[en] Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens that cause human diseases ranging from diarrhea to life-threatening complications including hemolytic-uremic syndrome. Virulence of STEC strains and their ability to cause severe diseases are associated with the activity of prophage-encoded Shiga toxins (Stxs). The first objective of this work was to isolate and characterize the Stx2d phage from STEC O80:H2 and to study the transfer of this phage in non-STEC strains. The second objective was to assess the survival of Galleria mellonella larvae inoculated with these transduced strains. Firstly, one bacteriophage isolated from a STEC O80:H2 strain was used to infect six non-STEC strains, resulting in the conversion of three strains. Then, stability assays were performed, showing that this phage was stable in the new STEC strains after three successive subculturing steps, as confirmed by a combination of short and long read genome sequencing approaches. This phage, vB_EcoS_ULI-O80_Stx2d, is resistant to moderate temperature and pH. It belongs to a currently unclassified genus and family within the Caudoviricetes class, shares 98% identity with Stx2_112808 phage and encodes several proteins involved in the lysogenic cycle. The yecE gene was identified at the insertion site. Finally, G. mellonella experiments showed that the transduced strains caused significantly higher mortality rates than the corresponding non-STEC strains. In conclusion, this study showed that stx2d gene from O80:H2 E. coli can be transferred to non-STEC strains and contributes to their virulence.
Disciplines :
Veterinary medicine & animal health
Author, co-author :
Habets, Audrey ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH)
Antoine, Céline ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH)
Wagemans, Jeroen; Laboratory of Gene Technology, Department of Biosystems, KU Leuven, 3000, Leuven, Belgium
Vermeersch, Marjorie; Electron Microscopy Laboratory, Center for Microscopy and Molecular Imaging, ULB, 6041, Gosselies, Belgium
Laforêt, Fanny ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH)
Diderich, Jacob ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Santé publique vétérinaire
Lavigne, Rob; Laboratory of Gene Technology, Department of Biosystems, KU Leuven, 3000, Leuven, Belgium
Mainil, Jacques ; Université de Liège - ULiège > Département des maladies infectieuses et parasitaires (DMI)
Thiry, Damien ; Université de Liège - ULiège > Département des maladies infectieuses et parasitaires (DMI) > Bactériologie vétérinaire et maladies bactériennes animales
Language :
English
Title :
Impact of Shiga-toxin encoding gene transduction from O80:H2 Shiga toxigenic Escherichia coli (STEC) on non-STEC strains.
The authors thank David Perez-Morga (CMMI, ULB) for the collaboration for the transmission electron microscopy. The CMMI is supported by the European Regional Development Fund and the Walloon Region. The authors also thank Dr Marc Saulmont from ARSIA for providing O80:H2 E. coli strains. The university of Liège provided financial support (‘Fonds Spéciaux de la Recherche Project HYBRID_COLI_O80’).
Mcallister, L. J. et al. Genomic comparison of two O111:H− enterohemorrhagic Escherichia coli isolates from a historic hemolytic-uremic syndrome outbreak in Australia. Infect. Immun. 84(3), 775–781. 10.1128/IAI.01229-15 (2016). DOI: 10.1128/IAI.01229-15
Bloch, S. et al. UV-sensitivity of Shiga toxin-converting bacteriophage birions Φ 24B, 933W, P22, P27 and P32. Toxins 7(9), 3727–3739. 10.3390/toxins7093727 (2015). DOI: 10.3390/toxins7093727
Smith, D. L. et al. Comparative genomics of Shiga toxin encoding bacteriophages. BMC Genom. 13, 311. 10.1186/1471-2164-13-311 (2012). DOI: 10.1186/1471-2164-13-311
Scheutz, F. et al. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J. Clin. Microbiol. 50(9), 2951–2963. 10.1128/JCM.00860-12 (2012). DOI: 10.1128/JCM.00860-12
Bai, X., Scheutz, F., Dahlgren, H. M., Hedenström, I. & Jernberg, C. Characterization of clinical Escherichia coli strains producing a novel Shiga toxin 2 subtype in Sweden and Denmark. Microorganisms 17(9), 2374. 10.3390/microorganisms9112374 (2021). DOI: 10.3390/microorganisms9112374
Paul, J. H. & Weinbauer, M. Detection of lysogeny in marine environments. Man. Aquat. Viral Ecol. 4, 30–33. 10.4319/mave.2010.978-0-9845591-0-7.30 (2010). DOI: 10.4319/mave.2010.978-0-9845591-0-7.30
Imamovic, L. & Muniesa, M. Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment. PLoS ONE 7, 2. 10.1371/journal.pone.0032393 (2012). DOI: 10.1371/journal.pone.0032393
Allison, H. E. et al. Immunity profiles of wild-type and recombinant Shiga-like toxin-encoding bacteriophages and characterization of novel double lysogens. Infect. Immun. 71(6), 3409–3418. 10.1128/IAI.71.6.3409 (2003). DOI: 10.1128/IAI.71.6.3409
Toth, I. et al. Transduction of porcine enteropathogenic Escherichia coli with a derivative of a Shiga toxin 2-encoding bacteriophage in a porcine ligated ileal loop system. Appl. Environ. Microbiol. 69(12), 7242–7247. 10.1128/AEM.69.12.7242 (2003). DOI: 10.1128/AEM.69.12.7242
Fang, Y., Mercer, R. G., Mcmullen, L. M. & Gänzle, M. G. Induction of Shiga toxin-encoding prophage by abiotic environmental stress in food. Appl. Environ. Microbiol. 83(19), 1–13. 10.1128/AEM.01378-17 (2017). DOI: 10.1128/AEM.01378-17
Plunkett, G. III., Rose, D. J., Durfee, T. J. & Blattner, F. R. Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181, 1767–1778. 10.1128/jb.181.6.1767-1778.1999 (1999). DOI: 10.1128/jb.181.6.1767-1778.1999
Creuzburg, K. et al. The Shiga toxin 1-converting bacteriophage BP-4795 encodes an NleA-like type III effector protein. J. Bacteriol. 187, 8494–8498. 10.1128/JB.187.24.8494-8498 (2005). DOI: 10.1128/JB.187.24.8494-8498
De Greve, H., Qizhi, C., Deboeck, F. & Hernalsteens, J. P. The Shiga-toxin VT2-encoding bacteriophage phi297 integrates at a distinct position in the Escherichia coli genome. Biochim. Biophys. Acta 1579, 196–202. 10.1016/S0167-4781(02)00539-0 (2002). DOI: 10.1016/S0167-4781(02)00539-0
Wick, L. M., Weihong, Q., Lacher, D. W. & Whittam, T. S. Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7. J. Bacteriol. 187(5), 1783–1791. 10.1128/JB.187.5.1783 (2005). DOI: 10.1128/JB.187.5.1783
Brzuszkiewicz, E. et al. Genome sequence analyses of two isolates from the recent Escherichia coli outbreak in Germany reveal the emergence of a new pathotype: Entero-aggregative-haemorrhagic Escherichia coli (EAHEC). Arch. Microbiol. 193(12), 883–891. 10.1007/s00203-011-0725-6 (2011). DOI: 10.1007/s00203-011-0725-6
Bielaszewska, M. et al. Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans. Appl. Environ. Microbiol. 73(10), 3144–3150. 10.1128/AEM.02937-06 (2007). DOI: 10.1128/AEM.02937-06
Soysal, N. et al. Enterohemorrhagic Escherichia coli hybrid pathotype O80:H2 as a new therapeutic challenge. Emerg. Infec. Dis. 22(9), 1604–1612. 10.3201/eid2209.160304 (2016). DOI: 10.3201/eid2209.160304
Wijnsma, K. L. et al. Unusual severe case of hemolytic uremic syndrome due to Shiga toxin 2d-producing E. coli O80:H2. Pediatr. Nephrol. 32(7), 1263–1268. 10.1007/s00467-017-3642-3 (2017). DOI: 10.1007/s00467-017-3642-3
Cointe, A. et al. Emerging multidrug-resistant hybrid pathotype Shiga toxin–producing Escherichia coli O80 and related strains of clonal complex 165. Eur. Emerg. Infect. Dis. 24(12), 2262–2269. 10.3201/eid2412.180272 (2018). DOI: 10.3201/eid2412.180272
Mariani-Kurkdjian, P. et al. Haemolytic-uraemic syndrome with bacteremia caused by a new hybrid Escherichia coli pathotype. New Microbes New Infect. 2(4), 127–131. 10.1002/nmi2.49 (2014). DOI: 10.1002/nmi2.49
Bruyand, M. et al. Paediatric haemolytic uraemic syndrome related to Shiga toxin-producing Escherichia coli, an overview of 10 years of surveillance in France, 2007 to 2016. Eurosurveillance 24, 8. 10.2807/1560-7917.ES.2019.24.8.1800068 (2019). DOI: 10.2807/1560-7917.ES.2019.24.8.1800068
Blanco, M. et al. Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-ξ). J. Clin. Microbiol. 42(2), 645–651. 10.1128/JCM.42.2.645-651.2004 (2004). DOI: 10.1128/JCM.42.2.645-651.2004
Habets, A. et al. Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2. J. Clin. Microbiol. 42(2), 645–651. 10.1111/jam.14759 (2020). DOI: 10.1111/jam.14759
De Rauw, K. et al. Characteristics of Shiga toxin producing and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium. Clin. Microbiol. Infect. 25, 23–26. 10.1016/j.cmi.2018.07.023 (2019). DOI: 10.1016/j.cmi.2018.07.023
Thiry, D. et al. Enteropathogenic Escherichia coli O80:H2 in young calves with diarrhea. Belgium. Emerg. Infect. Dis. 23(12), 2093–2095. 10.3201/eid2312.170450 (2017). DOI: 10.3201/eid2312.170450
Nüesch-Inderbinen, M. et al. Serotypes and virulence profiles of Shigatoxin-producing Escherichia coli strains isolated during 2017 from human infections in Switzerland. Int. J. Med. Microbiol. 308(7), 933–939. 10.1016/j.ijmm.2018.06.011 (2018). DOI: 10.1016/j.ijmm.2018.06.011
European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union one health 2019 zoonoses report. EFSA J. 19, 2. 10.2903/j.efsa.2021.6406 (2021). DOI: 10.2903/j.efsa.2021.6406
Donnenberg, M. S. & Whittam, T. S. Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J. Clin. Investig. 107(5), 539–548 (2001). DOI: 10.1172/JCI12404
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. Eur. J. Clin. Microbiol. Infect. Dis. 27(3), 227–232. 10.1007/s10096-007-0420-3 (2008). DOI: 10.1007/s10096-007-0420-3
Orth, D. et al. The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome. Diagn. Microbiol. Infect. Dis. 59(3), 235–242. 10.1016/j.diagmicrobio.2007.04.013 (2007). DOI: 10.1016/j.diagmicrobio.2007.04.013
Zimmermann, L. et al. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 430(15), 2237–2243. 10.1016/j.jmb.2017.12.007 (2018). DOI: 10.1016/j.jmb.2017.12.007
Feiner, R. et al. A new perspective on lysogeny: Prophages as active regulatory switches of bacteria. Nat. Rev. Microbiol. 13(10), 641–650. 10.1038/nrmicro3527 (2015). DOI: 10.1038/nrmicro3527
Yue, W. F., Du, M. & Zhu, M. J. High temperature in combination with UV irradiation enhances horizontal transfer of Stx2 gene from E. coli O157:H7 to non-pathogenic E. coli. PLoS ONE 7, 2. 10.1371/journal.pone.0031308 (2012). DOI: 10.1371/journal.pone.0031308
Krüger, A. & Lucchesi, P. M. A. Shiga toxins and Stx phages: Highly diverse entities. Microbiology 161(3), 1–12. 10.1099/mic.0.000003 (2015). DOI: 10.1099/mic.0.000003
Rakhuba, D. V., Kolomiets, E. I., Dey, E. S. & Novik, G. I. Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell. Pol. J. Microbiol. 59(3), 145–155 (2010). DOI: 10.33073/pjm-2010-023
Asmamaw, M. & Zawdie, B. Mechanism and applications of CRISPR/Cas-9-mediated genome editing. Biologics 21(15), 353–361. 10.2147/BTT.S326422 (2021). DOI: 10.2147/BTT.S326422
Goldfarb, T. et al. BREX is a novel phage resistance system widespread in microbioal genomes. Embo J. 34, 169–183. 10.15252/embj.201489455 (2015). DOI: 10.15252/embj.201489455
Tozzoli, R. Shiga toxin-converting phages and the emergence of new pathogenic Escherichia coli: A world in motion. Front. Cell. Infect. Microbiol 4, 80. 10.3389/fcimb.2014.00080 (2014). DOI: 10.3389/fcimb.2014.00080
Cointe, A. et al. Emergence of new ST301 Shiga toxin-producing Escherichia coli clones harboring extra-intestinal virulence traits in Europe. Toxins 13, 10. 10.3390/toxins13100686 (2021). DOI: 10.3390/toxins13100686
Rodwell, E. V. et al. Epidemiology and genomic analysis of Shiga toxin-producing Escherichia coli clonal complex 165 in the UK. J. Med. Microbiol. 70, 001471. 10.1099/jmm.0.001471 (2021). DOI: 10.1099/jmm.0.001471
Egido, J. E., Costa, A. R., Aparicio-Maldonado, C., Haas, P.-J. & Brouns, S. J. J. Mechanisms and clinical importance of bacteriophage resistance. FEMS Microbiol. Rev. 10.1093/femsre/fuab048 (2022). DOI: 10.1093/femsre/fuab048
Carbonari, C. C. et al. The importance of Shiga toxin-producing Escherichia coli O145:NM[H28]/H28 infections in Argentina, 1998–2020. Microorganisms 7, 10. 10.3390/microorganisms10030582 (2022). DOI: 10.3390/microorganisms10030582
Balding, C., Bromley, S. A., Pickup, R. W. & Saunders, J. R. Diversity of phage integrases in Enterobacteriaceae: Development of markers for environmental analysis of temperate phages. Environ. Microbiol. 7, 1558–1567. 10.1111/j.1462-2920.2005.00845.x (2005). DOI: 10.1111/j.1462-2920.2005.00845.x
Colavecchio, A. et al. Prophage integrase typing is a useful indicator of genomic diversity in Salmonella enterica. Front. Microbiol. 8, 1283. 10.3389/fmicb.2017.01283 (2017). DOI: 10.3389/fmicb.2017.01283
Nyambe, S., Burgess, C., Whyte, P. & Bolton, D. An investigation of vtx2 bacteriophage transduction to different Escherichia coli patho-groups in food matrices and nutrient broth. Food. Microbiol. 68, 1–6. 10.1016/j.fm.2017.06.004 (2017). DOI: 10.1016/j.fm.2017.06.004
Reckentenwald, J. & Schmidt, H. The nucleotide sequence of Shiga toxin (Stx) 2e-encoding phage φP27 is not related to other Stx phage genomes, but the modular genetic structure is conserved. Am. Soc. Microbiol. 70(4), 1896–1908 (2002).
Serra-Moreno, R., Jofre, J. & Munesia, M. Insertion site occupancy by Stx2 bacteriophages depends on the locus availability of the host strain chromosome. J. Bact. 189(18), 6645–6654. 10.1128/JB.00466-07 (2007). DOI: 10.1128/JB.00466-07
Fogg, P. C. M. et al. Identification of multiple integration sites for Stx-phage Phi24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor. Microbiology 153, 4098–4110. 10.1099/mic.0.2007/011205-0 (2007). DOI: 10.1099/mic.0.2007/011205-0
Saile, N. et al. Growth advantage of Escherichia coli O104:H4 strains on 5-N-acetyl-9-O-acetyl neuraminic acid as a carbon source is dependent on heterogeneous phage-Borne nanS-p esterases. Int. J. Med. Microbiol. 308(4), 459–468. 10.1016/j.ijmm.2018.03.006 (2018). DOI: 10.1016/j.ijmm.2018.03.006
Khalil, R. K. S., Skinner, C., Patfield, S. & He, X. Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains. Pathog. Dis. 74, 5. 10.1093/femspd/ftw037 (2016). DOI: 10.1093/femspd/ftw037
Iguchi, A. et al. Escherichia coli O-genotyping PCR: A comprehensive and practical platform for molecular O serogrouping. J. Clin. Microbiol. 53, 2427–2432. 10.1128/JCM.00321-15 (2003). DOI: 10.1128/JCM.00321-15
Park, D.-S. & Park, J.-H. Characteristics of bacteriophage isolates and expression of Shiga-toxin genes transferred to non Shiga toxin-producing E. coli by transduction. J. Microbiol. Technol. 31(5), 1–7. 10.4014/jmb.2102.02040 (2021). DOI: 10.4014/jmb.2102.02040
R: A Language and Environment for Statistical Computing. Available online: https://www.gbif.org/fr/tool/81287/r-a-language-and-environment-for-statistical-computing.
Verstraete, K. et al. A qPCR assay to detect and quantify Shiga toxin-producing E. coli (STEC) in cattle and on farms: A potential predictive tool for STEC culture-positive farms. Toxins 6(4), 1201–1221. 10.3390/toxins6041201 (2014). DOI: 10.3390/toxins6041201
Eskenazi, A. et al. Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae. Nature 13, 302. 10.1038/s41467-021-27656-z (2022). DOI: 10.1038/s41467-021-27656-z
Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. BioRxiv 10.1101/096412 (2016). DOI: 10.1101/096412
Altschul, S. Basic local alignment search tool (BLAST). J. Mol Biol. 215(3), 403–410. 10.1006/jmbi.1990.9999 (1990). DOI: 10.1006/jmbi.1990.9999
Nishimura, Y. et al. ViPTree: The viral proteomic tree server. Bioinformatics 33(15), 2379–2380. 10.1093/bioinformatics/btx157 (2017). DOI: 10.1093/bioinformatics/btx157
Moraru, C., Varsani, A. & Kropinski, A. M. VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12(11), 1268. 10.3390/v12111268 (2020). DOI: 10.3390/v12111268
Brettin, T. et al. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci. Rep. 5, 8365. 10.1038/srep08365 (2015). DOI: 10.1038/srep08365
Wattam, A. R. et al. Improvements to PATRIC, the all-bacterial bioinformatics database and analysis resource center. Nucleic Acids. Res. 45(D1), D535–D542. 10.1093/nar/gkw1017 (2017). DOI: 10.1093/nar/gkw1017
Söding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, 244–248. 10.1093/nar/gki408 (2005). DOI: 10.1093/nar/gki408
Sullivan, M. J., Petty, N. K. & Beatson, S. A. Easyfig: A genome comparison visualizer. Bioinformatics 27(7), 1009–1010. 10.1093/bioinformatics/btr039 (2011). DOI: 10.1093/bioinformatics/btr039
Wick, R. R., Judd, L. M. & Holt, K. E. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol. 20(1), 129. 10.1186/s13059-019-1727-y (2019). DOI: 10.1186/s13059-019-1727-y
Wick, R. R., Schultz, M. B., Zobel, J. & Holt, K. E. Bandage: Interactive visualization of de novo genome assemblies. Bioinformatics 31(20), 3350–3352. 10.1093/bioinformatics/btv383 (2015). DOI: 10.1093/bioinformatics/btv383
Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 15(29), 1072–1075. 10.1093/bioinformatics/btt086 (2013). DOI: 10.1093/bioinformatics/btt086
