A Probiotic Friend

We read with interest the publication by Nougayrède and coworkers who described that Escherichia coli strain Nissle 1917 (EcN) is mutagenic by means of its expressed colibactin (1). The contribution of this work on the biological activity of colibactin is of high significance and increases our knowledge on this enigmatic toxin. The use of mouse models is an important methodology to study the effect of colibactin in vivo. Nougayrède et al. provided evidence that EcN has genotoxic potential in the models they applied (1). The topic of colibactin is of extreme importance to our company, as patient and consumer safety has our utmost priority: the use of probiotic products that contain EcN as an active ingredient should not have health risks. That has been the basis of our company’s strategy in the past, and it remains our focus in the present and future. For this reason, databases on drug side effects, such as EudraVigilance (the European database of suspected adverse drug reaction reports, www.adrreports.eu) are closely being monitored. So far, these show no evidence of a potential cancer risk from intake of EcN. As this matter is of high concern to us, we had conducted genotoxicity tests that are compliant with FDA Recommendations (2) and OECD Guidelines (3, 4). These guidelines have been defined to detect genotoxicity of a given compound, and commercial companies are obliged to follow such guidelines. This included use of the Ames test, performed according to the original test protocol and additionally with slight modification for testing live EcN bacteria (5). No mutagenic or cytotoxic effect of live EcN or its metabolites were found with these tests (5). Needless to say, tests were performed according to good manufacturing practicing (GMP) standards with inclusion of all appropriate controls. These controls identified that the number of spontaneous revertants in the Ames test were in accordance with the frequency range reported in the literature and with in-house historical data. Compared to this control, the presence of EcN did not raise the number of revertant colonies of the Ames strains and it did not reduce the number of the Ames strain colonies by killing them, a conclusion that was unfortunately inaccurately drawn from our work by Nougayrède and colleagues (1). The background lawns obtained in the Ames tests indicated the absence of any cytotoxic effect by EcN, an observation that we possibly did not point out clearly enough in our publication (5). We observed a background lawn of the EcN samples that in all cases was identical to that of the H2O control. Thus, a possible killing effect by microcins or any other antagonistic influence of EcN on the used Salmonella Ames reporter strains can safely be excluded. The second OECD-approved technique we used was the in vivo mammalian alkaline comet assay using rats, which is also recommended by the FDA (2) for detection of DNA strand breaks. In contrast, Nougayrède et al. used two alternative mouse models (1). One model utilized axenic mice that were monoassociated once with EcN and sacrificed 7 days later. Their second model used 8-day-old mouse pups that were sacrificed only 6 h after a single exposure to EcN. Colon tissue from animals of both models was assessed immunohistologically for the presence of histonegH2AX. In the rodent model we used, 7to 9-week-old SPF rats were fed EcN daily for 2 days or 28 days. Despite this much longer exposure, histological examination and comet assay of the specified Editor Alfredo G. Torres, UTMB Copyright © 2021 Dubbert and von Bünau. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Silke Dubbert, silke.dubbert@ardeypharm.de. The authors declare a conflict of interest. The authors are employees of Ardeypharm GmbH. For the author reply, see https://doi.org/10 . 1128/mSphere.00906-21. Published