Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids

English version

Autoři: Stephen F. Fitzgerald ^aff001; Amy E. Beckett ^aff001; Javier Palarea-Albaladejo ^aff003; Sean McAteer ^aff001; Sharif Shaaban ^aff001; Jason Morgan ^aff001; Nur Indah Ahmad ^aff004; Rachel Young ^aff001; Neil A. Mabbott ^aff001; Liam Morrison ^aff001; James L. Bono ^aff005; David L. Gally ^aff001; Tom N. McNeilly ^aff002
Působiště autorů: Division of Immunity and Infection, The Roslin Institute and R(D)SVS, The University of Edinburgh, Midlothian, United Kingdom ^aff001; Moredun Research Institute, Penicuik, United Kingdom ^aff002; Biomathematics and Statistics Scotland, Edinburgh, United Kingdom ^aff003; Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia ^aff004; United States Department of Agriculture, Agricultural Research Service, Nebraska, United States of America ^aff005
Vyšlo v časopise: Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids. PLoS Pathog 15(10): e32767. doi:10.1371/journal.ppat.1008003
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008003

Souhrn

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>10³ cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a⁺/stx2c⁺ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.

Klíčová slova:

Bacteriophages – Cattle – Excretion – Gastrointestinal tract – Lysis (medicine) – Molting – Toxins – Organoids

Zdroje

1. Obrig TG, Karpman D. Shiga toxin pathogenesis: kidney complications and renal failure. Current topics in microbiology and immunology. 2012;357:105–36. doi: 10.1007/82_2011_172 21983749

2. Garcia A, Fox JG, Besser TE. Zoonotic enterohemorrhagic Escherichia coli: A One Health perspective. ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 2010;51(3):221–32. doi: 10.1093/ilar.51.3.221 21131723.

3. Majowicz SE, Scallan E, Jones-Bitton A, Sargeant JM, Stapleton J, Angulo FJ, et al. Global incidence of human Shiga toxin-producing Escherichia coli infections and deaths: a systematic review and knowledge synthesis. Foodborne pathogens and disease. 2014;11(6):447–55. doi: 10.1089/fpd.2013.1704 24750096

4. Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982–2002. Emerging infectious diseases. 2005;11(4):603–9. doi: 10.3201/eid1104.040739 15829201

5. Melton-Celsa A, Mohawk K, Teel L, O’Brien A. Pathogenesis of Shiga-toxin producing Escherichia coli. Current topics in microbiology and immunology. 2012;357:67–103. doi: 10.1007/82_2011_176 21915773.

6. Melton-Celsa AR. Shiga Toxin (Stx) Classification, Structure, and Function. Microbiology spectrum. 2014;2(2). doi: 10.1128/microbiolspec.EHEC-0024-2013 25530917

7. Scheutz F, Teel LD, Beutin L, Pierard D, Buvens G, Karch H, et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. Journal of clinical microbiology. 2012;50(9):2951–63. doi: 10.1128/JCM.00860-12 22760050

8. Brandal LT, Wester AL, Lange H, Lobersli I, Lindstedt BA, Vold L, et al. Shiga toxin-producing Escherichia coli infections in Norway, 1992–2012: characterization of isolates and identification of risk factors for haemolytic uremic syndrome. BMC infectious diseases. 2015;15:324. doi: 10.1186/s12879-015-1017-6 26259588

9. Buvens G, De Gheldre Y, Dediste A, de Moreau AI, Mascart G, Simon A, et al. Incidence and virulence determinants of verocytotoxin-producing Escherichia coli infections in the Brussels-Capital Region, Belgium, in 2008–2010. Journal of clinical microbiology. 2012;50(4):1336–45. doi: 10.1128/JCM.05317-11 22238434

10. Dallman TJ, Ashton PM, Byrne L, Perry NT, Petrovska L, Ellis R, et al. Applying phylogenomics to understand the emergence of Shiga-toxin-producing Escherichia coli O157: H7 strains causing severe human disease in the UK. Microbial Genomics. 2015;1(3).

11. Fuller CA, Pellino CA, Flagler MJ, Strasser JE, Weiss AA. Shiga toxin subtypes display dramatic differences in potency. Infection and immunity. 2011;79(3):1329–37. doi: 10.1128/IAI.01182-10 21199911

12. Louise CB, Obrig TG. Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells. The Journal of infectious diseases. 1995;172(5):1397–401. doi: 10.1093/infdis/172.5.1397 7594687.

13. Russo LM, Melton-Celsa AR, O’Brien AD. Shiga Toxin (Stx) Type 1a Reduces the Oral Toxicity of Stx Type 2a. The Journal of infectious diseases. 2016;213(8):1271–9. doi: 10.1093/infdis/jiv557 26743841

14. Kawano K, Okada M, Haga T, Maeda K, Goto Y. Relationship between pathogenicity for humans and stx genotype in Shiga toxin-producing Escherichia coli serotype O157. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 2008;27(3):227–32. doi: 10.1007/s10096-007-0420-3 18071766.

15. Fogg PC, Saunders JR, McCarthy AJ, Allison HE. Cumulative effect of prophage burden on Shiga toxin production in Escherichia coli. Microbiology. 2012;158(Pt 2):488–97. doi: 10.1099/mic.0.054981-0 22096150.

16. de Sablet T, Bertin Y, Vareille M, Girardeau JP, Garrivier A, Gobert AP, et al. Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C. Microbiology. 2008;154(Pt 1):176–86. doi: 10.1099/mic.0.2007/009704-0 18174136.

17. Ogura Y, Mondal SI, Islam MR, Mako T, Arisawa K, Katsura K, et al. The Shiga toxin 2 production level in enterohemorrhagic Escherichia coli O157:H7 is correlated with the subtypes of toxin-encoding phage. Scientific reports. 2015;5:16663. doi: 10.1038/srep16663 26567959

18. Robinson CM, Sinclair JF, Smith MJ, O’Brien AD. Shiga toxin of enterohemorrhagic Escherichia coli type O157:H7 promotes intestinal colonization. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(25):9667–72. doi: 10.1073/pnas.0602359103 16766659

19. Liu B, Yin X, Feng Y, Chambers JR, Guo A, Gong J, et al. Verotoxin 2 enhances adherence of enterohemorrhagic Escherichia coli O157:H7 to intestinal epithelial cells and expression of {beta}1-integrin by IPEC-J2 cells. Appl Environ Microbiol. 2010;76(13):4461–8. doi: 10.1128/AEM.00182-10 20453145

20. Gobert AP, Coste A, Guzman CA, Vareille M, Hindre T, de Sablet T, et al. Modulation of chemokine gene expression by Shiga-toxin producing Escherichia coli belonging to various origins and serotypes. Microbes and infection. 2008;10(2):159–65. doi: 10.1016/j.micinf.2007.10.018 18248761.

21. Gobert AP, Vareille M, Glasser AL, Hindre T, de Sablet T, Martin C. Shiga toxin produced by enterohemorrhagic Escherichia coli inhibits PI3K/NF-kappaB signaling pathway in globotriaosylceramide-3-negative human intestinal epithelial cells. Journal of immunology. 2007;178(12):8168–74. doi: 10.4049/jimmunol.178.12.8168 17548655.

22. Hoffman MA, Menge C, Casey TA, Laegreid W, Bosworth BT, Dean-Nystrom EA. Bovine immune response to shiga-toxigenic Escherichia coli O157:H7. Clinical and vaccine immunology: CVI. 2006;13(12):1322–7. doi: 10.1128/CVI.00205-06 17050743

23. Menge C, Wieler LH, Schlapp T, Baljer G. Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro. Infection and immunity. 1999;67(5):2209–17. 10225876

24. Stamm I, Mohr M, Bridger PS, Schropfer E, Konig M, Stoffregen WC, et al. Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1. Infection and immunity. 2008;76(11):5381–91. doi: 10.1128/IAI.00553-08 18765725

25. Steinberg KM, Levin BR. Grazing protozoa and the evolution of the Escherichia coli O157:H7 Shiga toxin-encoding prophage. Proceedings Biological sciences. 2007;274(1621):1921–9. doi: 10.1098/rspb.2007.0245 17535798

26. Schmidt CE, Shringi S, Besser TE. Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages. PloS one. 2016;11(1):e0147270. doi: 10.1371/journal.pone.0147270 26824472

27. Chase-Topping ME, McKendrick IJ, Pearce MC, MacDonald P, Matthews L, Halliday J, et al. Risk factors for the presence of high-level shedders of Escherichia coli O157 on Scottish farms. Journal of clinical microbiology. 2007;45(5):1594–603. doi: 10.1128/JCM.01690-06 17360845

28. Matthews L, Reeve R, Gally DL, Low JC, Woolhouse ME, McAteer SP, et al. Predicting the public health benefit of vaccinating cattle against Escherichia coli O157. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(40):16265–70. doi: 10.1073/pnas.1304978110 24043803

29. Munns KD, Selinger LB, Stanford K, Guan L, Callaway TR, McAllister TA. Perspectives on super-shedding of Escherichia coli O157:H7 by cattle. Foodborne pathogens and disease. 2015;12(2):89–103. doi: 10.1089/fpd.2014.1829 25514549.

30. Munns KD, Zaheer R, Xu Y, Stanford K, Laing CR, Gannon VP, et al. Comparative Genomic Analysis of Escherichia coli O157:H7 Isolated from Super-Shedder and Low-Shedder Cattle. PloS one. 2016;11(3):e0151673. doi: 10.1371/journal.pone.0151673 27018858

31. Spencer SE, Besser TE, Cobbold RN, French NP. ‘Super’ or just ‘above average’? Supershedders and the transmission of Escherichia coli O157:H7 among feedlot cattle. Journal of the Royal Society, Interface / the Royal Society. 2015;12(110):0446. doi: 10.1098/rsif.2015.0446 26269231

32. Chase-Topping M, Gally D, Low C, Matthews L, Woolhouse M. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nature reviews Microbiology. 2008;6(12):904–12. doi: 10.1038/nrmicro2029 19008890.

33. Omisakin F, MacRae M, Ogden ID, Strachan NJ. Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter. Applied and environmental microbiology. 2003;69(5):2444–7. doi: 10.1128/AEM.69.5.2444-2447.2003 12732509

34. Low JC, McKendrick IJ, McKechnie C, Fenlon D, Naylor SW, Currie C, et al. Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle. Applied and environmental microbiology. 2005;71(1):93–7. doi: 10.1128/AEM.71.1.93-97.2005 15640175

35. Stephens TP, McAllister TA, Stanford K. Perineal swabs reveal effect of super shedders on the transmission of Escherichia coli O157:H7 in commercial feedlots. Journal of animal science. 2009;87(12):4151–60. doi: 10.2527/jas.2009-1967 19684276.

36. Matthews L, Low JC, Gally DL, Pearce MC, Mellor DJ, Heesterbeek JA, et al. Heterogeneous shedding of Escherichia coli O157 in cattle and its implications for control. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(3):547–52. doi: 10.1073/pnas.0503776103 16407143

37. Cobbold RN, Hancock DD, Rice DH, Berg J, Stilborn R, Hovde CJ, et al. Rectoanal junction colonization of feedlot cattle by Escherichia coli O157:H7 and its association with supershedders and excretion dynamics. Applied and environmental microbiology. 2007;73(5):1563–8. doi: 10.1128/AEM.01742-06 17220263

38. Matthews L, McKendrick IJ, Ternent H, Gunn GJ, Synge B, Woolhouse ME. Super-shedding cattle and the transmission dynamics of Escherichia coli O157. Epidemiology and infection. 2006;134(1):131–42. doi: 10.1017/S0950268805004590 16409660

39. Arthur TM, Ahmed R, Chase-Topping M, Kalchayanand N, Schmidt JW, Bono JL. Characterization of Escherichia coli O157:H7 strains isolated from supershedding cattle. Appl Environ Microbiol. 2013;79(14):4294–303. doi: 10.1128/AEM.00846-13 23645203

40. Pearce MC, Chase-Topping ME, McKendrick IJ, Mellor DJ, Locking ME, Allison L, et al. Temporal and spatial patterns of bovine Escherichia coli O157 prevalence and comparison of temporal changes in the patterns of phage types associated with bovine shedding and human E. coli O157 cases in Scotland between 1998–2000 and 2002–2004. BMC microbiology. 2009;9:276. doi: 10.1186/1471-2180-9-276 20040112

41. Shaaban S. C, L A., McAteer SP., Jenkins C., Dallman TJ., Bono JL., and Gally DL. Evolution of a zoonotic pathogen: investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing. Microbial Genomics. 2016. doi: 10.1099/mgen.0.000096 28348836

42. Xu X, McAteer SP, Tree JJ, Shaw DJ, Wolfson EB, Beatson SA, et al. Lysogeny with Shiga toxin 2-encoding bacteriophages represses type III secretion in enterohemorrhagic Escherichia coli. PLoS pathogens. 2012;8(5):e1002672. doi: 10.1371/journal.ppat.1002672 22615557

43. Lupolova N, Dallman T, Bono J, Gally D. Support Vector Machine applied to predict the zoonotic potential of E. coli O157 cattle isolates. Proceedings of the National Academy of Sciences. 2016. doi: 10.1073/pnas.1606567113 27647883

44. Gallegos KM, Conrady DG, Karve SS, Gunasekera TS, Herr AB, Weiss AA. Shiga toxin binding to glycolipids and glycans. PloS one. 2012;7(2):e30368. doi: 10.1371/journal.pone.0030368 22348006

45. Karve SS, Weiss AA. Glycolipid binding preferences of Shiga toxin variants. PloS one. 2014;9(7):e101173. doi: 10.1371/journal.pone.0101173 24983355

46. Hoey DE, Currie C, Else RW, Nutikka A, Lingwood CA, Gally DL, et al. Expression of receptors for verotoxin 1 from Escherichia coli O157 on bovine intestinal epithelium. J Med Microbiol. 2002;51(2):143–9. doi: 10.1099/0022-1317-51-2-143 11865842.

47. Hoey DE, Sharp L, Currie C, Lingwood CA, Gally DL, Smith DG. Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysosomes and abrogation of toxicity. Cellular microbiology. 2003;5(2):85–97. 12580945.

48. Hamilton CA, Young R, Jayaraman S, Sehgal A, Paxton E, Thomson S, et al. Development of in vitro enteroids derived from bovine small intestinal crypts. Vet Res. 2018;49(1):54. doi: 10.1186/s13567-018-0547-5 29970174

49. McNeilly TN, Naylor SW, Mahajan A, Mitchell MC, McAteer S, Deane D, et al. Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infection and immunity. 2008;76(6):2594–602. doi: 10.1128/IAI.01452-07 18362130

50. McNeilly TN, Mitchell MC, Rosser T, McAteer S, Low JC, Smith DG, et al. Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine. 2010;28(5):1422–8. doi: 10.1016/j.vaccine.2009.10.076 19903545.

51. Naylor SW, Low JC, Besser TE, Mahajan A, Gunn GJ, Pearce MC, et al. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host. Infection and immunity. 2003;71(3):1505–12. doi: 10.1128/IAI.71.3.1505-1512.2003 12595469

52. Meeusen EN, Brandon MR. Antibody secreting cells as specific probes for antigen identification. J Immunol Methods. 1994;172(1):71–6. doi: 10.1016/0022-1759(94)90379-4 8207267.

53. Soderlund R, Jernberg C, Ivarsson S, Hedenstrom I, Eriksson E, Bongcam-Rudloff E, et al. Molecular typing of Escherichia coli O157:H7 isolates from Swedish cattle and human cases: population dynamics and virulence. Journal of clinical microbiology. 2014;52(11):3906–12. doi: 10.1128/JCM.01877-14 25143581

54. Pianciola L, D’Astek BA, Mazzeo M, Chinen I, Masana M, Rivas M. Genetic features of human and bovine Escherichia coli O157:H7 strains isolated in Argentina. International journal of medical microbiology: IJMM. 2016;306(2):123–30. doi: 10.1016/j.ijmm.2016.02.005 26935026.

55. Ashton PM, Perry N, Ellis R, Petrovska L, Wain J, Grant KA, et al. Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing. PeerJ. 2015;3:e739. doi: 10.7717/peerj.739 25737808

56. Park D, Stanton E, Ciezki K, Parrell D, Bozile M, Pike D, et al. Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157:H7 strains. Applied and environmental microbiology. 2013;79(5):1563–72. doi: 10.1128/AEM.03158-12 23275514

57. Tree JJ, Granneman S, McAteer SP, Tollervey D, Gally DL. Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli. Molecular cell. 2014;55(2):199–213. doi: 10.1016/j.molcel.2014.05.006 24910100

58. Stanford K, Bach SJ, Stephens TP, McAllister TA. Effect of rumen protozoa on Escherichia coli O157:H7 in the rumen and feces of specifically faunated sheep. Journal of food protection. 2010;73(12):2197–202. doi: 10.4315/0362-028x-73.12.2197 21219736.

59. Sehgal A, Donaldson DS, Pridans C, Sauter KA, Hume DA, Mabbott NA. The role of CSF1R-dependent macrophages in control of the intestinal stem-cell niche. Nature communications. 2018;9(1):1272. doi: 10.1038/s41467-018-03638-6 29593242

60. Naylor SW, Roe AJ, Nart P, Spears K, Smith DG, Low JC, et al. Escherichia coli O157: H7 forms attaching and effacing lesions at the terminal rectum of cattle and colonization requires the LEE4 operon. Microbiology. 2005;151(Pt 8):2773–81. doi: 10.1099/mic.0.28060-0 16079353.

61. Kolling GL, Matthews KR. Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7. Applied and environmental microbiology. 1999;65(5):1843–8. 10223967

62. Yokoyama K, Horii T, Yamashino T, Hashikawa S, Barua S, Hasegawa T, et al. Production of shiga toxin by Escherichia coli measured with reference to the membrane vesicle-associated toxins. FEMS microbiology letters. 2000;192(1):139–44. doi: 10.1111/j.1574-6968.2000.tb09372.x 11040442.

63. Bielaszewska M, Ruter C, Bauwens A, Greune L, Jarosch KA, Steil D, et al. Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury. PLoS pathogens. 2017;13(2):e1006159. doi: 10.1371/journal.ppat.1006159 28158302

64. Pruimboom-Brees IM, Morgan TW, Ackermann MR, Nystrom ED, Samuel JE, Cornick NA, et al. Cattle lack vascular receptors for Escherichia coli O157:H7 Shiga toxins. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(19):10325–9. doi: 10.1073/pnas.190329997 10973498

65. Blasche S, Mortl M, Steuber H, Siszler G, Nisa S, Schwarz F, et al. The E. coli effector protein NleF is a caspase inhibitor. PloS one. 2013;8(3):e58937. doi: 10.1371/journal.pone.0058937 23516580

66. Scott NE, Giogha C, Pollock GL, Kennedy CL, Webb AI, Williamson NA, et al. The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD). J Biol Chem. 2017;292(42):17337–50. doi: 10.1074/jbc.M117.805036 28860194

67. Wong AR, Pearson JS, Bright MD, Munera D, Robinson KS, Lee SF, et al. Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements. Molecular microbiology. 2011;80(6):1420–38. doi: 10.1111/j.1365-2958.2011.07661.x 21488979.

68. Loukiadis E, Nobe R, Herold S, Tramuta C, Ogura Y, Ooka T, et al. Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli. Journal of bacteriology. 2008;190(1):275–85. doi: 10.1128/JB.00844-07 17873042

69. Samba-Louaka A, Nougayrede JP, Watrin C, Oswald E, Taieb F. The enteropathogenic Escherichia coli effector Cif induces delayed apoptosis in epithelial cells. Infection and immunity. 2009;77(12):5471–7. doi: 10.1128/IAI.00860-09 19786559

70. Iwai H, Kim M, Yoshikawa Y, Ashida H, Ogawa M, Fujita Y, et al. A bacterial effector targets Mad2L2, an APC inhibitor, to modulate host cell cycling. Cell. 2007;130(4):611–23. doi: 10.1016/j.cell.2007.06.043 17719540.

71. Kim M, Ogawa M, Fujita Y, Yoshikawa Y, Nagai T, Koyama T, et al. Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment. Nature. 2009;459(7246):578–82. doi: 10.1038/nature07952 19489119.

72. Balasubramanian S, Osburne MS, BrinJones H, Tai AK, Leong JM. Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection. PLoS pathogens. 2019;15(1):e1007494. doi: 10.1371/journal.ppat.1007494 30629725.

73. Loftsdottir H, Soderlund R, Jinnerot T, Eriksson E, Bongcam-Rudloff E, Aspan A. Dynamics of insertion sequence element IS629 inactivation of verotoxin 2 genes in Escherichia coli O157:H7. FEMS microbiology letters. 2017. doi: 10.1093/femsle/fnx074 28402463.

74. Kusumoto M, Nishiya Y, Kawamura Y. Reactivation of insertionally inactivated Shiga toxin 2 genes of Escherichia coli O157:H7 caused by nonreplicative transposition of the insertion sequence. Applied and environmental microbiology. 2000;66(3):1133–8. doi: 10.1128/aem.66.3.1133-1138.2000 10698782

75. Merlin C, McAteer S, Masters M. Tools for characterization of Escherichia coli genes of unknown function. Journal of bacteriology. 2002;184(16):4573–81. doi: 10.1128/JB.184.16.4573-4581.2002 12142427

76. Corbishley A, Ahmad NI, Hughes K, Hutchings MR, McAteer SP, Connelley TK, et al. Strain-dependent cellular immune responses in cattle following Escherichia coli O157:H7 colonization. Infection and immunity. 2014;82(12):5117–31. doi: 10.1128/IAI.02462-14 25267838

77. Borten MA, Bajikar SS, Sasaki N, Clevers H, Janes KA. Automated brightfield morphometry of 3D organoid populations by OrganoSeg. Scientific reports. 2018;8(1):5319. doi: 10.1038/s41598-017-18815-8 29593296

78. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PloS one. 2010;5(6):e11147. doi: 10.1371/journal.pone.0011147 20593022

79. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC genomics. 2011;12:402. doi: 10.1186/1471-2164-12-402 21824423

80. Stajich JE, Block D, Boulez K, Brenner SE, Chervitz SA, Dagdigian C, et al. The Bioperl toolkit: Perl modules for the life sciences. Genome research. 2002;12(10):1611–8. doi: 10.1101/gr.361602 12368254

81. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MT, et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 2015;31(22):3691–3. doi: 10.1093/bioinformatics/btv421 26198102

82. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate—a Practical and Powerful Approach to Multiple Testing. J Roy Stat Soc B Met. 1995;57(1):289–300.

83. Team RC. R: A language and environment for statistical computing. R foundation for statistical computing, Vienna, Austria. 2014.