# Reply to Dubbert and von Bünau, “A Probiotic Friend”

Jean-Philippe Nougayrède, Eric Oswald

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Jean-Philippe Nougayrède, Eric Oswald. Reply to Dubbert and von Bünau, “A Probiotic Friend”. MSphere, 2021, 6 (6), 10.1128/msphere.00906-21. hal-03827352

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Submitted on 24 Oct 2022

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# Reply to Dubbert and von Bünau, “A Probiotic Friend”

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Jean-Philippe Nougayrède,ª Eric Oswaldª

ªIRSD, INSERM, INRAE, Université de Toulouse, ENVT, Toulouse, France

We read with great interest the comment letter to the editor by Dubbert and von Bünau (1) from Ardeypharm regarding our work, recently published in *mSphere*, on the production of the genotoxin colibactin by the Nissle 1917 probiotic (2). We fully understand and acknowledge the strong commitment made by Ardeypharm—which markets Nissle 1917 under the trade name Mutaflor—with regard to the safety of its customers and the improvement of patient health.

In their letter (1), the authors question our results on the *in vitro* and *in vivo* genotoxicity of Nissle 1917 (2) with respect to their previous publication in which they stated that this *E. coli* strain did not exert such genotoxicity in standard tests (3). These standard tests, which are recommended by the FDA and the OECD, are suitable for testing the mutagenic effect of chemicals on human health. However, we continue to believe that the tests used by the authors are not the most suitable for testing the mutagenic effect of the Nissle 1917 bacteria. The first test they used is an Ames mutation reversion test in *Salmonella*, exposed to a supernatant of Nissle 1917 (3). However, no genotoxic activity associated with colibactin could be observed in the supernatant (4). It is also well-known that *Salmonella* is killed by the siderophore microcins produced by Nissle 1917 (5). These microcins are responsible for the antagonistic activity of Nissle 1917 at the heart of its probiotic property, which is linked to the production of colibactin (6, 7). Although Dubbert and von Bünau write in their letter that all the appropriate controls were performed and that bacterial lawns were observed on the control plates, no quantitative data pertaining to these important controls is mentioned in the publication. The reader therefore has no way of assessing whether the microcin activity of Nissle 1917 generated a false-negative result or not. We agree with the authors that it is important to also test the contact-dependent mutagenic activity of Nissle 1917. Dubbert et al. used a second modified Ames test in which an antibiotic was added so as to inhibit the growth of Nissle 1917, but not that of *Salmonella* (3). However, it is known that inhibiting growth with an antibiotic impairs the production of colibactin (4). Without using a purified and stable form of colibactin, it is impossible to use the Ames test. We therefore maintain that these Ames tests could not work to test the mutagenicity of Nissle 1917 bacteria. Similarly, the Ames test could not demonstrate the mutagenicity of *Helicobacter pylori* (8), which is a well-recognized oncomicrobe.

We agree with the authors that it is crucial to test *in vivo* the genotoxicity of Nissle 1917. We observed histone H2AX phosphorylation (in response to DNA damage) in 8-day-old mice or in axenic mice which had been monocolonized with Nissle 1917, but not with a mutant unable to produce colibactin (2), which is similar to the results reported in young rats or monoxenic mice colonized with other *pks*⁺ *E. coli* strains (9, 10). Dubbert et al. reported a contradictory result in another Nissle 1917 genotoxicity test in rats where no detectable DNA damage was demonstrated by a standard comet assay (3). This comet assay allows for the detection of DNA breaks inflicted by colibactin at very high doses, as observed in cells exposed to a very high number of *pks*⁺ *E. coli* (4). We now know that these lesions are derived from the generation of DNA interstand cross-links (ICLs) by colibactin, which can break by way of

Copyright © 2021 Nougayrède and Oswald.
This is an open-access article distributed under
the terms of the Creative Commons
Attribution 4.0 International license.

Address correspondence to
Jean-Philippe Nougayrède,
Jean-Philippe.Nougayrede@inrae.fr.

The authors declare no conflict of interest.

This is a response to a letter by Dubbert and
von Bünau https://doi.org/10.1128/mSphere
.00856-21.

Published 22 December 2021
depurination and, consequently, generate DNA breaks (11, 12). However, at a more
moderate dose of colibactin, ICL lesions remain in the majority, and the comet
assay remains negative because ICLs inhibit DNA electrophoretic migration (13).
Accordingly, it is quite possible that the negative result the authors obtained arises
from the low sensitivity of the comet assay to colibactin-induced DNA damage at a
realistic dose in vivo.

We agree with the authors that murine tests are not an adequate representation of
the use of Nissle 1917 in humans, which exhibit in particular normal or, on the con-
trary, dysbiotic microbiota and altered barrier functions in inflammatory bowel disease
patients. However, the recent detection of the colibactin mutational signature in colo-
rectal tumor banks indicates that colibactin produced by pks⁺ *E. coli* is indeed produced,
and genotoxic, in humans (14, 15). We agree with Dubbert and von Bünau who stated in
their letter (1) that these pks⁺ *E. coli* bacteria are frequently found in young children.
Previous work has shown that pks⁺ *E. coli* induces DNA damage during the perinatal col-
onization phase in a rat model, with persistent changes to the intestinal tissue homeo-
stasis and cell turnover (10). In addition, the mutational signature of colibactin has also
been observed in the intestinal crypts of healthy young humans (16), suggesting that
colibactin can imprint damage during childhood, thus leading to potential long-term
consequences (17).

The detection of the colibactin mutational signature in colorectal tumors, the
understanding of the mechanism of DNA damage, mutagenesis, and transformation,
as well as various epidemiological studies showing more *E. coli* pks⁺ in colorectal
cancer (CRC) patients and finally the experimental reproduction of colibactin-aggra-
vated CRC in mouse models all concur to implicate colibactin in this type of cancer
(18). However, we agree with Dubbert and von Bünau (1) that CRC is a multifactorial
disease and that other risk factors—in particular, diet—are very important. The
authors submit that the mutational signature of colibactin is found in only 5% of the
analyzed tissues. Indeed, this signature was identified in 5.3% of the mutations in
the adenomatous polyposis coli (APC) gene, mutated in colorectal tumors (14).
However, care should be taken not to minimize and misinterpret this figure. First,
the colibactin-specific mutation in the APC gene suggests a causal role in CRC.
Second, although a significant fraction of driver mutations is induced by other muta-
genic processes (including endogenous), a tumor typically harbors between 2 and
10 driver mutations, which can be induced by various environmental genotoxins
such as colibactin. This may be sufficient to induce full malignancy (19). The colibac-
tin mutational signature is found in genes other than APC, and another study identi-
fies it in nearly 10% of CRCs (15). In addition, colibactin has been shown to also
induce large-scale DNA damage and chromosome instability, which can result in
nonspecific mutations and cell transformation (18, 20, 21). Consequently, colibactin
may be responsible for inducing a larger fraction of cancers than the fraction of
mutations attributed to colibactin in the APC gene.

We again fully agree with Dubbert and von Bünau (1) that CRC is multifactorial, diet and intestinal inflammation contributing in a major way. Indeed, we have shown that food contaminants may participate in conjunction with colibactin to the mutagenesis process (22). As observed by the authors, intestinal inflammation is a well-known factor in CRC, and promotes colibactin expression by pks⁺ *E. coli* and tumorigenesis (9, 23). It is clear that Nissle 1917 has interesting properties with regard to reducing intestinal inflammation, but to the best of our knowledge, no study has yet thoroughly examined whether its use was associated with a decreased or increased risk of developing colorectal cancer. On the other hand, certain molecules with real potential—such as mesalamine—decrease both inflammation and the production of colibactin by pks⁺ *E. coli*, and therefore reduce the risk of CRC (24, 25).

In conclusion, the impact of colibactin in cancer can still be discussed and needs to
be further studied. However, in light of our results (2) and of the arguments presented
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above, it would seem sensible to reevaluate the use of colibactin-producing *E. coli* for therapeutic use in humans.

REFERENCES

1. Dubbert S, von Bünau R. 2021. A probiotic friend. mSphere 6:e00856-21. https://doi.org/10.1128/mSphere.00856-21.

2. Nougayrède J-P, Chagneau CV, Motta J-P, Bossuet-Greif N, Belloy M, Taieb F, Gratadoux J-J, Thomas M, Langella P, Oswald E. 2021. A toxic friend: genotoxic and mutagenic activity of the probiotic strain Escherichia coli Nissle 1917. mSphere 6:e00624-21. https://doi.org/10.1128/mSphere.00624-21.

3. Dubbert S, Klinkert B, Schimiczek M, Wassenaar TM, von Bünau R. 2020. No genotoxicity is detectable for Escherichia coli strain Nissle 1917 by standard in vitro and in vivo tests. Eur J Microbiol Immunol 10:11-19. https://doi.org/10.1556/1886.2019.00025.

4. Nougayrède J-P, Homburg S, Taieb F, Boury M, Bruszzkiewicz E, Gottschalk G, Buchrieser C, Hacker J, Dobrindt U, Oswald E. 2006. Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science 313:848-851. https://doi.org/10.1126/science.1127059.

5. Sassone-Corsi M, Nuccio S-P, Liu H, Hernandez D, Vu CT, Takahashi AA, Edwards RA, Raffatellu M. 2016. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature 540:280-283. https://doi.org/10.1038/nature20557.

6. Olier M, Marcq I, Salvador-Cartier C, Secher T, Dobrindt U, Boury M, Bacquié V, Pénary M, Gaultier E, Nougayrède J-P, Fioramonti J, Oswald E. 2012. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity. Gut Microbes 3:501-509. https://doi.org/10.4161/gmic.21737.

7. Massip C, Branchu P, Bossuet-Greif N, Chagneau CV, Gaillard D, Martin P, Boury M, Sécher T, Dubois D, Nougayrède J-P, Oswald E. 2019. Deciphering the interplay between the genotoxic and probiotic activities of Escherichia coli Nissle 1917. PLoS Pathog 15:e1008029. https://doi.org/10.1371/journal.ppat.1008029.

8. Kaneko T, Kawakami Y, Akaimatsu T, Kiyosawa K, Katsuyama T. 2000. Mutagenicity of Helicobacter pylori in the Ames test using Salmonella typhimurium TA100. J Int Med Res 28:222-228. https://doi.org/10.1177/147323000002800504.

9. Arthur JC, Perez-Chanona E, Mühlbauer M, Tomkovich S, Uronis JM, Fan T-J, Campbell BJ, Abujamel T, Dogan B, Rogers AB, Rhodes JM, Stintzi A, Simpson KW, Hansen JJ, Keku TO, Fodor AA, Jobin C. 2012. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 338: 120-123. https://doi.org/10.1126/science.1224820.

10. Payros D, Secher T, Boury M, Brehin C, Ménard S, Salvador-Cartier C, Cuevas-Ramos G, Watrin C, Marcq I, Nougayrède J-P, Dubois D, Bedu A, Garnier F, Clermont O, Denamur E, Plaisancié P, Theodorou V, Fioramonti J, Olier M, Oswald E. 2014. Maternally acquired genotoxic Escherichia coli alters offspring's intestinal homeostasis. Gut Microbes 5:313-325. https://doi.org/10.4161/gmic.28932.

11. Bossuet-Greif N, Vignard J, Taieb F, Mirey G, Dubois D, Petit C, Oswald E, Nougayrède J-P. 2018. The colibactin genotoxin generates DNA inter-strand cross-links in infected cells. mBio 9:e02393-17. https://doi.org/10.1128/mBio.02393-17.

12. Xue M, Wernke KM, Herzon SB. 2020. Depurination of colibactin-derived interstrand cross-links. Biochemistry 59:892-900. https://doi.org/10.1021/acs.biochem.9b01070.

13. Wilson MR, Jiang Y, Villalta PW, Stornetta A, Boudreau PD, Carrá A, Brennan CA, Chun E, Ngo L, Samson LD, Engelward BP, Garrett WS, Balbo S, Balskus EP. 2019. The human gut bacterial genotoxin colibactin alkylates DNA. Science 363:eaar7785. https://doi.org/10.1126/science.aar7785.

14. Pleguezuelos-Manzano C, Puschhof J, Rosendahl Huber A, van Hoeck A, Wood HM, Nomburg J, Gurjao C, Manders F, Dalmasso G, Stege PB, Paganelli FL, Geurts MH, Beumer J, Mizutani T, Miao Y, van der Linden R, van der Elst S, Genomics England Research Consortium, Garcia KC, Top J, Willems RJL, Giannakis M, Bonnet R, Quirke P, Meyerson M, Cuppen E, van Boxtel R, Clevers H. 2020. Mutational signature in colorectal cancer caused by genotoxic pks + E. coli. Nature 580:269-273. https://doi.org/10.1038/s41586-020-2080-8.

15. Dziubańska-Kusibab PJ, Berger H, Battistini F, Bouwman BAM, Iftekhar A, Katainen R, Cajuso T, Crosetto N, Orozco M, Aaltonen LA, Meyer TF. 2020. Colibactin DNA-damage signature indicates mutational impact in colorectal cancer. Nat Med 26:1063-1069. https://doi.org/10.1038/s41591-020-0908-2.

16. Lee-Six H, Olafsson S, Ellis P, Osborne RJ, Sanders MA, Moore L, Georgakopoulos N, Torrente F, Noorani A, Goddard M, Robinson P, Coorens THH, O'Neill L, Alder C, Wang J, Fitzgerald RC, Zilbauer M, Coleman N, Saeb-Parsy K, Martincorena I, Campbell PJ, Stratton MR. 2019. The landscape of somatic mutation in normal colorectal epithelial cells. Nature 574:532-537. https://doi.org/10.1038/s41586-019-1672-7.

17. Secher T, Brehin C, Oswald E. 2016. Early settlers: which E. coli strains do you not want at birth? Am J Physiol Gastrointest Liver Physiol 311: G123-G129. https://doi.org/10.1152/ajpgi.00091.2016.

18. Berger H, Meyer TF. 2021. Mechanistic dissection unmasks colibactin as a prevalent mutagenic driver of cancer. Cancer Cell 39:1439-1441. https://doi.org/10.1016/j.ccell.2021.10.010.

19. Rosendahl Huber A, Van Hoeck A, Van Boxtel R. 2021. The mutagenic impact of environmental exposures in human cells and cancer: imprints through time. Front Genet 12:760039. https://doi.org/10.3389/fgene.2021.760039.

20. Cuevas-Ramos G, Petit CR, Marcq I, Boury M, Oswald E, Nougayrède J-P. 2010. Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc Natl Acad Sci U S A 107:11537-11542. https://doi.org/10.1073/pnas.1001261107.

21. Iftekhar A, Berger H, Bouznad N, Heuberger J, Boccellato F, Dobrindt U, Hermeking H, Sigal M, Meyer TF. 2021. Genomic aberrations after short-term exposure to colibactin-producing E. coli transform primary colon epithelial cells. Nat Commun 12:1003. https://doi.org/10.1038/s41467-021-21162-y.

22. Payros D, Dobrindt U, Martin P, Secher T, Bracarense APFL, Boury M, Laffitte J, Pinton P, Oswald E, Oswald IP. 2017. The food contaminant deoxynivalenol exacerbates the genotoxicity of gut microbiota. mBio 8:e00007-17. https://doi.org/10.1128/mBio.00007-17.

23. Yang Y, Gharaibeh RZ, Newsome RC, Jobin C. 2020. Amending microbiota by targeting intestinal inflammation with TNF blockade attenuates development of colorectal cancer. Nat Cancer 1:723-734. https://doi.org/10.1038/s43018-020-0078-7.

24. Tang-Fichaux M, Chagneau CV, Bossuet-Greif N, Nougayrède J-P, Oswald É, Branchu P. 2020. The polyphosphate kinase of Escherichia coli is required for full production of the genotoxin colibactin. mSphere 5:e01195-20. https://doi.org/10.1128/mSphere.01195-20.

25. Bonovas S, Fiorino G, Lytras T, Nikolopoulos G, Peyrin-Biroulet L, Danese S. 2017. Systematic review with meta-analysis: use of 5-aminosalicylates and risk of colorectal neoplasia in patients with inflammatory bowel disease. Aliment Pharmacol Ther 45:1179-1192. https://doi.org/10.1111/apt.14023.