR reaction was repeated at least three times to confirm reproducibility. This publication made use of the Bacillus cereus Multi Locus Sequence Type website (pubmlst.org/bcereus) developed by Jolley et al. (2004) analyzing the seven housekeeping genes (glp, gmk, ilvD, pta, pur, pycA and tpi) employing PCR conditions as described in the MLST database. PCR products from MLST genes and emetic toxin gene (be) were purified using the GFX-PCR DNA Kit (GE Healthcare Life Sciences). PCR fragments were sequenced in both directions, using the amplification primers to provide unambiguous sequence data, by use of the BigDye Ready Reaction mix (ABI Corp) and reaction products were analyzed on a Prism 3700 automated DNA analyser (ABI Corp). All the sequencing procedures were performed as published by Otto et al. (2008). Data analysis - Sequencher 4.8 software (Genecodes) was used for assembly of contigs and determination of consensus sequences. The nucleotide sequences of each housekeeping gene were trimmed to the appropriate length (348-504 bp), as previously described (Jolley et al. 2004), and then queried to the MLST database. STs were assigned based on the combination of the seven alleles in a specific order. New alleles and allele sets were deposited in the Bacillus MLST database. Designation of new alleles and ST was determined by MLST database curators. A phylogenetic tree was constructed based on the multiple alignments of the concatenated sequences of the seven MLST genes through the neighbour-joining method using Mega 4.0 software (Tamura et al. 2007). Split decomposition analysis of allelic profiles was carried out with the SplitsTree 4.0 software (Huson 1998). Some known strains from the MLST Bacillus databank were included to assess the relationships of those isolates with the ones identified in our study. […]

Pipeline specifications

Software tools Sequencher, MEGA, SplitsTree
Databases PubMLST
Application Phylogenetics
Organisms Bacillus thuringiensis, Bacillus cereus