Fifteen strains of Bacillus spp. (three B. cereus strains, one B. subtilis, B. macerans, B. pumilus, and B. mycoides strains, and eight unknown isolates from the food-industry) were identified on species level with three designed pairs of primers for B. cereus, B. licheniformis, and B. subtilis. Primers designed for B. licheniformis (BlichF and BlichR) and B. subtilis (BsubF and BsubR) allowed specific species identification, whereas the designed pair of primers for B. cereus (BcerF2 and BcerR2) showed specificity for B. cereus sensu lato, because both B. cereus strains and B. mycoides gave positive reaction. Bacilli identification was also carried out with routine API method with unsatisfactory results. The (GTG)5-PCR method was used for strain characterization, enabling bacilli classification into separate clusters according to their taxonomic designations.
Bottone, E.J. (2010): Bacillus cereus, a volatile human pathogen. Clin. Microbiol., 23(2), 382–398.
Gupta, R., Beg, Q.K. & Lorenz, P. (2002): Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biot., 59, 15–32.
Heyrman, J., Rodríguez-Díaz, M., Devos, J., Felske, A., Logan, N.A. & De Vos, P. (2005): Bacillus arenosi sp. nov., Bacillus arvi sp. nov. and Bacillus humi sp. nov., isolated from soil. Int. J. Syst. Evol. Micr., 55, 111–117.
Jeyaram, K., Romi, W., Singh, T.A., Adewumi, G.A., Basanti, K. & Oguntoyinbo, F.A. (2011): Distinct differentiation of closely related species of Bacillus subtilis group with industrial importance. J. Microbiol. Meth., 87(2), 161–164.
Lee, J., Kwon, G.H., Park, J.Y., Park, C.S., Kwon, D.Y., … & Kim, J.H. (2011): A RAPD-PCR method for the rapid detection of Bacillus cereus. J. Microbiol. Biotechnol., 21(3), 274–276.
Logan, N.A. & Berkley, R.C. (1984): Identification of Bacillus strains using the API system. J. Gen. Microbiol., 130, 1871–1882.
Ludwig, W., Schleifer, K.H. & Whitman, W.B. (2009): Revised road map to the phylum Firmicutes. -in: De Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., … & Whitman, W.B. (Eds): Bergey's Manual of systematic bacteriology. Springer Science + Business Media, New York. pp. 1–8.
Manzano, M., Cristina, C., Mędrala, D., Antoni, C. & Comi, G. (2003): Optimization of DNA extraction to detect Bacillus cereus from food using a PCR technique – short communication. Pol. J. Food Nutr. Sci., 53, S69–S71.
MultAlin software (2018): Available at http://multalin.toulouse.inra.fr/multalin (last accessed 14 October 2018).
NCBI (2018): Available at http://www.ncbi.nlm.nih.gov/sites/gquery (last accessed 19 September 2018).
Netprimer software (2018): Available at http://www.premierbiosoft.com/netprimer (last accessed 14 October 2018)
Němečková, I., Solichová, K., Roubal, P., Uhrová, B. & Šveráková, E. (2011): Methods for detection of Bacillus sp., B. cereus and B. licheniformis in raw milk. Czech J. Food Sci., 29, S55–S60.
Pepe, O., Blaiotta, G., Moschetti, G., Greco, T. & Villani, F. (2003): Rope-producing strains of Bacillus sp. from wheat bread and strategy for their control by lactic acid bacteria. Appl. Environ. Microbiol., 69, 2321–2329.
Rasko, D.A., Altherr, M.R., Han, C.S. & Ravel, J. (2005): Genomics of the Bacillus cereus group of organisms. FEMS Microbiol. Rev., 29, 303–329.
Rowan, N.J., Deans, K., Anderson, J.G., Gemmell, C.G., Hunter, I.S. & Chaithong, T. (2001): Putative virulence factor expression by clinical and food isolates of Bacillus spp. after growth in reconstituted infant milk formulae. Appl. Environ. Microbiol., 67(9), 3873–3881.
Scheldeman, P., Rodríguez-Díaz, M., Goris, J., Pil, A., De Clerck, E., … & Heyndrickx, M. (2004): Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms. Int. J. Syst. Evol. Micr., 54, 1355–1364.
Sorokulova, I.B., Reva, O.N., Smirnov, V.V., Pinchuk, I.V., Lapa, S.V. & Urdaci, M.C. (2003): Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread. Lett. Appl. Microbiol., 37(2), 169–173.
Temmerman, R., Huys, G. & Swings, J. (2004): Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends Food Sci. Tech. (NFIF part 2), 15(7–8), 348–359.
Thorsen, L., Abdelgadir, W.S., Rønsbo, M.H., Abban, S., Hamad, S.H., … & Jacobsen, M. (2011): Identification and safety evaluation of Bacillus species occurring in high numbers during spontaneous fermentations to produce Gergoush, a traditional Sudanese bread snack. Int. J. Food Microbiol., 146, 244–252.
Tran, H.T.M., Cheirsilpa, B., Hodgson, B. & Umsakul, K. (2010): Potential use of Bacillus subtilis in a co-culture with Clostridium butyricum for acetone–butanol–ethanol production from cassava starch. Biochem. Eng. J., 48, 260–267.
Wang, L.T., Lee, F.L., Tai, C.J. & Kasai, H. (2007): Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. Int. J. Syst. Evol. Micr., 57, 1846–1850.
Wu, X.Y. Walker, M.J., Hornitzky, M. & Chin, J. (2006): Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance. J. Microbiol. Meth., 64(1), 107–119.
Wunschel, D., Fox, K.F., Black, G.E. & Fox, A. (1995): Discrimination among the B. cereus group, in comparison to B. subtilis, by structural carbohydrate profiles and ribosomal RNA spacer region PCR. Syst. Appl. Microbiol., 17(4), 625–635.