Dear Authors,

First of all, please allow me to introduce myself, my name is Guillermo Jiménez, DVM, Technical Manager of the company Rubinum, S.A., manufacturer and EU authorization holder of the feed additive strain Bacillus cereus var. toyoi (Toyocerin®).

We have just read the article “Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus” published in the PLOS ONE web page in April 2013, Vol. 8, Issue 4, eNumber: e61404, and according to your results, the probiotic strain B. cereus var. toyoi revealed similar cytoxicity on the intestinal epithelial cells Ptk6 IEC compared to B. cereus NHV 0391-98 isolated from a food borne outbreack with high patient mortality or to the nhe reference strain B. cereus NVH 0075-95.

On the other hand, there are other published articles demonstrating the absence of any cytotoxicity of Bacillus cereus var. toyoi that we would like to show you:
- in the article “The use of a porcine intestinal cell model system for evaluating the food safety risk of Bacillus cereus probiotics and the implications for assessing enterotoxigenicity” published in the journal APMIS 119:877-884 in July 2011 by Trapecar et al. (© 2011 APMIS http://onlinelibrary.wile...), the probiotic strain Bacillus cereus var. toyoi showed absence of any cytoxicity in culture supernatants tested on the porcine intestinal cell lines IPEC-J2-9 and PSI and on the human foetal cell line H4-1 when compared to B. cereus strain NVH 75/95, among other B. cereus strains. Such results were consistent with in vivo trials showing the absence of any deleterious effects in pigs and in some instances actually conferring some protection against diarrheal disease. Furthermore, the authors conclude that “the use of the pig small intestinal epithelial cell line PSI is appropriate for identification of potential toxicity in B. cereus strains and sets a low threshold for risk of enterotoxicity to humans.”
- in the article “Literature review on the safety of Toyocerin®, a non-toxigenic and non-pathogenic Bacillus cereus var. toyoi preparation” published in the Regulatory Toxicology and Pharmacology in November 2009, Vol. 55, Issue 2, 236-246 by Williams et al., (http://dx.doi.org/10.1016...), an acute toxicity test with mice where a suspension of Bacillus cereus var. toyoi was administered via intraperitoneal injection at doses ranging from 10^5 to 10^8 spores per animal (approx. equivalent to 5x10^6 – 5x10^9 spores/kg body weight) resulted in no death nor adverse effects at any of the doses tested. Several in vivo intestinal ileal loop studies were conducted which evaluated the potential entertoxocity of Bacillus cereus var. toyoi in rabbits. In these studies isolated ileal loop were injected with Bacillus cereus var. toyoi preparations containing up to 1 x 10^10 spores/ml or concentrated up to 100-fold and observed for signs of enterotoxicicy for up to 25 h. There were not reported adverse effects or signs of enterotoxicity in the rabbit ileal loops. Furthermore, in an eight-day human clinical trial, Toyocerin® did not cause any adverse effects in healthy male and female subjects at 1 x10^9 and 1 x 10^10 spores/kg bw/day once daily (1 h following breakfast for 7 days), followed by a final dose of a preparation containing 6 x 10^11 B. cereus var. toyoi spores (approximately equivalent to 1 x 10^10 B. cereus var.toyoi spores/kg bw/day) 1 h after breakfast on the eighth day.

At the same time, we do recognize the excellency of your study and we understand that the main objective was to demonstrate the relevant role of SMase in the Bacillus cereus virulence, but we would have appreciated that the above mentioned articles would have been taken into account in the discussion part of your paper, apart from the fact that our strain has long history of safety use in the animal nutrition worldwide for more than 30 years now, without any food poisoning outbreak associated with our strain (also see attached “Report of the Scientific Committee of Animal Nutrition on Product Toyocerin for use as feed additive”-http://ec.europa.eu/food/fs/sc/scan/out72_en.pdf - and “Certificate on Determination of Generally Recognized as Safe – GRAS- status for Bacillus cereus var. toyoi as an animal feed ingredient”- http://rubinum.es/en/news... -).

Rubinum, S.A., as manufacturer and registration holder of B. cereus var. toyoi, is the most interested party to clarify and demonstrate the absence of any enterotoxic activity in the probiotic strain, we would like to pose the following questions/comments regarding your recent published study:
1. Did your research team verify which substance contained in the supernatant of Bacillus cereus var. toyoi was actually causing the cytotoxicity effect on Ptk6 cells?
2. Did your research team confirm the presence in the supernatant of Bacillus cereus var. toyoi of the same substances found in the supernatant of the pathogenic Bacillus cereus strains? On table S3 in your publication, the toxin gene profile is showed for all the used strains. Have you performed any sequence comparison or transcription analyses?
3. If the probiotic strain Bacillus cereus var. toyoi has revealed to have similar cytotoxicity compared to the nhe reference strain B. cereus NHV 0075-95 when tested on Ptk6 IEC, why the same probiotic strain does not show any cytoxicity compared to the same nhe reference strain when tested on IPEC-J2-9, on PSI or on the human foetal cell line H4-1?
4. The results obtained in your study revealed cytoxicity of the tested probiotic strain Bacillus cereus var. toyoi on the intestinal epithelial cells Ptk6 IEC, however, there are in vitro evidences of non-cytoxixity activity of the same probiotic strain in other eukaryotic cell lines including Vero cells, Hep-2 cells, IPEC-J2-9, PSI and human foetal cell line. Accordingly, the sentence “Even B. cereus strains isolated from probiotic formulae are cytotoxic to eukaryotic cells in vitro at a very low MOI of 1, whereas usually high doses of 10^5-10^8 enterotoxigenic B. cereus cells or spores are required for food poisoning” should have been more accurately as “Even B. cereus strains isolated from probiotic formulae are cytotoxic to colon epithelial cells from Ptk6 null mice in vitro at a very low MOI of 1, whereas usually high doses of 10^5-10^8 enterotoxigenic B. cereus cells or spores are required for food poisoning”

Thank you very much in advance for your kind attention.

We look forward to your earliest expertise feedback on our comments and we would appreciate your recognition of the proven safety of the probiotic strain B. cereus var. toyoi when used in animal nutrition.

Yours sincerely,

Guillermo Jiménez DVM, M.Sc.
Technical Manager
Rubinum, S.A.