{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T00:24:49Z","timestamp":1793406289775,"version":"4.5.0"},"reference-count":46,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T00:00:00Z","timestamp":1767744000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004725","name":"Ministry of Economic Affairs","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004725","name":"Ministry of Economic Affairs","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004725","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001663","name":"Volkswagen Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001663","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    Food safety continues to be an important issue for consumer protection and public health globally. Chicken meat is considered a primary source of\n                    <jats:italic>Salmonella<\/jats:italic>\n                    and\n                    <jats:italic>E. coli<\/jats:italic>\n                    infections in humans. In recent years, phage-based biocontrol has attracted attention as a promising approach to combat these foodborne pathogens due to its advantages over traditional methods and its biological properties as a natural bactericide. Using phage-based control as a decontamination method to ensure microbial safety of food aligns with the One Health strategy for sustainable pathogen control and prevention of foodborne infections. This study aimed to develop and evaluate the effectiveness of a three-phage cocktail with optimized efficacy for simultaneously controlling\n                    <jats:italic>Salmonella<\/jats:italic>\n                    and\n                    <jats:italic>E. coli<\/jats:italic>\n                    on raw chicken filets during cold storage. To optimize the efficacy of the final phage cocktail, three phages were selected according to their host ranges and the efficiency of plating (EOP) values. They were combined in a cocktail, and the host range was expanded using the Appelmans protocol for 30 training cycles. The antibacterial efficacy of the trained three-phage cocktail was evaluated in liquid culture using a planktonic killing assay (PKA) and on raw chicken filets stored at 4\u202f\u00b1\u202f0.5\u202f\u00b0C for 72\u202fh, employing multiplicities of infection (MOI) of 1, 10, and 100 for targeting filets contaminated with single\n                    <jats:italic>Salmonella<\/jats:italic>\n                    and\n                    <jats:italic>E. coli<\/jats:italic>\n                    strains and a mixture of both. After training according to the Appelmans protocol, the cocktail showed an expanded host range, covering 62.5% (5\/8 after 30 training cycles) instead of 37.5% (3\/8 before training) of the tested bacteria. The planktonic killing assay demonstrated that the trained three-phage cocktail had a significant inhibitory effect on bacterial growth of the\n                    <jats:italic>Salmonella<\/jats:italic>\n                    strains (4\/4, 100%) from 3 to 6\u202fh, while the non-trained initial three-phage cocktail\u2019s effect was less pronounced (1\/4, 25%) and lasted only 3\u202fh. However, three of four\n                    <jats:italic>E. coli<\/jats:italic>\n                    strains (75%) were not sensitive to the three-phage cocktail after 30\u202fcycles of the Appelmans protocol compared to two out of four strains (50%) with the non-trained initial three-phage cocktail. On raw chicken filets, significant bacterial reduction was observed when using MOI 10 and 100 of the trained three-phage cocktail. A maximum reduction of 1.56 log\n                    <jats:sub>10<\/jats:sub>\n                    CFU\/mL of\n                    <jats:italic>Salmonella<\/jats:italic>\n                    BfR 20-SA00418 and 1.48 log\n                    <jats:sub>10<\/jats:sub>\n                    CFU\/mL of\n                    <jats:italic>E. coli<\/jats:italic>\n                    19\/302\/1\/A after 72\u202fh compared to placebo-treated controls were achieved using an MOI of 100. We observed a synergistic effect of the three-phage cocktail compared to single treatment, with a stronger effect on\n                    <jats:italic>Salmonella<\/jats:italic>\n                    than on\n                    <jats:italic>E. coli<\/jats:italic>\n                    strains. Using the Appelmans protocol improved the effects of the developed three-phage cocktail, leading to broader pathogen coverage. The efficacy of the developed three-phage cocktail under cold storage conditions and its ability to reduce the bacterial load in raw chicken filets highlighted its potential for extending shelf life and reducing risks for the consumer. The findings of this study demonstrate that the developed and optimized three-phage cocktail is a promising biocontrol agent for enhancing safety in raw chicken meat production.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2025.1696826","type":"journal-article","created":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T06:59:25Z","timestamp":1767769165000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Phage-based biocontrol of Salmonella and E. coli in raw chicken filets: optimizing phage-based solutions to enhance food safety under cooled storing conditions"],"prefix":"10.3389","volume":"16","author":[{"given":"Oleksandra","family":"Berhilevych","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elisa","family":"Peh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Johanna Charlotte","family":"Vahle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madeleine","family":"Pl\u00f6tz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophie","family":"Kittler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,1,7]]},"reference":[{"key":"ref9001","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/ph14101019","article-title":"Phage Cocktail Development for Bacteriophage Therapy: Toward Improving Spectrum of Activity Breadth and Depth","volume":"14","author":"Abedon","year":"2021"},{"key":"ref9002","doi-asserted-by":"publisher","first-page":"779","DOI":"10.3389\/fmicb.2020.00779","article-title":"Examination of the Use of Bacteriophage as an Additive and Determining Its Best Application Method to Control Listeria monocytogenes in a Cooked-Meat Model System","volume":"11","author":"Ahmadi","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref1","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1089\/phage.2021.0017","article-title":"Using phages to reduce Salmonella prevalence in chicken meat: a systematic review","volume":"3","author":"Almutairi","year":"2022","journal-title":"Phage (New Rochelle, N.Y.)"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"2348","DOI":"10.3390\/v13122348","article-title":"Bacteriophages as an alternative method for control of zoonotic and foodborne pathogens","volume":"13","author":"Alomari","year":"2021","journal-title":"Viruses"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"549","DOI":"10.3390\/v17040549","article-title":"Harnessing the activity of lytic bacteriophages to foster the sustainable development goals and the \u201cone health\u201d strategy","volume":"17","author":"\u00c1lvarez","year":"2025","journal-title":"Viruses"},{"key":"ref4","doi-asserted-by":"publisher","DOI":"10.1073\/pnas","article-title":"How to train your bacteriophage","volume":"118","author":"Borges","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref5","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2104592118","article-title":"Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance","volume":"118","author":"Borin","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref9003","doi-asserted-by":"publisher","DOI":"10.1515\/ijafr-2016-0007","article-title":"Effect of a bacteriophage cocktail in combination with modified atmosphere packaging in controlling Listeria monocytogenes on fresh-cut spinach","volume":"55","author":"Boyacioglu","year":"2016","journal-title":"Ir. j. agric. food res."},{"key":"ref6","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics11121709","article-title":"Modeling the directed evolution of broad host range phages","volume":"11","author":"Bull","year":"2022","journal-title":"Antibiotics (Basel, Switzerland)"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"2734","DOI":"10.3390\/foods12142734","article-title":"Bacteriophages for the targeted control of foodborne pathogens","volume":"12","author":"Bumunang","year":"2023","journal-title":"Foods (Basel, Switzerland)"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"241","DOI":"10.3390\/v1103024","article-title":"Directed in vitro evolution of therapeutic bacteriophages: the Appelmans protocol","volume":"11","author":"Burrowes","year":"2019","journal-title":"Viruses"},{"key":"ref9004","doi-asserted-by":"publisher","DOI":"10.4161\/bact.22825","article-title":"Bacteriophage cocktail significantly reduces Escherichia coli O157: H7 contamination of lettuce and beef, but does not protect against recontamination","volume":"2","author":"Carter","year":"2012","journal-title":"Bacteriophage."},{"key":"ref9005","doi-asserted-by":"publisher","first-page":"769","DOI":"10.2217\/fmb.13.47","article-title":"Phage cocktails and the future of phage therapy","volume":"8","author":"Chan","year":"2013","journal-title":"Future Microbiol."},{"key":"ref9006","doi-asserted-by":"publisher","first-page":"94","DOI":"10.3390\/microorganisms7040094","article-title":"Efficiency of single phage suspensions and phage cocktail in the inactivation of escherichia coli and salmonella typhimurium: an in vitro preliminary study","volume":"7","author":"Costa","year":"2019","journal-title":"Microorganisms."},{"key":"ref9","volume-title":"Eliava Institute of Bacteriophage, microbiology and virology, Tbilisi, Georgia. A literature review of the practical application of bacteriophage research","author":"Chanishvili","year":"2009"},{"key":"ref9007","doi-asserted-by":"publisher","first-page":"185","DOI":"10.1016\/j.copbio.2017.09.004","article-title":"Phages as friends and enemies in food processing","volume":"49","author":"de Melo","year":"2018","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"96","DOI":"10.1016\/j.copbio.2020.10.003","article-title":"Looking into the future of phage-based control of zoonotic pathogens in food and animal production","volume":"68","author":"Gambino","year":"2021","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"1354696","DOI":"10.3389\/fmicb.2024.1354696","article-title":"A bacteriophage cocktail can efficiently reduce five important Salmonella serotypes both on chicken skin and stainless steel","volume":"15","author":"Gvaladze","year":"2024","journal-title":"Front. Microbiol."},{"key":"ref12","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.613529","article-title":"Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria","volume":"12","author":"Haines","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"1324","DOI":"10.3390\/microorganisms10071324","article-title":"Phage products for fighting antimicrobial resistance","volume":"10","author":"Huang","year":"2022","journal-title":"Microorganisms"},{"key":"ref14","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics11111536","article-title":"Recent advances in the application of bacteriophages against common foodborne pathogens","volume":"11","author":"Hyla","year":"2022","journal-title":"Antibiotics (Basel, Switzerland)"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"841","DOI":"10.3390\/v11090841","article-title":"Application of a phage cocktail for control of Salmonella in foods and reducing biofilms","volume":"11","author":"Islam","year":"2019","journal-title":"Viruses"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"2153","DOI":"10.1007\/s11274-014-1655-7","article-title":"Natural solution to antibiotic resistance: bacteriophages 'the living drugs'","volume":"30","author":"Jassim","year":"2014","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref17","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.011855","article-title":"Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy","volume":"10","author":"Khan Mirzaei","year":"2015","journal-title":"PLoS One"},{"key":"ref9010","doi-asserted-by":"publisher","first-page":"1594533","DOI":"10.3389\/fmicb.2025.1594533","article-title":"Bacteriophage cocktail LEC2-LEC10 for broad-spectrum control of pathogenic and uncharacterized Escherichia coli in fresh produce","volume":"16","author":"Kim","year":"2025","journal-title":"Front. Microbiol."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"526","DOI":"10.1111\/1750-3841.15042","article-title":"Characterization of Salmonella spp.-specific bacteriophages and their biocontrol application in chicken breast meat","volume":"85","author":"Kim","year":"2020","journal-title":"J. Food Sci."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"293","DOI":"10.3390\/pathogens9040293","article-title":"Impact of bacteriophage-supplemented drinking water on the E. coli population in the chicken gut","volume":"9","author":"Kittler","year":"2020","journal-title":"Pathogens (Basel, Switzerland)"},{"key":"ref20","doi-asserted-by":"publisher","first-page":"1470","DOI":"10.3390\/v12121470","article-title":"In vitro evaluation of a phage cocktail controlling infections with Escherichia coli","volume":"12","author":"Korf","year":"2020","journal-title":"Viruses"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"562","DOI":"10.56093\/ijans.v87i5.70222","article-title":"Isolation and characterization of Salmonella bacteriophages from poultry and pig sewage wastes","volume":"87","author":"Kumar","year":"2017","journal-title":"Indian J. Anim. Sci."},{"key":"ref22","doi-asserted-by":"publisher","DOI":"10.1093\/femsml\/uqae003","article-title":"Multireceptor phage cocktail against Salmonella enterica to circumvent phage resistance","volume":"5","author":"Martinez-Soto","year":"2024","journal-title":"MicroLife"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"339","DOI":"10.1016\/j.lwt.2016.04.013","article-title":"Isolation and bio-control of extended spectrum beta-lactamase (ESBL)-producing Escherichia coli contamination in raw chicken meat by using lytic bacteriophages","volume":"71","author":"Minh","year":"2016","journal-title":"Food Sci Technol"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"209","DOI":"10.1186\/s12866-023-02963-1","article-title":"Bacteriophage cocktail application for Campylobacter mitigation - from in vitro to in vivo","volume":"23","author":"Peh","year":"2023","journal-title":"BMC Microbiol."},{"key":"ref9008","doi-asserted-by":"publisher","DOI":"10.3934\/microbiol.2019.4.324","article-title":"Bacteriophages-a new hope or a huge problem in the food industry","volume":"5","author":"Po\u0142aska","year":"2019","journal-title":"AIMS Microbiol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"270","DOI":"10.22099\/IJVR.2022.41490.6030","article-title":"Biocontrol of Escherichia coli and Salmonella in poultry meat using phage cocktail","volume":"23","author":"Poojari","year":"2022","journal-title":"Iran J Vet Res."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"178","DOI":"10.3390\/v10040178","article-title":"Expert opinion on three phage therapy related topics: bacterial phage resistance, phage training and prophages in bacterial production strains","volume":"10","author":"Rohde","year":"2018","journal-title":"Viruses"},{"key":"ref27","doi-asserted-by":"publisher","first-page":"186","DOI":"10.5851\/kosfa.2016.36.2.186","article-title":"Inhibiting the growth of Escherichia coli O157:H7 in beef, pork, and chicken meat using a bacteriophage","volume":"36","author":"Seo","year":"2016","journal-title":"Korean J. Food Sci. Anim. Resour."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"e0142421","DOI":"10.1128\/AEM.01424-21","article-title":"The lytic Siphophage vB_StyS-LmqsSP1 reduces the number of Salmonella enterica serovar typhimurium isolates on chicken skin","volume":"87","author":"Shakeri","year":"2021","journal-title":"Appl. Environ. Microbiol."},{"key":"ref9009","doi-asserted-by":"publisher","first-page":"e14870","DOI":"10.1016\/j.heliyon.2023.e14870","article-title":"A systematic review and modeling of the effect of bacteriophages on Salmonella spp. Reduction in chicken meat","volume":"9","author":"Shahdadi","year":"2023","journal-title":"Heliyon"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"8","DOI":"10.1016\/j.ijfoodmicro.2015.04.025","article-title":"Reduction of Salmonella on chicken meat and chicken skin by combined or sequential application of lytic bacteriophage with chemical antimicrobials","volume":"207","author":"Sukumaran","year":"2015","journal-title":"Int. J. Food Microbiol."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"1505805","DOI":"10.3389\/fmicb.2024.1505805","article-title":"Evaluating the effectiveness and safety of a novel phage cocktail as a biocontrol of Salmonella in biofilm, food products, and broiler chicken","volume":"15","author":"Torkashvand","year":"2024","journal-title":"Front. Microbiol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"199272","DOI":"10.1016\/j.virusres.2023.199272","article-title":"Appelmans protocol - a directed in vitro evolution enables induction and recombination of prophages with expanded host range","volume":"339","author":"Vu","year":"2024","journal-title":"Virus Res."},{"key":"ref9011","doi-asserted-by":"publisher","first-page":"e25697","DOI":"10.4161\/bact.25697","article-title":"Bacteriophages lytic for rapidly reduce contamination on glass and stainless steel surfaces","volume":"3","author":"Woolston","year":"2013","journal-title":"Bacteriophage."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"327","DOI":"10.3389\/fmicb.2020.00327","article-title":"Development of a bacteriophage cocktail to constrain the emergence of phage-resistant Pseudomonas aeruginosa","volume":"11","author":"Yang","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref33","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.01258-23","article-title":"Designing phage cocktails to combat the emergence of bacteriophage-resistant mutants in multidrug-resistant Klebsiella pneumoniae","volume":"12","author":"Yoo","year":"2024","journal-title":"Microbiol. Spectr."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"2055","DOI":"10.1111\/eva.13260","article-title":"Overcoming the growth-infectivity trade-off in a bacteriophage slows bacterial resistance evolution","volume":"14","author":"Zhang","year":"2021","journal-title":"Evol. Appl."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"286","DOI":"10.3390\/v14020286","article-title":"A polyvalent broad-spectrum Escherichia phage tequatrovirus EP01 capable of controlling Salmonella and Escherichia coli contamination in foods","volume":"14","author":"Zhou","year":"2022","journal-title":"Viruses"}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1696826\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T06:59:27Z","timestamp":1767769167000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2025.1696826\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,7]]},"references-count":46,"alternative-id":["10.3389\/fmicb.2025.1696826"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2025.1696826","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,7]]},"article-number":"1696826"}}