Bacteriophage cocktails and bacterial coevolution: a systematic review of resistance prevention strategies against multidrug-resistant pathogens in sewage water
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Abstract
Background and Objectives: This systematic review aimed to determine whether phage cocktails and co-evolutionarily trained phages prevent or delay the emergence of resistance in multidrug-resistant (MDR) bacteria more effectively than single-phage therapy in sewage and wastewater-derived systems, and to characterize the underlying mechanisms and the maturity of the current evidence base.
Materials and Methods: Following PRISMA guidelines, a systematic review was performed using the PubMed and Scopus databases, with screening conducted via Rayyan Premium.
Results: Among 4,795 records, 20 studies (≈0.4%) fulfilled the inclusion criteria; this low fraction reflects a deliberately broad search combined with strict eligibility criteria targeting original experimental studies of phage activity against MDR pathogens specifically isolated from sewage or wastewater. Phage cocktails showed a wider range of host coverage, a delay in resistance development, and improved penetration of biofilms, particularly when paired with depolymerases or antibiotics. Phage co-evolution training resulted in a more prolonged suppression of multidrug-resistant bacteria.
Conclusion: Phage cocktails, especially those that are co-evolved and enhanced with enzymes, show potential for applications in wastewater; however, additional long-term and large-scale research is necessary.
1. Alaoui Mdarhri H, Benmessaoud R, Yacoubi H, Seffar L, Guennouni Assimi H, Hamam M, et al. Alternatives Therapeutic Approaches to Conventional Antibiotics: Advantages, Limitations and Potential Application in Medicine. Antibiotics (Basel) 2022; 11: 1826.
2. Singha B, Singh V, Soni V. Alternative therapeutics to control antimicrobial resistance: a general perspective. Front Drug Discov 2024; 4: 1385460.
3. Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022; 399: 629-655.
4. Lin P, Liu S, Cao Z, Zeng Y, Zhao Y, Li T, et al. An experimental study on the lytic bacteriophage MSP15 with wide-spectrum targeting methicillin-resistant Staphylococcus aureus. Virology 2025; 605: 110452.
5. Lin DM, Koskella B, Lin HC. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther 2017; 8: 162-173.
6. Wittebole X, De Roock S, Opal SM. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 2014; 5: 226-235.
7. Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med 2019; 25: 730-733.
8. Aranaga C, Pantoja LD, Martínez EA, Falco A. Phage Therapy in the Era of Multidrug Resistance in Bacteria: A Systematic Review. Int J Mol Sci 2022; 23: 4577.
9. Strathdee SA, Hatfull GF, Mutalik VK, Schooley RT. Phage Therapy: From Biological Mechanisms to Future Directions. Cell 2023; 186: 17-31.
10. Aghaee BL, Khan Mirzaei M, Alikhani MY, Mojtahedi A, Maurice CF. Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics. Viruses 2021; 13: 334.
11. Mayorga-Ramos A, Carrera-Pacheco SE, Barba-Ostria C, Guamán LP. Bacteriophage-mediated approaches for biofilm control. Front Cell Infect Microbiol 2024; 14: 1428637.
12. Nilsson AS. Phage therapy--constraints and possibilities. Ups J Med Sci 2014; 119: 192-198.
13. Bernheim A, Sorek R. The Pan-Immune System of Bacteria: Antiviral Defence as a Community Resource. Nat Rev Microbiol 2020; 18: 113-119.
14. Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, Keren M, et al. Systematic Discovery of Antiphage Defense Systems in the Microbial Pangenome. Science 2018; 359(6379): eaar4120.
15. Podlacha M, Gaffke L, Grabowski Ł, Mantej J, Grabski M, Pierzchalska M, et al. Bacteriophage DNA induces an interrupted immune response during phage therapy in a chicken model. Nat Commun 2024; 15: 2274.
16. Sadeqi S, Hashemi Shahraki A, Nikkhahi F, Javadi A, Amin Marashi SM. Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater. Iran J Microbiol 2022; 14: 395-401.
17. Reisoglu Ş, Aydin S. Bacteriophages as a promising approach for the biocontrol of antibiotic resistant pathogens and the reconstruction of microbial interaction networks in wastewater treatment systems: A review. Sci Total Environ 2023; 890: 164291.
18. Jansen M, Wahida A, Latz S, Krüttgen A, Häfner H, Buhl EM, et al. Enhanced Antibacterial Effect of the Novel T4-Like Bacteriophage KARL-1 in Combination with antibiotics against Multi-Drug-Resistant Acinetobacter baumannii. Sci Rep 2018; 8: 14140.
19. Borin JM, Lee JJ, Gerbino KR, Meyer JR. Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages. Evol Appl 2022; 16: 152-162.
20. Wang M, Wei J, Jiang L, Jiang L, Zhang J, He X, et al. Coevolutionary phage training and Joint application delays the emergence of phage resistance in Pseudomonas aeruginosa. Virus Evol 2023; 9(2): vead067.
21. Oechslin F. Resistance Development to Bacteriophages Occurring During Bacteriophage Therapy. Viruses 2018; 10: 351.
22. Friman VP, Soanes-Brown D, Sierocinski P, Molin S, Johansen HK, Merabishvili M, et al. Pre-Adapting Parasitic Phages to a Pathogen Leads to Increased Pathogen Clearance and Lowered Resistance Evolution with Pseudomonas aeruginosa Cystic Fibrosis Bacterial Isolates. J Evol Biol 2016; 29: 188-198.
23. Schmerer M, Molineux IJ, Bull JJ. Synergy as a Rationale for Phage Therapy Using Phage Cocktails. PeerJ 2014; 2: e590.
24. Pu M, Han P, Zhang G, Liu Y, Li Y, Li F, et al. Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae. Int J Mol Sci 2022; 23: 8040.
25. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 Statement: An Updated Guideline for Reporting Systematic Reviews. BMJ 2021; 373: n71.
26. Natarajan SP, Teh SH, Lin LC, Lin NT. In Vitro and In Vivo Assessments of Newly Isolated N4-Like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1. Int J Mol Sci 2024; 25: 9595.
27. Fang Q, Zong Z. Lytic Phages against ST11 K47 Carbapenem-Resistant Klebsiella pneumoniae and the Corresponding Phage Resistance Mechanisms. mSphere 2022; 7(2): e0008022.
28. Zaki BM, Fahmy NA, Aziz RK, Samir R, El-Shibiny A. Characterization and comprehensive genome analysis of novel bacteriophage, vB_Kpn_ZCKp20p, with lytic and anti-biofilm potential against clinical multidrug-resistant Klebsiella pneumoniae. Front Cell Infect Microbiol 2023; 13: 1077995.
29. Sun Y, Feng JQ, Tan YR, Zhou L, Lan T, Ma JY. Genomic and Biological Characterization of vB_PvuS_Pm34, a Novel Lytic Bacteriophage that Infects Proteus vulgaris. Genomics 2022; 114: 38-44.
30. Yang P, Shan B, Hu X, Xue L, Song G, He P, et al. Identification of a novel phage depolymerase against ST11 K64 carbapenem-resistant Klebsiella pneumoniae and its therapeutic potential. J Bacteriol 2025; 207(4): e0038724.
31. Fikadu A, Amankwah S, Alemu B, Alemu Y, Naga A, Tekle E, et al. Isolation and Phenotypic Characterization of Virulent Bacteriophages Against Multidrug-Resistant Escherichia coli and Its Phage-Resistant Variant from Sewage Sources. Infect Drug Resist 2024; 17: 293-303.
32. Nour El-Din HT, Kettal M, Granados Maciel JC, Beaudoin G, Oktay U, Hrapovic S, et al. Isolation, Characterization, and Genomic Analysis of Bacteriophages against Pseudomonas aeruginosa Clinical Isolates from Early and Chronic Cystic Fibrosis Patients for Potential Phage Therapy. Microorganisms 2025; 13: 511.
33. Senhaji-Kacha A, Bernabéu-Gimeno M, Domingo-Calap P, Aguilera-Correa JJ, Seoane-Blanco M, Otaegi-Ugartemendia S, et al. Isolation and characterization of two novel bacteriophages against carbapenem-resistant Klebsiella pneumoniae. Front Cell Infect Microbiol 2024; 14: 1421724.
34. Wang Q, Chen R, Liu H, Liu Y, Li J, Wang Y, et al. Isolation and characterization of lytic bacteriophage vB_KpnP_23: A promising antimicrobial candidate against carbapenem-resistant Klebsiella pneumoniae. Virus Res 2024; 350: 199473.
35. Yazdi M, Bouzari M, Ghaemi EA, Shahin K. Isolation, Characterization and Genomic Analysis of a Novel Bacteriophage VB_EcoS-Golestan Infecting Multidrug-Resistant Escherichia coli Isolated from Urinary Tract Infection. Sci Rep 2020; 10: 7690.
36. Badawy S, Baka ZAM, Abou-Dobara MI, El-Sayed AKA, Skurnik M. Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate. Arch Virol 2022; 167: 1333-1341.
37. Akremi I, Merabishvili M, Jlidi M, Haj Brahim A, Ben Ali M, Karoui A, et al. Isolation and Characterization of Lytic Pseudomonas aeruginosa Bacteriophages Isolated from Sewage Samples from Tunisia. Viruses 2022; 14: 2339.
38. Mirza KA, Tchatchiashvili T, Marquet M, Nietzsche S, Pletz MW, Makarewicz O. Characterization and genome analysis of novel Klebsiella pneumoniae phage vbKpUKJ_2 isolated from hospital sewage water. BMC Microbiol 2025; 25: 96.
39. Ponsecchi G, Olimpieri T, Poerio N, Antonelli A, Coppi M, Di Lallo G, et al. Characterization of four novel bacteriophages targeting multi-drug resistant Klebsiella pneumoniae strains of sequence type 147 and 307. Front Cell Infect Microbiol 2024; 14: 1473668.
40. Menon ND, Kumar MS, Satheesh Babu TG, Bose S, Vijayakumar G, Baswe M, et al. A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates. mSphere 2021; 6(1): e01215-20.
41. Oduor JMO, Kiljunen S, Kadija E, Mureithi MW, Nyachieo A, Skurnik M. Genomic characterization of four novel Staphylococcus myoviruses. Arch Virol 2019; 164: 2171-2173.
42. Chakraborty S, Rohit A, Prasanthi SJ, Chauhan A. A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates. Pharmaceutics 2024; 16: 904.
43. Chen H, Liu H, Gong Y, Dunstan RA, Ma Z, Zhou C, et al. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes 2024; 10: 127.
2. Singha B, Singh V, Soni V. Alternative therapeutics to control antimicrobial resistance: a general perspective. Front Drug Discov 2024; 4: 1385460.
3. Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022; 399: 629-655.
4. Lin P, Liu S, Cao Z, Zeng Y, Zhao Y, Li T, et al. An experimental study on the lytic bacteriophage MSP15 with wide-spectrum targeting methicillin-resistant Staphylococcus aureus. Virology 2025; 605: 110452.
5. Lin DM, Koskella B, Lin HC. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther 2017; 8: 162-173.
6. Wittebole X, De Roock S, Opal SM. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 2014; 5: 226-235.
7. Dedrick RM, Guerrero-Bustamante CA, Garlena RA, Russell DA, Ford K, Harris K, et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med 2019; 25: 730-733.
8. Aranaga C, Pantoja LD, Martínez EA, Falco A. Phage Therapy in the Era of Multidrug Resistance in Bacteria: A Systematic Review. Int J Mol Sci 2022; 23: 4577.
9. Strathdee SA, Hatfull GF, Mutalik VK, Schooley RT. Phage Therapy: From Biological Mechanisms to Future Directions. Cell 2023; 186: 17-31.
10. Aghaee BL, Khan Mirzaei M, Alikhani MY, Mojtahedi A, Maurice CF. Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics. Viruses 2021; 13: 334.
11. Mayorga-Ramos A, Carrera-Pacheco SE, Barba-Ostria C, Guamán LP. Bacteriophage-mediated approaches for biofilm control. Front Cell Infect Microbiol 2024; 14: 1428637.
12. Nilsson AS. Phage therapy--constraints and possibilities. Ups J Med Sci 2014; 119: 192-198.
13. Bernheim A, Sorek R. The Pan-Immune System of Bacteria: Antiviral Defence as a Community Resource. Nat Rev Microbiol 2020; 18: 113-119.
14. Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, Keren M, et al. Systematic Discovery of Antiphage Defense Systems in the Microbial Pangenome. Science 2018; 359(6379): eaar4120.
15. Podlacha M, Gaffke L, Grabowski Ł, Mantej J, Grabski M, Pierzchalska M, et al. Bacteriophage DNA induces an interrupted immune response during phage therapy in a chicken model. Nat Commun 2024; 15: 2274.
16. Sadeqi S, Hashemi Shahraki A, Nikkhahi F, Javadi A, Amin Marashi SM. Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater. Iran J Microbiol 2022; 14: 395-401.
17. Reisoglu Ş, Aydin S. Bacteriophages as a promising approach for the biocontrol of antibiotic resistant pathogens and the reconstruction of microbial interaction networks in wastewater treatment systems: A review. Sci Total Environ 2023; 890: 164291.
18. Jansen M, Wahida A, Latz S, Krüttgen A, Häfner H, Buhl EM, et al. Enhanced Antibacterial Effect of the Novel T4-Like Bacteriophage KARL-1 in Combination with antibiotics against Multi-Drug-Resistant Acinetobacter baumannii. Sci Rep 2018; 8: 14140.
19. Borin JM, Lee JJ, Gerbino KR, Meyer JR. Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages. Evol Appl 2022; 16: 152-162.
20. Wang M, Wei J, Jiang L, Jiang L, Zhang J, He X, et al. Coevolutionary phage training and Joint application delays the emergence of phage resistance in Pseudomonas aeruginosa. Virus Evol 2023; 9(2): vead067.
21. Oechslin F. Resistance Development to Bacteriophages Occurring During Bacteriophage Therapy. Viruses 2018; 10: 351.
22. Friman VP, Soanes-Brown D, Sierocinski P, Molin S, Johansen HK, Merabishvili M, et al. Pre-Adapting Parasitic Phages to a Pathogen Leads to Increased Pathogen Clearance and Lowered Resistance Evolution with Pseudomonas aeruginosa Cystic Fibrosis Bacterial Isolates. J Evol Biol 2016; 29: 188-198.
23. Schmerer M, Molineux IJ, Bull JJ. Synergy as a Rationale for Phage Therapy Using Phage Cocktails. PeerJ 2014; 2: e590.
24. Pu M, Han P, Zhang G, Liu Y, Li Y, Li F, et al. Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae. Int J Mol Sci 2022; 23: 8040.
25. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 Statement: An Updated Guideline for Reporting Systematic Reviews. BMJ 2021; 373: n71.
26. Natarajan SP, Teh SH, Lin LC, Lin NT. In Vitro and In Vivo Assessments of Newly Isolated N4-Like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1. Int J Mol Sci 2024; 25: 9595.
27. Fang Q, Zong Z. Lytic Phages against ST11 K47 Carbapenem-Resistant Klebsiella pneumoniae and the Corresponding Phage Resistance Mechanisms. mSphere 2022; 7(2): e0008022.
28. Zaki BM, Fahmy NA, Aziz RK, Samir R, El-Shibiny A. Characterization and comprehensive genome analysis of novel bacteriophage, vB_Kpn_ZCKp20p, with lytic and anti-biofilm potential against clinical multidrug-resistant Klebsiella pneumoniae. Front Cell Infect Microbiol 2023; 13: 1077995.
29. Sun Y, Feng JQ, Tan YR, Zhou L, Lan T, Ma JY. Genomic and Biological Characterization of vB_PvuS_Pm34, a Novel Lytic Bacteriophage that Infects Proteus vulgaris. Genomics 2022; 114: 38-44.
30. Yang P, Shan B, Hu X, Xue L, Song G, He P, et al. Identification of a novel phage depolymerase against ST11 K64 carbapenem-resistant Klebsiella pneumoniae and its therapeutic potential. J Bacteriol 2025; 207(4): e0038724.
31. Fikadu A, Amankwah S, Alemu B, Alemu Y, Naga A, Tekle E, et al. Isolation and Phenotypic Characterization of Virulent Bacteriophages Against Multidrug-Resistant Escherichia coli and Its Phage-Resistant Variant from Sewage Sources. Infect Drug Resist 2024; 17: 293-303.
32. Nour El-Din HT, Kettal M, Granados Maciel JC, Beaudoin G, Oktay U, Hrapovic S, et al. Isolation, Characterization, and Genomic Analysis of Bacteriophages against Pseudomonas aeruginosa Clinical Isolates from Early and Chronic Cystic Fibrosis Patients for Potential Phage Therapy. Microorganisms 2025; 13: 511.
33. Senhaji-Kacha A, Bernabéu-Gimeno M, Domingo-Calap P, Aguilera-Correa JJ, Seoane-Blanco M, Otaegi-Ugartemendia S, et al. Isolation and characterization of two novel bacteriophages against carbapenem-resistant Klebsiella pneumoniae. Front Cell Infect Microbiol 2024; 14: 1421724.
34. Wang Q, Chen R, Liu H, Liu Y, Li J, Wang Y, et al. Isolation and characterization of lytic bacteriophage vB_KpnP_23: A promising antimicrobial candidate against carbapenem-resistant Klebsiella pneumoniae. Virus Res 2024; 350: 199473.
35. Yazdi M, Bouzari M, Ghaemi EA, Shahin K. Isolation, Characterization and Genomic Analysis of a Novel Bacteriophage VB_EcoS-Golestan Infecting Multidrug-Resistant Escherichia coli Isolated from Urinary Tract Infection. Sci Rep 2020; 10: 7690.
36. Badawy S, Baka ZAM, Abou-Dobara MI, El-Sayed AKA, Skurnik M. Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate. Arch Virol 2022; 167: 1333-1341.
37. Akremi I, Merabishvili M, Jlidi M, Haj Brahim A, Ben Ali M, Karoui A, et al. Isolation and Characterization of Lytic Pseudomonas aeruginosa Bacteriophages Isolated from Sewage Samples from Tunisia. Viruses 2022; 14: 2339.
38. Mirza KA, Tchatchiashvili T, Marquet M, Nietzsche S, Pletz MW, Makarewicz O. Characterization and genome analysis of novel Klebsiella pneumoniae phage vbKpUKJ_2 isolated from hospital sewage water. BMC Microbiol 2025; 25: 96.
39. Ponsecchi G, Olimpieri T, Poerio N, Antonelli A, Coppi M, Di Lallo G, et al. Characterization of four novel bacteriophages targeting multi-drug resistant Klebsiella pneumoniae strains of sequence type 147 and 307. Front Cell Infect Microbiol 2024; 14: 1473668.
40. Menon ND, Kumar MS, Satheesh Babu TG, Bose S, Vijayakumar G, Baswe M, et al. A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates. mSphere 2021; 6(1): e01215-20.
41. Oduor JMO, Kiljunen S, Kadija E, Mureithi MW, Nyachieo A, Skurnik M. Genomic characterization of four novel Staphylococcus myoviruses. Arch Virol 2019; 164: 2171-2173.
42. Chakraborty S, Rohit A, Prasanthi SJ, Chauhan A. A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates. Pharmaceutics 2024; 16: 904.
43. Chen H, Liu H, Gong Y, Dunstan RA, Ma Z, Zhou C, et al. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes 2024; 10: 127.
| Files | ||
| Issue | Vol 18 No 4 (2026) | |
| Section | Review Article(s) | |
| Keywords | ||
| Bacteriophages Drug resistance Wastewater Sewage Biofilms Klebsiella pneumoniae Anti-bacterial agents | ||
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How to Cite
1.
Badakshi F, Zahid K. Bacteriophage cocktails and bacterial coevolution: a systematic review of resistance prevention strategies against multidrug-resistant pathogens in sewage water. Iran J Microbiol. 2026;18(4):460-472.
