Enterohemorrhagic Escherichia coli (EHEC) is a Shiga toxin producing pathogenic E. coli. Infection caused by EHEC results in broad spectrum of disease ranging from mild diarrhea to severe hemorrhagic colitis and hemolytic uremic syndrome (HUS); characterized by thrombocytopenia, hemolytic anemia and kidney failure. Shiga toxin is the key virulence factor of EHEC, and the gene encoding toxin is located in a phage genome integrated into the bacterial chromosome. Antibiotics are prohibited to treat EHEC infection due to undesirable induction of Shiga toxin expression. However, if the toxin production is inhibited, antibiotics are available to treat the disease. So, our aim is to find chemical compounds which can inhibit Shiga toxin production in EHEC. Here we screened a chemical library named Natural Product Depository (NPDepo) from RIKEN, Japan. There are two types of libraries, authentic library and natural product depository library. Authentic library comprises 80 compounds, which are well reported compounds having multiple biological activities. The natural product library contains 25,000 compounds. These compounds were clusterized by their chemical structure, and 400 compounds representing the cluster were chosen for the first screening. Then, compounds having similar structure to the hit compounds in the first screening were used in the second screening. As the wild type EHEC strain is pathogenic, phage 933W from pathogenic strain E. coli O157 H:7 EDL933 was introduced into E. coli K-12 MG1655 as a laboratory strain, and it’s designated as MG1655 933W. The lysogenized phage contains toxin genes, but the gene is replaced by green fluorescence protein (GFP) gene in the strain (MG1655 933W Δstx::gfp). The strain was cultured with mitomycin C to induce phage production during the culture, and OD600 and GFP expression were determined. OD600 is for the bacterial growth and cell lysis, and GFP is a reporter for Shiga toxin expression. Chemical compounds from the libraries were also added to the culture. Potential chemical compounds capable to inhibit Shiga toxin production can be identified by both low GFP expression and inhibition of bacterial lysis. From authentic library screening, we found three hit compounds showing low GFP expression. From the first screening of natural product library, 11 compounds showed significant reduction of GFP expression. These 11 compounds were representing 160 compounds with common sub-structures. So, I screened those 160 compounds as the second screening of natural product library, and found five hit compounds with significantly reduced GFP expression. Further experiment and analysis can reveal the potentiality of these compounds to inhibit Shiga toxin production as well as to reduce the severity of EHEC symptom. Compound A; an oral anti-helminthic drug used to treat parasitic infection, is one of the hit compounds found from authentic library screening. We demonstrated that compound A inhibits Shiga toxin and phage production under MMC treatment in E. coli O157:H7 EDL933.
Key words: Enterohemorrhagic Escherichia coli, Shiga toxin, phage.

1. Nataro, J.P. and J.B. Kaper, Diarrheagenic escherichia coli. Clinical microbiology reviews, 1998. 11(1): p. 142-201.
2. Karmali, M.A., Infection by verocytotoxin-producing Escherichia coli. Clinical microbiology reviews, 1989. 2(1): p. 15-38.
3. Hunter, P.R., Drinking water and diarrhoeal disease due to Escherichia coli. Journal of water and health, 2003. 1(2): p. 65-72.
4. Riley, L.W., et al., Hemorrhagic colitis associated with a rare Escherichia coli serotype. New England Journal of Medicine, 1983. 308(12): p. 681-685.
5. Meng, J., et al., Enterohemorrhagic Escherichia coli. Food microbiology: Fundamentals and frontiers, 2012: p. 287-309.
6. Furukawa, I., et al., An outbreak of enterohemorrhagic Escherichia coli O157: H7 infection associated with minced meat cutlets in Kanagawa, Japan. Japanese journal of infectious diseases, 2018: p. JJID. 2017.495.
7. Diaz-Aviles, E. and A. Stewart. Tracking Twitter for epidemic intelligence: Case study: EHEC/HUS outbreak in Germany, 2011. in Proceedings of the 4th annual ACM web science conference. 2012.
8. Tack, D.M., et al., Shiga Toxin-Producing Escherichia coli Outbreaks in the United States, 2010–2017. Microorganisms, 2021. 9(7): p. 1529.
9. Konowalchuk, J., J. Speirs, and S. Stavric, Vero response to a cytotoxin of Escherichia coli. Infection and immunity, 1977. 18(3): p. 775-779.
10. Dini, C. and P.J. De Urraza, Isolation and selection of coliphages as potential biocontrol agents of enterohemorrhagic and Shiga toxin‐producing E. coli (EHEC and STEC) in cattle. Journal of applied microbiology, 2010. 109(3): p. 873-887.
11. Scotland, S., H. Smith, and B. Rowe, Two distinct toxins active on Vero cells from Escherichia coli O157. Lancet (London, England), 1985. 2(8460): p. 885-886.
12. Nakao, H. and T. Takeda, Escherichia coli Shiga toxin. Journal of natural toxins, 2000. 9(3): p. 299-313.
13. Cohen, S.E., et al., Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli. Proceedings of the National Academy of Sciences, 2010. 107(35): p. 15517-15522.
14. Friedberg, E.C., et al., DNA repair and mutagenesis. 2005: American Society for Microbiology Press.
15. Little, J.W. and M. Gellert, The SOS regulatory system: control of its state by the level of RecA protease. Journal of molecular biology, 1983. 167(4): p. 791-808.
16. Galkin, V.E., et al., Cleavage of bacteriophage λ cI repressor involves the RecA C-terminal domain. Journal of molecular biology, 2009. 385(3): p. 779-787.
17. Sperandio, V. and A.R. Pacheco, Shiga toxin in enterohemorrhagic E. coli: regulation and novel anti-virulence strategies. Frontiers in cellular and infection microbiology, 2012. 2: p. 81.
18. McGannon, C.M., C.A. Fuller, and A.A. Weiss, Different classes of antibiotics differentially influence Shiga toxin production. Antimicrobial agents and chemotherapy, 2010. 54(9): p. 3790-3798.
19. Wong, C.S., et al., The risk of the hemolytic–uremic syndrome after antibiotic treatment of Escherichia coli O157: H7 infections. New England Journal of Medicine, 2000. 342(26): p. 1930-1936.
20. Hong, Y., et al., Post-stress bacterial cell death mediated by reactive oxygen species. Proceedings of the National Academy of Sciences, 2019. 116(20): p. 10064-10071.
21. Barini, E., et al., The anthelmintic drug niclosamide and its analogues activate the Parkinson's disease associated protein kinase PINK1. ChemBioChem, 2018. 19(5): p. 425.
22. Wu, C.-J., et al., Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrobial agents and chemotherapy, 2004. 48(7): p. 2693-2696.
23. Rajamuthiah, R., et al., Repurposing salicylanilide anthelmintic drugs to combat drug resistant Staphylococcus aureus. PloS one, 2015. 10(4): p. e0124595.
24. Chowdhury, M.K.H., et al., Niclosamide reduces glucagon sensitivity via hepatic PKA inhibition in obese mice: Implications for glucose metabolism improvements in type 2 diabetes. Scientific reports, 2017. 7(1): p. 1-9.
25. Wang, A.M., et al., The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells. Journal of cellular biochemistry, 2009. 106(4): p. 682-692.
26. Kadri, H., O.A. Lambourne, and Y. Mehellou, Niclosamide, a drug with many (re) purposes. ChemMedChem, 2018. 13(11): p. 1088.
27. Zhu, P.J., et al., Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death. Bioorganic & medicinal chemistry, 2009. 17(14): p. 5139-5145.