Synergisric effects of bacteriophages and antibiotics against Shiga toxigenic Escherichia Coli isolatrd from diarrheic calves

Document Type : Research Article

Authors

Aseem Keder Albaker ¹

Maher Saleh ¹

Ashraf Alaaleh ²

¹ Department of Public Health and Preventive Medicine, Faculty of Veterinary Medicine, University of Hama, Hama, Syria.

² Department of microbiology, Faculty of Veterinary Medicine, University of Hama, Hama, Syria.

10.22067/ijvst.2026.96481.1617

Abstract

This study investigated the potential of coliphages as a means of decreasing the amount of antibiotics needed for treatment. Shigatoxigenic E.Coli (STEC) strains were isolated from diarrheic calves within the Hama governorate, Syria. Isolation was done with differential and selective media and biochemical confirmation. The presence of genes encoding Shiga toxins (stx1 and stx2) was confirmed through multiplex polymerase chain reaction (PCR). Antibiotic susceptibility testing of the bacterial isolates was also done. In addition, bacteriophages from wastewater from cattle barns were isolated and their synergistic potential was assessed when combined with the antibiotics streptomycin, oxytetracycline, and neomycin. The STEC isolates were resistant to antibiotics. The highest resistance rates were observed against amoxicillin (88%) and penicillin G (92%). Notably, the phage-antibiotic combination therapy proved highly effective since the addition of bacteriophages reduced the minimum inhibitory concentration (MIC) of streptomycin, oxytetracycline, and neomycin. In streptomycin, the combination MIC was significantly lower than that of streptomycin alone (40 µg/mL streptomycin + 6 log₁₀ PFU/mL phages versus 250 µg/mL streptomycin alone). Similarly, the MIC for the oxytetracycline-phage combination (40 µg/mL + phages) was markedly lower than that of oxytetracycline alone (250 µg/mL). For neomycin, the combination (10 µg/mL + phages) also showed a significant MIC reduction compared to neomycin alone (200 µg/mL).

Keywords

coli phage

PCR

MIC

Antibiotic Resistance

Virulence Factors

Subjects

• Microbiology

---

References

1.    Foster DM, Smith GW. Pathophysiology of diarrhea in calves. Vet Clin North Am Food Anim Pract. 2009;25:13–36. Doi:10.1016/j.cvfa.2008.10.013.
2.    Abdulridha RN, Ibrahim OMS. Activity Of Bacterial Antibiotics Against Some Pathogenic Bacteria Isolated From Calves Diarrhoea In Baghdad (PartI). Iraqi Journal of Agricultural Sciences. 2018;49(5):248-54. Doi:10.36103/ijas.v49i5.45.
3.    Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr. 2016;4(2):4-2. Doi: 10.1128/microbiolspec.VMBF-0016-2015.
4.    Sobierajski T, Mazińska B, Chajęcka-Wierzchowska W, Śmiałek M, Hryniewicz W. Antimicrobial and antibiotic resistance from the perspective of Polish veterinary students: an inter-university study. Antibiotics (Basel). 2022;11(1):115. Doi:10.3390/antibiotics11010115. 
5.    OIE. Antimicrobial Resistance. World Organization for Animal Health. 2015. www.oie.int. Accessed 22 May 2016.
6.    Al wendawi SH, Al Rekaby SM. The Effeciency Of Enteric Lactobacillus In Preventing Hemorrhagic Colitis And Blocking Shiga Toxins Productions In Rats Models Infected With Enterohemorrhagic Escherichia Coli (EHEC). Iraqi Journal of Agricultural Sciences. 2021;52(6):1346-55. Doi:10.36103/ijas.v52i6.1474.
7.    Majda B, Barchan AA, Aarab A, Bakkali M, Arakrak A, Laglaoui A. Evaluation Of The Antibacterial Activity Of Essential Oils Against E. Coli Isolated From Rabbits. Iraqi Journal of Agricultural Sciences. 2022;53(4):802-9. Doi:10.36103/ijas.v53i4.1592.
8.    Oechslin F. Resistance development to bacteriophages occurring during bacteriophage therapy. Viruses. 2018;10(7):1-23. Doi:10.3390/v10070351.
9.    Marwa Sh, Ibrahim MAH. Correlation Between Antibiotic Resistance Phenotypes With Mobile Genetic Elements (Integron Class I) In Uropathogenic E. Coli. Iraqi Journal of Agricultural Sciences. 2024;55(4):1280-91. Doi:10.36103/shxqpf87.
10.    Keen EC. A century of phage research: bacteriophages and the shaping of modern biology. Bioessays. 2015;37:6–9. Doi: 10.1002/bies.201400152.
11.    Hesse S, Adhya S. Phage therapy in the twenty-first century: facing the decline of the antibiotic era; is it finally time for the age of the phage? Annu Rev Microbiol. 2019;73:155–74. Doi:10.1146/annurev-micro-090817-062535.
12.    McCallin S, Sacher JC, Zheng J, Chan BK. Current state of compassionate phage therapy. Viruses. 2019;11:1–14. Doi: 10.3390/v11040343.
13.    Payne RJ, Phil D, Jansen VA. Phage therapy: the peculiar kinetics of self-replicating pharmaceuticals. Clin Pharmacol Ther. 2000;68:225–30. Doi:10.1067/mcp.2000.109520.
14.    FDA (US Food and Drug Administration). Food additives permitted for direct addition to food for human consumption; bacteriophage preparation. Silver Spring, MD: US Food and Drug Administration; 2006. 
15.    Wang L, Bai Y, Shi X, Zheng X, Liu Y, Zhang Y, et al. The Virulence Gene and Drug Resistance Analysis of Shiga Toxin-Producing Escherichia coli from Diarrhea Calves in Xinjiang, China. Foodborne Pathog Dis. 2025. Doi:10.1089/fpd.2024.0056.
16.    Nguyen TD, Vo TT, Vu-Khac H. Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam. J Vet Sci. 2011;12(2):159-64. Doi:10.4142/jvs.2011.12.2.159.
17.    Cengiz S, Adigüzel MC. Determination of virulence factors and antimicrobial resistance of E. coli isolated from calf diarrhea, part of eastern Turkey. Ankara Univ Vet Fak Derg. 2020;67(3):365–71. Doi:10.33988/auvfd.640990.
18.    Wang Z, Sun M, Guo S, Wang Y, Meng L, Shi J, et al. Detection of drug resistance in Escherichia coli from calves with diarrhea in the Tongliao region: an analysis of multidrug-resistant strains. Front Vet Sci. 2024;11:1466690. Doi:10.3389/fvets.2024.1466690.
19.    Ibrahim HS, Abdelhamed GS, Elgohary AH, Fathi M. Relationship between virulence and antibiotic resistant genes in some Gram negative bacteria causing diarrhea in calves. J Adv Vet Res. 2025;15(1):6-12. Available from: https://advetresearch.com/index.php/AVR/article/view/2055.
20.    Narulita E, Aji GP, Wahono B, Murdiyah S, Yulian R. Synergism of Phage фPT1b and Antibiotics for Reducing Infection of Escherichia coli. Biogenesis. 2020;8:22-8. Doi:10.24252/bio.v8i1.11280.
21.    Moradpour Z, Yousefi N, Sadeghi D, Ghasemian A. Synergistic bactericidal activity of a naturally isolated phage and ampicillin against urinary tract infecting Escherichia coli O157. Iran J Basic Med Sci. 2020;23:257-63. Doi:10.22038/ijbms.2019.37561.8989.
22.    Shamsuzzaman M, Kim S, Kim J. Therapeutic potential of novel phages with antibiotic combinations against ESBL-producing and carbapenem-resistant Escherichia Coli. J Glob Antimicrob Resist. 2025;43:86–97. DOI: https://doi.org/10.1016/j.jgar.2025.04.005.
23.    Supina BSI, Dennis JJ. The Current Landscape of Phage–Antibiotic Synergistic (PAS) Interactions. Antibiotics. 2025;14:545. Doi:10.3390/antibiotics14060545.
24.    Chan BK, Sistrom M, Wertz JE, Kortright KE, Narayan D, Turner PE. Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa. Sci Rep. 2016;6:26717. Doi:10.1038/srep26717. 
25.    Chen Q, Dharmaraj T, Cai PC, Burgener EB, Haddock NL, Spakowitz AJ, et al. Bacteriophage and bacterial susceptibility, resistance, and tolerance to antibiotics. Pharmaceutics. 2022;14(7):1425. Doi:10.3390/pharmaceutics14071425.
26.    Ruest MK, Supina BSI, Dennis JJ. Bacteriophage steering of Burkholderia cenocepacia toward reduced virulence and increased antibiotic sensitivity. J Bacteriol. 2023;205(12):e00196-23. Doi:10.1128/jb.00196-23. 
27.    Wang X, Loh B, Gordillo Altamirano F, Yu Y, Hua X, Leptihn S. Colistin-phage combinations decrease antibiotic resistance in Acinetobacter baumannii via changes in envelope architecture. Emerg Microbes Infect. 2021;10(1):2205-19. Doi:10.1080/22221751.2021.2002671. 
28.    Qin K, Shi X, Yang K, Xu Q, Wang F, Chen S, et al. Phage-antibiotic synergy suppresses resistance emergence of Klebsiella pneumoniae by altering the evolutionary fitness. mBio. 2024;15(2):e01393-24. Doi:10.1128/mbio.01393-24. 
29.    Gibbons JF, Boland F, Buckley JF, et al. Patterns of antimicrobial resistance in pathogenic Escherichia coli isolates from cases of calf enteritis during the spring-calving season. Vet Microbiol. 2014;170:73-80. Doi:10.1016/j.vetmic.2014.01.010.
30.    Franck SM, Bosworth BT, Moon HW. Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves. J Clin Microbiol. 1998;36(6):1795–7. Doi:10.1128/jcm.36.6.1795-1797.1998.
31.    CLSI. Performance standards for antimicrobial susceptibility testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standard Institute; 2020.
32.    Beaudoin R, DeCesaro D, Durkee D, Barbaro S. Isolation of a Bacteriophage From Sewage Sludge and characterization of its bacterial host cell. Rivier Academic Journal. 2007;3(1):1-8. 
33.    Alexyuk P, Bogoyavlenskiy A, Alexyuk M, Akanova K, Moldakhanov Y, Berezin V. Isolation and Characterization of Lytic Bacteriophages Active against Clinical Strains of E. coli and Development of a Phage Antimicrobial Cocktail. Viruses. 2022;14(11):2381. Doi:10.3390/v14112381.
34.    Kobayashi DY, Palumbo JD. Bacterial endophytes and their effects on plants and uses in agriculture. In: Bacon CW, White JF, editors. Microbial Endophytes. New York, USA: Marcel Dekker, Inc.; 2000.
35.    Pallavali RR, Degati VL, Lomada D, Reddy MC, Durbaka VRP. Isolation and in vitro evaluation of bacteriophages against MDR bacterial isolates from septic wound infections. PLoS One. 2017;12(7):1-16. Doi:10.1371/journal.pone.0179245.
36.    Santos SB, Carvalho CM, Sillankorva S, Nicolau A, Ferreira EC, Azeredo J. The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique. BMC Microbiol. 2009;9(1):1-10. Doi:10.1186/1471-2180-9-148.
37.    Gu Liu C, Green SI, Min L, Clark JR, Salazar KC, Terwilliger AL, et al. Phageantibiotic synergy is driven by a unique combination of antibacterial mechanism of action and stoichiometry. mBio. 2020;11:e01462-20. Doi:10.1128/mbio.01462-20.
38.    Cheesbrough M. Medical Laboratory Manual for Tropical Countries. 1st ed. London: English Language Book Society; 1987. p. 400–480.
39.    Moody, J. Synergism testing: Broth microdilution checkerboard and broth macrodilution method. In Clinical Microbiology Procedures Handbook; ASM Press: Washington, DC, USA, 2004; 1–28.
40.    Labrie SJ, Samson JE, Moineau S. Bacteriophage resistance mechanisms. Nat Rev Microbiol. 2010;8(5):317-27. 
41.    Andrade GI, Coura FM, Santos EL, Ferreira MG, Galinari GC, Facury Filho EJ, et al. Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil. Trop Anim Health Prod. 2012;44(7):1783-90. Doi:10.1007/s11250-012-0139-8.