Systematic and Applied Microbiology 36 (2013) 383–391 Contents lists available at SciVerse ScienceDirect Systematic and Applied Microbiology jo ur nal ho mepage: www.elsevier.de/syapm Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations Guillermo Jiménez a,∗ , Mercedes Urdiain b , Ana Cifuentes b , Aránzazu López-López b , Anicet R. Blanch c , Javier Tamames d , Peter Kämpfer e , Anne-Brit Kolstø f , Daniel Ramón g , Juan F. Martínez g , Francisco M. Codo ˜ ner g , Ramon Rosselló-Móra b a Rubinum S.A., Avda. de La Llana 123, 08191 Rubí, Catalonia, Spain b Marine Microbiology Group, Department of Ecology and Marine Resources, Institut Mediterrani d’Estudis Avanc ¸ ats IMEDEA (CSIC-UIB), C/Miquel Marqués 21, 07190 Esporles, Illes Balears, Spain c Department of Microbiology, University of Barcelona, Diagonal 643, 08028 Barcelona, Catalonia, Spain d Systems Biology Department, Centro Nacional de Biotecnología-CSIC, C/Darwin 3, 28049 Madrid, Spain e Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany f Department of Pharmaceutical Biosciences, University of Oslo, NO-0316 Oslo, Norway g Lifesequencing S.L., C/Catedrático A. Escardino Belloch 9, 46980 Paterna, Valencia, Spain a r t i c l e i n f o Article history: Received 25 February 2013 Received in revised form 16 April 2013 Accepted 18 April 2013 Keywords: Bacillus cereus group ANI Polyphasic taxonomy Probiotic Genome comparisons a b s t r a c t Strain BCT-7112 T was isolated in 1966 in Japan from a survey designed to obtain naturally occurring microorganisms as pure cultures in the laboratory for use as probiotics in animal nutrition. This strain, which was primarily identified as Bacillus cereus var toyoi, has been in use for more than 30 years as the active ingredient of the preparation TOYOCERIN ® , an additive for use in animal nutrition (e.g. swine, poultry, cattle, rabbits and aquaculture). Despite the fact that the strain was initially classified as B. cereus, it showed significant genomic differences from the type strains of the B. cereus group that were large enough (ANI values below 92%) to allow it to be considered as a different species within the group. The polyphasic taxonomic study presented here provides sufficient discriminative parameters to classify BCT-7112 T as a new species for which the name Bacillus toyonensis sp. nov. is proposed, with BCT-7112 T (=CECT 876 T ; =NCIMB 14858 T ) being designated as the type strain. In addition, a pairwise comparison between the available genomes of the whole B. cereus group by means of average nucleotide identity (ANI) calculations indicated that besides the eight classified species (including B. toyonensis), additional genomospecies could be detected, and most of them also had ANI values below 94%. ANI values were on the borderline of a species definition only in the cases of representatives of B. cereus versus B. thuringiensis, and B. mycoides and B. weihenstephanensis. © 2013 Elsevier GmbH. All rights reserved. Introduction The “Bacillus cereus group” of Gram-positive, spore-forming bacteria forms a homogeneous independent branch within the Bacillus genus and comprises seven closely related species: B. cereus [9,35], B. thuringiensis [3,35], B. anthracis [5,35], B. mycoides [7,35], B. pseudomycoides [27], B. weihenstephanensis [20], and B. cytotoxicus [11]. Some of the species of this group (i.e. B. cereus, B. thuringiensis, B. anthracis and B. mycoides) were classified about one century ago, ∗ Corresponding author. Tel.: +34 935860310. E-mail addresses: g.jimenez@rubinum.es, rubinum@rubinum.es (G. Jiménez). much earlier than the discovery of DNA as hereditable material. At that time, the classification of bacteria was generally made on the basis of their habitats, pathogenicity for mammals or insects, and their morphological and physiological characteristics [1,29]. The large extent of synteny among their chromosomes and other genomic similarities led some scientists to propose that these four species could represent a single taxon [29]. The species of the B. cereus group are soil-dwelling saprophytes that are of medical and economic importance because of their pathogenicity in humans (e.g. B. cereus and B. anthracis) or insects (e.g. B. thuringiensis, which is used industrially as a biological pesticide and considered harm- less to humans [40]). Other species, such as B. mycoides, B. weihen- stephanensis and B. pseudomycoides, are considered harmless given 0723-2020/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2013.04.008