Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) strains are the causative agents of severe foodborne diseases in both humans and animals. In this study, porcine pathogenic E. coli strains (n = 277) as well as porcine commensal strains (n = 188) were tested for their susceptibilities to 34 bacteriocin monoproducers to identify the most suitable bacteriocin types inhibiting porcine pathogens. Under in vitro conditions, the set of pathogenic E. coli strains was found to be significantly more susceptible to the majority of tested bacteriocins than commensal E. coli. Based on the production of bacteriocins with specific activity against pathogens, three potentially probiotic commensal E. coli strains of human origin were selected. These strains were found to be able to outcompete ETEC strains expressing F4 or F18 fimbriae in liquid culture and also decreased the severity and duration of diarrhea in piglets during experimental ETEC infection as well as pathogen numbers on the last day of in vivo experimentation. While the extents of the probiotic effect were different for each strain, the cocktail of all three strains showed the most pronounced beneficial effects, suggesting synergy between the tested E. coli strains. IMPORTANCE Increasing levels of antibiotic resistance among bacteria also increase the need for alternatives to conventional antibiotic treatment. Pathogenic Escherichia coli represents a major diarrheic infectious agent of piglets in their postweaning period; however, available measures to control these infections are limited. This study describes three novel E. coli strains producing antimicrobial compounds (bacteriocins) that actively inhibit a majority of toxigenic E. coli strains. The beneficial effect of three potentially probiotic E. coli strains was demonstrated under both in vitro and in vivo conditions. The novel probiotic candidates may be used as prophylaxis during piglets' postweaning period to overcome common infections caused by E. coli.

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Faculty of Informatics Masaryk University Brno Czech Republic

RECETOX Center Faculty of Science Masaryk University Brno Czech Republic

Veterinary Research Institute Brno Czech Republic

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Rhouma M, Fairbrother JM, Beaudry F, Letellier A. 2017. Post weaning diarrhea in pigs: risk factors and non-colistin-based control strategies. Acta Vet Scand 59:31. 10.1186/s13028-017-0299-7. PubMed DOI PMC

Luppi A, Gibellini M, Gin T, Vangroenweghe F, Vandenbroucke V, Bauerfeind R, Bonilauri P, Labarque G, Hidalgo Á. 2016. Prevalence of virulence factors in enterotoxigenic PubMed DOI PMC

Fairbrother JM, Nadeau É, Gyles CL. 2005. PubMed DOI

Tseng M, Fratamico PM, Manning SD, Funk JA. 2014. Shiga toxin-producing PubMed DOI PMC

Luppi A. 2017. Swine enteric colibacillosis: diagnosis, therapy and antimicrobial resistance. Porcine Health Manag 3:16. 10.1186/s40813-017-0063-4. PubMed DOI PMC

Sun Y, Kim SW. 2017. Intestinal challenge with enterotoxigenic PubMed DOI PMC

Matise I, Sirinarumitr T, Bosworth BT, Moon HW. 2000. Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing PubMed DOI

Casanova NA, Redondo LM, Dailoff GC, Arenas D, Fernández Miyakawa ME. 2018. Overview of the role of Shiga toxins in porcine edema disease pathogenesis. Toxicon 148:149–154. 10.1016/j.toxicon.2018.04.019. PubMed DOI

Bourgeois AL, Wierzba TF, Walker RI. 2016. Status of vaccine research and development for enterotoxigenic PubMed DOI

Majowicz SE, Scallan E, Jones-Bitton A, Sargeant JM, Stapleton J, Angulo FJ, Yeung DH, Kirk MD. 2014. Global incidence of human Shiga toxin-producing PubMed DOI PMC

Newell DG, La Ragione RM. 2018. Enterohaemorrhagic and other Shiga toxin-producing PubMed DOI

Marshall BM, Levy SB. 2011. Food animals and antimicrobials: impacts on human health. Clin Microbiol Rev 24:718–733. 10.1128/CMR.00002-11. PubMed DOI PMC

Maron DF, Smith TJS, Nachman KE. 2013. Restrictions on antimicrobial use in food animal production: an international regulatory and economic survey. Global Health 9:48. 10.1186/1744-8603-9-48. PubMed DOI PMC

EMA Committee for Medicinal Products for Veterinary Use, EFSA Panel on Biological Hazards, Murphy D, Ricci A, Auce Z, Beechinor JG, Bergendahl H, Breathnach R, Bureš J, Duarte Da Silva JP, Hederová J, Hekman P, Ibrahim C, Kozhuharov E, Kulcsár G, Lander Persson E, Lenhardsson JM, Mačiulskis P, Malemis I, Markus‐Cizelj L, Michaelidou-Patsia A, Nevalainen M, Pasquali P, Rouby J-C, Schefferlie J, Schlumbohm W, Schmit M, Spiteri S, Srčič S, Taban L, Tiirats T, Urbain B, Vestergaard E, Wachnik-Święcicka A, Weeks J, Zemann B, Allende A, Bolton D, Chemaly M, Fernandez Escamez PS, Girones R, Herman L, Koutsoumanis K, Lindqvist R, Nørrung B, Robertson L, Ru G, Sanaa M, Simmons M, Skandamis P, Snary E, Speybroeck N, et al. 2017. EMA and EFSA joint scientific opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union, and the resulting impacts on food safety (RONAFA). EFSA J 15:e04666. 10.2903/j.efsa.2017.4666. PubMed DOI PMC

Bosák J, Hrala M, Micenková L, Šmajs D. 2021. Non-antibiotic antibacterial peptides and proteins of PubMed DOI

Gillor O, Kirkup BC, Riley MA. 2004. Colicins and microcins: the next generation antimicrobials. Adv Appl Microbiol 54:129–146. 10.1016/S0065-2164(04)54005-4. PubMed DOI

Meade E, Slattery MA, Garvey M. 2020. Bacteriocins, potent antimicrobial peptides and the fight against multi drug resistant species: resistance is futile? Antibiotics (Basel) 9:32. 10.3390/antibiotics9010032. PubMed DOI PMC

Quigley EMM. 2019. Prebiotics and probiotics in digestive health. Clin Gastroenterol Hepatol 17:333–344. 10.1016/j.cgh.2018.09.028. PubMed DOI

Collado M, Isolauri E, Salminen S, Sanz Y. 2009. The impact of probiotic on gut health. Curr Drug Metab 10:68–78. 10.2174/138920009787048437. PubMed DOI

Schroeder B, Duncker S, Barth S, Bauerfeind R, Gruber AD, Deppenmeier S, Breves G. 2006. Preventive effects of the probiotic PubMed DOI

Krause DO, Bhandari SK, House JD, Nyachoti CM. 2010. Response of nursery pigs to a synbiotic preparation of starch and an anti- PubMed DOI PMC

Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev 71:158–229. 10.1128/MMBR.00036-06. PubMed DOI PMC

Duquesne S, Destoumieux-Garzón D, Peduzzi J, Rebuffat S. 2007. Microcins, gene-encoded antibacterial peptides from enterobacteria. Nat Prod Rep 24:708–734. 10.1039/b516237h. PubMed DOI

Micenková L, Bosák J, Štaudová B, Kohoutová D, Čejková D, Woznicová V, Vrba M, Ševčíková A, Bureš J, Šmajs D. 2016. Microcin determinants are associated with B2 phylogroup of human fecal PubMed DOI PMC

Bosák J, Hrala M, Pirková V, Micenková L, Čížek A, Smola J, Kučerová D, Vacková Z, Budinská E, Koláčková I, Šmajs D. 2019. Porcine pathogenic PubMed DOI

Setia A, Bhandari SK, House JD, Nyachoti CM, Krause DO. 2009. Development and PubMed DOI

Bhandari SK, Opapeju FO, Krause DO, Nyachoti CM. 2010. Dietary protein level and probiotic supplementation effects on piglet response to DOI

Khafipour E, Munyaka PM, Nyachoti CM, Krause DO, Rodriguez-Lecompte JC. 2014. Effect of crowding stress and PubMed DOI

Salajka E, Salajkova Z, Alexa P, Hornich M. 1992. Colonization factor different from K88, K99, F41 and 987P in enterotoxigenic PubMed DOI

Dubreuil JD, Isaacson RE, Schifferli DM. 8 September 2016, posting date. Animal enterotoxigenic PubMed DOI PMC

Luise D, Lauridsen C, Bosi P, Trevisi P. 2019. Methodology and application of PubMed DOI PMC

Wasteson Y. 2001. Zoonotic PubMed

Kaufmann M, Zweifel C, Blanco M, Blanco JE, Blanco J, Beutin L, Stephan R. 2006. PubMed DOI

Stahl CH, Callaway TR, Lincoln LM, Lonergan SM, Genovese KJ. 2004. Inhibitory activities of colicins against PubMed DOI PMC

Pilsl H, Šmajs D, Braun V. 1999. Characterization of colicin S4 and its receptor, OmpW, a minor protein of the PubMed DOI PMC

Šmajs D, Weinstock GM. 2001. The iron- and temperature-regulated PubMed DOI PMC

Šmajs D, Weinstock GM. 2001. Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes. J Bacteriol 183:3949–3957. 10.1128/JB.183.13.3949-3957.2001. PubMed DOI PMC

Bosák J, Micenková L, Doležalová M, Šmajs D. 2016. Colicins U and Y inhibit growth of PubMed DOI

Schamberger GP, Phillips RL, Jacobs JL, Diez-Gonzalez F. 2004. Reduction of PubMed DOI PMC

Budič M, Rijavec M, Petkovšek Ž, Žgur-Bertok D. 2011. PubMed DOI PMC

Mazurek-Popczyk J, Pisarska J, Bok E, Baldy-Chudzik K. 2020. Antibacterial activity of bacteriocinogenic commensal PubMed DOI PMC

Šmajs D, Pilsl H, Braun V. 1997. Colicin U, a novel colicin produced by PubMed DOI PMC

Riley MA, Cadavid L, Collett MS, Neely MN, Adams MD, Phillips CM, Neel JV, Friedman D. 2000. The newly characterized colicin Y provides evidence of positive selection in pore-former colicin diversification. Microbiology 146:1671–1677. 10.1099/00221287-146-7-1671. PubMed DOI

Šmajs D, Matějková P, Weinstock GM. 2006. Recognition of pore-forming colicin Y by its cognate immunity protein. FEMS Microbiol Lett 258:108–113. 10.1111/j.1574-6968.2006.00201.x. PubMed DOI

Šmajs D, Doležalová M, Macek P, Žídek L. 2008. Inactivation of colicin Y by intramembrane helix-helix interaction with its immunity protein. FEBS J 275:5325–5331. 10.1111/j.1742-4658.2008.06662.x. PubMed DOI

Carlos C, Pires MM, Stoppe NC, Hachich EM, Sato MIZ, Gomes TAT, Amaral LA, Ottoboni LMM. 2010. PubMed DOI PMC

Micenková L, Bosák J, Vrba M, Ševčíková A, Šmajs D. 2016. Human extraintestinal pathogenic PubMed DOI PMC

Gordon DM, Cowling A. 2003. The distribution and genetic structure of PubMed DOI

Scaldaferri F, Gerardi V, Mangiola F, Lopetuso LR, Pizzoferrato M, Petito V, Papa A, Stojanovic J, Poscia A, Cammarota G, Gasbarrini A. 2016. Role and mechanisms of action of PubMed DOI PMC

Cordonnier C, Thévenot J, Etienne-Mesmin L, Alric M, Livrelli V, Blanquet-Diot S. 2017. Probiotic and enterohemorrhagic PubMed DOI

Cutler SA, Lonergan SM, Cornick N, Johnson AK, Stahl CH. 2007. Dietary inclusion of colicin E1 is effective in preventing postweaning diarrhea caused by F18-positive PubMed DOI PMC

Sato T, Hamabata T, Takita E, Matsui T, Sawada K, Imaoka T, Nakanishi N, Nakayama K, Tsukahara T. 2017. Improved porcine model for Shiga toxin-producing PubMed DOI

Wang J, Ji H, Wang S, Liu H, Zhang W, Zhang D, Wang Y. 2018. Probiotic PubMed DOI PMC

Cheng MP, Domingo MC, Lévesque S, Yansouni CP. 2016. A case report of a deep surgical site infection with PubMed DOI PMC

Burrough ER, Arruda BL, Plummer PJ. 2017. Comparison of the luminal and mucosa-associated microbiota in the colon of pigs with and without swine dysentery. Front Vet Sci 4:139. 10.3389/fvets.2017.00139. PubMed DOI PMC

Kiros TG, Derakhshani H, Pinloche E, D’Inca R, Marshall J, Auclair E, Khafipour E, Van Kessel A. 2018. Effect of live yeast PubMed DOI PMC

Šmajs D, Bureš J, Šmarda J, Chaloupková E, Květina J, Förstl M, Kohoutová D, Kuneš M, Rejchrt S, Lesná J, Kopáčová M. 2012. Experimental administration of the probiotic PubMed DOI

Suzzi G, Tofalo R, Golí N, Veljoví K, Diní M, Lukí J, Mihajloví S, Tolinački M, Tolinački T, Živkoví M, Begoví J, Mrvaljeví I, Terzí C-Vidojeví A. 2017. Promotion of early gut colonization by probiotic intervention on microbiota diversity in pregnant sows. Front Microbiol 8:2028. 10.3389/fmicb.2017.02028. PubMed DOI PMC

Shin D, Chang SY, Bogere P, Won KH, Choi JY, Choi YJ, Lee HK, Hur J, Park BY, Kim Y, Heo J. 2019. Beneficial roles of probiotics on the modulation of gut microbiota and immune response in pigs. PLoS One 14:e0220843. 10.1371/journal.pone.0220843. PubMed DOI PMC

Clermont O, Bonacorsi S, Bingen E. 2000. Rapid and simple determination of the PubMed DOI PMC

Zajacova ZS, Konstantinova L, Alexa P. 2012. Detection of virulence factors of PubMed DOI

Šmarda J. 1963. Lysogenie a colicinogenie bakterií Escherichia coli. PhD thesis. Masaryk University, Brno, Czech Republic.

Šmarda J, Obdržálek V. 1966. Colicine Q. Zentralbl Bakteriol Orig 200:493–497. PubMed

Šmarda J, Šmajs D. 1998. Colicins—exocellular lethal proteins of PubMed DOI

Alexa P, Rychlík I, Nejezchleb A, Hamřík J. 1997. Identification of enterotoxin-producing strains of PubMed

Bosák J, Micenková L, Vrba M, Ševčíková A, Dědičová D, Garzetti D, Šmajs D. 2013. Unique activity spectrum of colicin F PubMed DOI PMC

Patzer SI, Baquero MR, Bravo D, Moreno F, Hantke K. 2003. The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 149:2557–2570. 10.1099/mic.0.26396-0. PubMed DOI

Eraso JM, Chidambaram M, Weinstock GM. 1996. Increased production of colicin E1 in stationary phase. J Bacteriol 178:1928–1935. 10.1128/jb.178.7.1928-1935.1996. PubMed DOI PMC

Kuhar I, Žgur-Bertok D. 1999. Transcription regulation of the colicin K PubMed DOI PMC

Moreno F, Gónzalez-Pastor JE, Baquero MR, Bravo D. 2002. The regulation of microcin B, C and J operons. Biochimie 84:521–529. 10.1016/S0300-9084(02)01452-9. PubMed DOI

Trckova M, Lorencova A, Hazova K, Sramkova Zajacova Z. 2016. Prophylaxis of post-weaning diarrhoea in piglets by zinc oxide and sodium humate. Vet Med (Praha) 60:351–360. 10.17221/8382-VETMED. DOI

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. 10.1093/bioinformatics/btu170. PubMed DOI PMC

Aronesty E. 2011. Command-line tools for processing biological sequencing data. https://expressionanalysis.github.io/ea-utils/.

Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461. 10.1093/bioinformatics/btq461. PubMed DOI

Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pe˜a AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. 10.1038/nmeth.f.303. PubMed DOI PMC

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. 2013. The SILVA ribosomal RNA gene database project: improved data processing and Web-based tools. Nucleic Acids Res 41:D590–D596. 10.1093/nar/gks1219. PubMed DOI PMC

Gentleman R. 2008. R programming for bioinformatics. CRC Press, Boca Raton, FL.

R Core Team. 2019. R: the R project for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.r-project.org/.

Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H. 2017. Vegan: community ecology package. R package version 2.4-3. https://cran.r-project.org/web/packages/vegan/index.html.

Wickham H. 2016. ggplot2: elegant graphics for data analysis. Springer-Verlag, New York, NY.

Complete genome sequences of five Escherichia coli strains with probiotic attributes