Latest studies supporting the safety of TOYOCERIN®
Here you can find the interview with Rubinum’s President, Mr. Lau Andersen, where he explains Toyocerin’s situation: the interview
List of published studies supporting the safety of Toyocerin:
The Identification of Intrinsic Chloramphenicol and Tetracycline Resistance Genes in Members of the Bacillus cereus Group (sensu lato). H. Glenwright et al. / Frontiers in Microbiology, Vol.7, Art. 2122 (2017). doi: 10.3389/fmicb.2016.02122 (Available upon request)
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. G. Jimenez et al./Sytematic and Applied Microbiology 36 (2013) 383-391. (Available upon request)
Complete genome sequence of the type strain of Bacillus toyonensis BCT-7112T, the active ingredient of the feed additive preparation TOYOCERIN. Genome Announcements November/December 2013 Vol 1, Issue 6, e01080-13: click here
Comparison of procedures for the extraction of supernatants and cytotoxicity tests in Vero cells, applied to assess the toxigenic potential of Bacillus spp. and Lactobacillus spp., intended for use as probiotic strains. A.R. Blanch et al. / Journal of Microbiological Methods 103 (2014) 64–69 (Available upon request)
Validation List no. 155 byThe ICSP (International Committee on Systematics of Prokaryotes: body that oversees the nomenclature of prokaryotes, determines the rules by which prokaryotes are named and whose Judicial Commission issues Opinions concerning taxonomic matters, revisions to the Bacteriological Code).List of new names and new combinations previously effectively, but not validly, published: click here
International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1–5 DOI 10.1099/ijs.0.060285-0(Available upon request)
The use of a porcine intestinal cell model system for evaluating the food safety risk of Bacillus cereusprobiotics and the implications for assessing enterotoxigenicity. M. Trapecar et al. (2011) APMIS 119:877-884(Available upon request)
Literature review on the safety of Toyocerin, a non-toxigenic and non-pathogenic Bacillus cereus var. toyoipreparation. L.D. Williams et al. Regulatory Toxicology and Pharmacology 55 (2009) 236–246 (Available upon request)
Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles. Siele Ceuppens, Nico Boon, Mieke Uyttendael: click here
HISTORY OF THE REGISTRATION OF TOYOCERIN IN THE EUROPEAN UNION (1994-2014)
Bacillus cereus var. toyoi NCIMB 40112/CNCM I-1012 (B. toyo ) (TOYOCERIN®) was authorized for the first time by the EU Commission in 1994 for use use as feed additive in sows, piglets and pigs. Subsequently, it was also authorized for use in poultry, cattle and rabbits.
In December 2001, the Scientific Committee of Animal Nutrition (SCAN) concluded that Toyocerin®:
- does not produce, nor has the capacity to produce the toxins found in other strains of cereus ;
- will not transfer the resistance to antibiotics to other bacteria;
- is safe for those handling the product;
- is safe for use with the target animals.
In 2009 Toyocerin was determined as “Generally Recognized as Safe” by the US GRAS Expert Panel (see GRAS Certificate).
In October 2012, EFSA FEEDAP Panel adopted a Scientific Opinion concluding that the strain of B. cereus in Toyocerin®:
- harbours resistance determinants to two antibiotics, one of which can be ascribed to an acquired resistance.
- harbours all the genes coding for the non-haemolytic and haemolytic enterotoxins. Since the two operons present the same organisation as pathogenic cereus strains and since no mutation affecting transcription or translation has been detected, it has to be assumed that the strain has the capacity to elaborate functional toxins, and thus to pose a hazard for those exposed to the organism.
Based on the EFSA’s Scientific Opinion above mentioned, the European Commission adopted the Implementing Regulation (EU) No. 288/2013 of 25 March 2013 concerning the suspension of the authorization of Bacillus cereus var. toyoi as feed additive for use in all target animal species.
However, the same Regulation states the possibility to provide supplementary data concerning the safety of use of Bacillus cereus var. toyoi that bring new elements to allow a re-consideration of the assessment carried out for that additive.
Accordingly, in December 2013 Rubinum S.A. provided to EFSA FEEDAP Panel supplementary data including new studies supporting:
- a new taxonomical classification of the micro-organism as a newBacillus species (i.e. Bacillus toyonensis)
- the non-transferability of the antibiotic resistance genes and
- the non-functionality of the enterotoxin genes present in the genome of the strain.
INTRINSIC NATURE AND NON-TRANSFERABILITY OF THE ANTIBIOTIC RESISTANCE GENES HARBORED IN THE GENOME OF BACILLUS TOYONENSIS
EFSA’S GUIDANCE ON ASSESSMENT OF BACTERIAL SUSCEPTIBILITY TO ANTIMICROBIALS
(EFSA Journal 2012;10(6):270)
|EVIDENCES PROVIDED TO EFSA IN DECEMBER 2013|
|If MIC > cut-off values:
Necessary to make distinction between acquired and intrinsic resistance. The structural nature and genetic basis of the antimicrobial resistance must be demonstrated by analysing a representative slection of strains belonging to the same taxonomical unit of the bacterial strain used as feed additive.
Since intrinsic resistance is specific for a bacterial species or genus, an indispensable pre-requisite is the correct identification of the strain at species level by means of molecular taxonomy methods.
|1. B. toyonensis BCT-7112 T is identified as a new species within the B. cereus group by means of ANI calculations. (See peer reviewed article 1)
2. B. toyonensis shows resistance to chloramphenicol & tetracycline (MIC > 8 mg/L)
3. Single copies of cat & tetM genes are present in a structural conserved region of the chromosome of B. toyonensis. (See genome information in article 2)
4. Neither of the two genes is flanked by any genetic mobile element nor other possible horizontal gene transfer mechanism. (See genome information in article 2)
5. Orthologous of cat & tetM genes are also present in all the genomes of the strains belonging to the same taxonomical unit of B. toyonensis BCT-7112 T , indicating a common ancestry.
6. The guanine-cytosine (G+C) content in the cat and tetM genes are in accordance with the overall G+C content of the genome of B. toyonensis.
7. The antibiotic resistance present in B. toyonensisBCT-7112 T is associated with an unknown putative mechanism in which a plamidial ger IC-nuc B intergenic region is involved but not related to any antibiotic resistant gene. (See genome information in article 2)
The use of B. toyonensis as feed additive does not increase significantly the quantity of resistant bacteria already present in the intestinal tract of target animals. (See article 3)
ABSENCE OR NON-FUNCTIONALITY OF THE GENES CODING FOR ENTEROTOXINS AND CEREULIDE SYNTHASE IN BACILLUS TOYONENSIS
|EFSA’S GUIDANCE ON ASSESSMENT OF THE TOXIGENIC POTENTIAL OF BACILLUS SPECIES USED IN ANIMAL NUTRITION
(EFSA Journal 2014;12(5):3665)
|EVIDENCES PROVIDED TO EFSA IN DECEMBER 2013|
|Assessment of species belonging to the Bacillus cereus group
Bacillus cereus toxins considered as the causative agents of gastro-intestinal diseases are: Nhe, Hbl, CytK and cereulide.
If strains belonging to the B. cereus taxonomic group are proposed for direct use in animal production then the full genome (including chromosome and plasmids) should be sequenced and bioinformatic analysis made to search for genes coding for enterotoxins and cereulide synthase. If there is evidence of homology, the non-functionality of the genes (e.g., mutation, deletion) should be demonstrated.
|1. The full genome of B. toyonensis has been completely sequenced and annotated (5.03-Mb in size; one single chromosome and two plasmids). (See genome information in article 2)
2. The bioinformatic analysis of the genome has revealed that:
– The enterotoxin gene CytK is not present
– The operon encoding for cereulide synthase lacks the genes cesD, cesH, cesP and cesT, thus it is not functional.
– The Nhe and Hbl operons show important changes in the amino acids of the encoded NheC and Hbl L2, which demonstrate the non-functionality of both enterotoxins
1. New In vitro evidences of the non-functionality of the Nhe & Hbl operons:
– Western blot results: B. toyonensis does not have the capacity to produce the L2 component of Hbl nor the NheC component of Nhe.
– Cytotoxicity test in Vero cells: B. toyonensis shows no cytotoxicity at the maximum supernatant concentration recommended by EFSA. (See peer reviewed article 4)
B. toyonensis showed absence of cytotoxicity in three intestinal cell lines (e.g. swine and human cell lines). (See peer reviewed article 5)
See the summary here in pdf file
In July 2014, after reviewing additional data submitted by Rubinum in Dec’13, a new opinion of EFSA again stated that B. toyonensis has the capacity to elaborate functional toxins and thus, to pose a risk to humans exposed to the organism, and that it poses a risk for the spread of genes coding for resistance to tetracycline & chloramphenicol, antibiotics of human and veterinary importance. (See EFSA Journal 2014;12(7):3766 [17 pp.]. DOI: 10.2903/j.efsa.2014.3766)
In August 2015, the European Commission revoked the authorizations of Toyocerin® for use in animal in the EU. (See OJ L 217, 17.8.2015, p. 1)
Since its first authorization in Japan in 1975, Toyocerin® has been used worldwide in animal nutrition for over 40 years without any known case on toxicity/safety ever reported by the competent authorities related to the use of Toyocerin® either at the level of the target species, personnel handling the product or environment.