Principle of identification method to a phylogenetic group :

The program compares the query nucleotide sequence(s) from panC gene to sequences in a database, in multiple steps, and calculates the % homology and the statistical significance of matches with all database sequences. The phylogenetic group containing the sequence for which the homology is the highest is selected.

Requirements:

The sequences AACAAAC or AACAGAC are needed to initiate alignments, so that one of theses two initial sequences must be present on the query sequence(s). The program then extends alignment to generate the final alignment.

Length of query sequence(s) should be over 450 bases.

Mathematic formula for % homology calculations and significance of matches :

The program calculates the percentage of homology, from initial sequence (AACAAAC or AACAGAC) to the end of the alignment (end of the database sequence or end of the query sequence if it is shorter than the database sequence).

* number of bases covering the alignment

When the highest % homology is < 90%, identification to phylogenetic groups is considered as not significant and rejected.

When the highest % homology is <75%, identification to the B. cereus Group is considered as not significant and rejected.

Group I

Mesophilic group corresponding to B. pseudomycoides species. Strains from this group are easily distinguished from B. mycoides by the absence of psychrotolerance associated signature on the cspA gene (signature defined by Francis et al.,1998). They are generally not or weakly cytotoxic.

Group II

This group includes 25% of intermediate to mesophilic strains and 75% of psychrotolerant strains. This is a mixed group made up of B. thuringiensis II and B. cereus II strains, depending on whether they contain parasporal crystal or not. psychrotolerant strains are easily distinguished from group VI psychrotolerant strains by the absence of psychrotolerance associated signature on the cspA gene (signature defined by Francis et al. ,1998). This group may include cytotoxic strains.

Groupe III

Mesophilic group corresponding to B. thuringiensis III, B. cereus III (emetic strains included) or B. anthracis species, depending on whether strains contain parasporal crystal or not, or whether they contain virulence factors associated with anthrax. There are not yet phenotypic features that could be exploited to easily and rapidly identify these strains. Strains from this group are generally cytotoxic, many of them being highly cytotoxic.

Group IV

Mesophilic group corresponding to B. thuringiensis IV or B. cereus IV species, depending on whether strains contain parasporal crystal or not. There are not yet phenotypic features that could be used to easily and rapidly identify these strains. Strains from this group are generally cytotoxic, some can be highly cytotoxic.

Group V

Intermediate group corresponding to B. thuringiensis V or B. cereus V strains, depending on whether strains contain parasporal crystal or not. There are not yet phenotypic features that could be used to easily and rapidly identify these strains. This group may include cytotoxic strains

Group VI

Psychrotolerant group corresponding to B. weihenstephanensis, B. mycoides or B. thuringiensis VI strains, depending on whether strains contain parasporal crystal or not and whether strains form rhizoidal colony or not. All strains in this group are easily identified by the presence of psychrotolerance associated signature on the cspA gene (signature defined by Francis et al., 1998). They are generally not or weakly cytotoxic.According to some authors, some strains frm this group could contain the ces gene. It is thus advised to check the absense of this gene (by using the test described by Ehling-Shulz et al. 2005) in order to assess non-pathogenicity.

Group VII

Thermotolerent group corresponding to a new species (B. cytotoxicus), not yet described. Strains of this group are easily identified by the absence of growth in minimal medium without tryptophan and growth at 50C (according to the method of Bergey's Manual). They are relatively rare but generally highly cytotoxic.

References

Claus, D. and R.C.W. Berkeley. 1986. Genus Bacillus Cohn 1872, 174AL. In Bergey's manual of systematic Bacteriology. Sneath P.H.A., Mair N.S., Sharpe M.E. and Holt J.G. (eds). Baltimore, Md: Williams & Wilkins, pp:1105-1139.

Ehling-Schulz M., N. Vukov, A. Schulz, R. Shaheen, M. Andersson, E. Martlbauer, and S. Scherer. 2005. Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus. Applied and Environmental Microbiology 71:105-113.

Francis, K.P., Mayr, R., von Stetten, F., Stewart, G., and S. Scherer. 1998. Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes. Appl. Environ. Microbiol. 64: 3525-3529.

Guinebretière M.H., F. L. Thompson, A. Sorokin, P. Normand, P. Dawyndt, M. Ehling-Schulz, B. Svensson, V. Sanchis, C. Nguyen-The, M. Heyndrickx, and P. De Vos. 2008. Ecological diversification in the Bacillus cereus Group. Environmental Microbiology 10:851-865.)

Guinebretière, M.H., P. Velge, O. Couvert, M.L. Debuyser and C. Nguyen-The. 2010. Ability of Bacillus cereus Group strains to cause food poisoning varies according to phylogenetic affiliation (groups I to VII) rather than species affiliation. Journal of Clinical Microbiolgy (in press).

Partial amplification of the panC gene and sequencing:

PCR amplification can be performed on cellular suspension or DNA issue from different preparation methods : frozen/defrosted bacterial cells and then heated up to 90°C during 10 min ; semi-purified DNA prepared with 'Instagene Matrix' kit (Biorad), purified DNA (an example of a rapid method is provided in Guinebretière et al., 2003).

The final PCR mixture (90 µl) may contain 300 ng of DNA template, 0.2 mM dNTP mix (Eurogentec, Seraing, Belgium), 2.5 mM MgCl2, 0.25 µM of each primer, 0.75 U of AmpliTaq polymerase (Perkin-Elmer, Courtaboeuf, France) and 9 µl of 10X AmpliTaq buffer without MgCl2 (Perkin-Elmer, Courtaboeuf, France). For all groups except group VII, the following primers can be used: 5'- TYGGTTTTGTYCCAACRATGG-3' (Forward degenerated primer) and 5'-CATAATCTACAGTGCCTTTCG-3' (Reverse). For group VII strains, reverse primer 5'- CATAATCAACTATACCGTTTG-3' can be used.

Thermal cycling can be carried out in a thermocycler with the following run: a starting cycle of 5 min at 94°C, followed by 30 cycles of 15 sec at 94°C, 30 sec at 55°C,and 30 sec at 72°C, and a final extension of 7 min at 72°C. PCR products can be purified using 'High Pure PCR Product' kit (Roche Diagnostics, Mannheim, Germany) and sent for sequencing, using primer 5'-ATAATCTACAGTGCCTTTCG-3'for all strains except group 7 strains and primer 5'-ATAATCAACTATACCGTTTG-3' for group 7 strains. As group VII strains are relatively rare, a strategy is to first use the primers recommended for all groups except group VII, and then if the reaction is negative, use primers that are recommended for group VII.

Sequence preparation:

The sequence should be in fasta format (reverse complemented if necessary) and its length must be over 250 bases. A, C, G, T are the only characters to be permitted. If no identification is obtained, presence of the initial sequence 5'-AACAAAC-3' or 5'-AACAGAC-3' may be checked.This sequence must be present on your sequence(s) to initiate the alignments (position 305-311 on panC sequence recorded for ATCC 14579). Absence of this sequence would signify that your sequence does not belong to the B. cereus Group or that an error occured during sequencing.

Your sequences must be in 'fasta' format, as shown below: