Isolation, molecular characterization, and therapeutic evaluation of bacteriophages targeting shiga toxin-producing Escherichia coli (STEC)

HILARI  DEBBARMA; MOON MOON  SATPATHY; SANJANA; AMBIKA  ARUN; ABHISHEK; BABLU  KUMAR

doi:10.56093/ijans.v95i12.170345

Authors

HILARI DEBBARMA ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India
MOON MOON SATPATHY ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India
SANJANA ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India
AMBIKA ARUN ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India
ABHISHEK ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India
BABLU KUMAR ICAR-Indian Veterinary Research Institute, Bareilly- 243 122, Uttar Pradesh, India

https://doi.org/10.56093/ijans.v95i12.170345

Keywords:

Bacteriophage, Environment, Food safety, Milk, Stability, STEC, Sewage

Abstract

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen responsible for severe gastrointestinal infections in humans and animals. The present study aimed to isolate and characterize bacteriophages targeting STEC and evaluate their potential for bacterial control. Twenty-two E. coli isolates were revived and confirmed using cultural, biochemical, and molecular methods. Molecular analysis showed that 86.36% of isolates carried the stx1 (Shiga toxin 1) gene and 50% carried the eae (Escherichia coli attaching and effacing) gene, while stx2 was not detected. Thirteen bacteriophages (Ecp1–Ecp13) were isolated from farm environments and hospital sewage, of which twelve exhibited lytic activity against STEC isolates. Five phages showing broader lytic spectra were further characterized. Transmission electron microscopy identified them as members of the order Caudovirales, belonging to the families Podoviridae and Siphoviridae. The phages remained stable across temperatures from −20°C to 37°C and within pH 4–9, with optimal stability at 4°C and pH 7.5 Time-kill analysis demonstrated rapid bacterial reduction by phage Ecp10, and its application in experimentally contaminated milk significantly reduced STEC counts compared to untreated controls. These findings highlight the potential of bacteriophages as natural and targeted biocontrol agents for reducing STEC contamination in food systems.

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References

Abdelrahman F, Rezk N, Fayez M S, Abdelmoteleb M, Atteya R, Elhadidy M and El-Shibiny A. 2022. Isolation, characterization, and genomic analysis of three novel E. coli bacteriophages that effectively infect E. coli O18. Microorganisms 10 (3): 589.

Abdelsattar A S, Safwat A, Nofal R, Elsayed A, Makky S and El-Shibiny A. 2021. Isolation and characterization of bacteriophage ZCSE6 against Salmonella spp.: Phage application in milk. Biologics 1 (2): 164–76.

Algammal A M, El-Kholy A W, Riad E M, Mohamed H E, Elhaig M M, Yousef S A A, Hozzein W N and Ghobashy M O. 2020. Genes encoding the virulence and the antimicrobial resistance in enterotoxigenic and shiga-toxigenic E. coli isolated from diarrheic calves. Toxins 12 (6): 383.

Ateba C N and Akindolire M A. 2019. Isolation and characterization of bacteriophages with lytic activity against virulent Escherichia coli O157:H7: Potential bio-control agents. African Journal of Biotechnology 18 (12): 355–365.

Chandra M, Thakur S, Narang D and Saxena H M. 2011. Isolation of a bacteriophage against Salmonella Dublin and determination of its physical resistance under varied in vitro conditions. African Journal of Microbiology Research 5(15): 2044-2047.

Chevallereau A, Pons BJ, van Houte S and Westra E R. 2022. Interactions between bacterial and phage communities in natural environments. Nature Reviews Microbiology 20: 49–62.

Fauconnier A. 2019. Phage therapy regulation: from night to dawn. Viruses 11 (4): 352.

García A, Fox JG, Besser TE. 2010. Zoonotic enterohemorrhagic Escherichia coli: A One Health perspective. ILAR Journal 51(3):221-232.

FAO/WHO. (2023). Shiga toxin-producing Escherichia coli (STEC) and food safety risk assessment. Food and Agriculture Organization / World Health Organization. Gordillo Altamirano F L and Barr J J. 2019. Phage therapy in the post antibiotic era. Clinical Microbiology Reviews 32(2):e00066-18.

Gonzalez G A and Cerqueira M A. 2020. Shiga toxin‐producing Escherichia coli in the animal reservoir and food in Brazil. Journal of Applied Microbiology 128 (6): 1568–1582.

Grygorcewicz B, Chajęcka-Wierzchowska W, Augustyniak A, Wasak A, Stachurska X, Nawrotek P and Dołęgowska B. 2020. In‐milk inactivation of Escherichia coli O157:H7 by the environmental lytic bacteriophage ECPS‐6. Journal of Food Safety 40 (2): e12747.

Hesse S, Adhya S. 2019. Phage therapy in the twenty-first century: facing the decline of the antibiotic era; Is it finally time for the age of the phage? Annual Review of Microbiology 73:155-174.

Hwang S B, Chelliah R, Kang J E, Rubab M, Banan-Mwine Daliri E, Elahi F and Oh D H. 2021. Role of recent therapeutic applications and the infection strategies of Shiga toxinproducing Escherichia coli. Frontiers in Cellular and Infection Microbiology 11: 614963.

Hyman P. 2019. Phages for phage therapy: isolation, characterization, and host range breadth. Pharmaceuticals 12 (1): 35.

Kim J S, Lee M S and Kim J H. 2020. Recent updates on outbreaks of Shiga toxin-producing Escherichia coli and its potential reservoirs. Frontiers in Cellular and Infection Microbiology

10: 273.

Korf I H, Meier-Kolthoff J P, Adriaenssens E M, Kropinski A M, Nimtz M, Rohde M, van Raaij M J and Wittmann J. 2019. Still something to discover: novel insights into Escherichia coli phage diversity and taxonomy. Viruses 11 (5): 454.

Lin D M, Koskella B and Lin H C. 2017. Phage therapy: An alternative to antibiotics in the age of multidrug resistance. World Journal of Gastrointestinal Pharmacology and Therapeutics 8(3): 162–173.

Mir R A, Schaut R G, Looft T, Allen H K, Sharma V K and Kudva I T. 2020. Recto-anal junction (RAJ) and fecal microbiomes of cattle experimentally challenged with Escherichia coli O157:H7. Frontiers in Microbiology 11: 522078.

Montso P K, Mlambo V and Ateba C N. 2019. Characterization of lytic bacteriophages infecting multidrug-resistant shiga toxigenic atypical Escherichia coli O177 isolates isolated from cattle feces. Frontiers in Public Health 7: 355.

Moye Z D, Woolston J and Sulakvelidze A. 2018. Bacteriophage applications for food production and processing. Viruses 10(4): 205.

Necel A, Bloch S, Nejman-Faleńczyk B, Grabski M, Topka G, Dydecka A, Kosznik-Kwaśnicka K, Grabowski Ł, Jurczak- Kurek A, Wołkowicz T and Węgrzyn G. 2020. Characterization of a bacteriophage, vB_Eco4M-7, that effectively infects many Escherichia coli O157 isolates. Scientific Reports 10 (1): 3743.

Newell D G and La Ragione R M. 2018. Enterohaemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): Where are we now regarding diagnostics and control strategies?.

Transboundary and Emerging Diseases 65: 49–71.

O’Sullivan L, Bolton D, McAuliffe O and Coffey A. 2020. The use of bacteriophages to control and detect pathogens in the dairy industry. International Journal of Dairy Technology 73 (1): 1–11.

Owusu-Kwarteng J, Akabanda F, Agyei D and Jespersen L. 2020. Microbial safety of milk production and fermented dairy products in Africa. Microorganisms 8 (5): 752.

Phongtang W, Choi G P, Chukeatirote E and Ahn J. 2019. Bacteriophage control of Salmonella Typhimurium in milk. Food Science and Biotechnology 28: 297–301.

Pinto G, Almeida C and Azeredo J. 2020. Bacteriophages to control Shiga toxin-producing E. coli–safety and regulatory challenges. Critical Reviews in Biotechnology 40 (8): 1081–1897.

Połaska M and Sokołowska B. 2019. Bacteriophages—a new hope or a huge problem in the food industry. AIMS Microbiology 5 (4): 324.

Połaska M and Sokołowska B. 2019. Bacteriophages—a new hope or a huge problem in the food industry. AIMS Microbiology 5(4): 324.

Poyil M M, Karuppiah P, Raja S S and Sasikumar P. 2022. Isolation, extraction, and characterization of Verotoxinproducing Escherichia coli O157:H7 from diarrheal stool samples. Sudan Journal of Medical Sciences 17 (1): 116–127.

Rehman S, Ali Z, Khan M, Bostan N and Naseem S. 2019. The dawn of phage therapy. Reviews in Medical Virology 29(4): e2041.

Sajeena T A M and Kalyanikutty S. 2024. Pathogenic Factors of Shiga Toxigenic Escherichia coli. Journal of Pure and Applied Microbiology 18(1) :46-63.

Sarowska J, Futoma-Koloch B, Jama-Kmiecik A, Frej-Madrzak M, Ksiazczyk M, Bugla-Ploskonska G and Choroszy-Krol I. 2019. Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathogens 11: 1-16.

Sjahriani T, Wasito E B and Tyasningsih W. 2021. Isolation and identification of Escherichia coli O157:H7 lytic bacteriophage from environment sewage. International Journal of Food Science 2021(1): 7383121.

Topka G, Bloch S, Nejman-Faleńczyk B, Gąsior T, Jurczak-Kurek A, Necel A, Dydecka A, Richert M, Węgrzyn G and Węgrzyn A. 2019. Characterization of bacteriophage vBEcoS-95, isolated from urban sewage and revealing extremely rapid lytic development. Frontiers in Microbiology 9 :3326.

Turner D, Kropinski AM and Adriaenssens EM. 2022. A Roadmap for Genome-Based Phage Taxonomy. Viruses 13(3):506.

Ullah A, Qamash T, Khan F A, Sultan A, Ahmad S, Abbas M, Khattak M A K, Begum N, Din S U, Jamil J and Kalsoom. 2021. Characterization of a coliphage AS1 isolated from sewage effluent in Pakistan. Brazilian Journal of Biology 82: e240943.

Yazdi M, Bouzari M, Ghaemi E A and Shahin K. 2020. Isolation, characterization and genomic analysis of a novel bacteriophage VB_EcoS-Golestan infecting multidrug-resistant Escherichia coli isolated from urinary tract infection. Scientific Reports 10 (1): 7690.

Zhu Z and Dimitrov D S. 2008. Construction of a large naïve human phage-displayed Fab library through one-step cloning. (In) Therapeutic Antibodies: Methods and Protocols (pp. 129- 142). Totowa, NJ: Humana Press.