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BMC veterinary research2025; 21(1); 568; doi: 10.1186/s12917-025-04989-1

Newly isolated bacteriophages show efficacy and phage-antibiotic synergy in vitro against the equine genital pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa.

Abstract: Bacterial infections of the genital tract are a severe problem in equine reproduction. Biofilms produced by Klebsiella (K.) pneumoniae and Pseudomonas (P.) aeruginosa present further concerns in such cases as they can limit the success of antibiotic treatments. Alternative treatment approaches are urgently needed for treating bacterial equine genital tract infections and thus, reduce antibiotic use. The present study reports on the bactericidal efficacy of both, novel K. pneumoniae- and P. aeruginosa-specific phages, in a biofilm model and in conjunction with antibiotic drugs as tested in vitro. Results: In total, 15 phages with lytic activity (K. pneumoniae: n = 6; P. aeruginosa: n = 9) were isolated and host ranges were determined. Four phages with a broad host range (K. pneumoniae: n = 3; P. aeruginosa: n = 1) were selected for further characterization, including electron microscopy and whole genome sequencing. Significant bacterial growth reduction was observed in planktonic killing assays at three multiplicities of infection (MOI 1, MOI 10, MOI 100), when the phages vB_KpnS_LmqsRe28-1, vB_KpnS_LmqsRe28-2, vB_KpnM_LmqsRe27-1 or vB_PaeS-LmqsRe25-1 were added. In addition, planktonic killing assays were used to examine the four phages in conjunction with gentamicin, enrofloxacin and trimethoprim-sulfadiazine in vitro. The results indicate synergistic activities between the four phages and the investigated antibiotics. However, bacterial growth was not affected in biofilm models after phage treatment, likely due the absence of depolymerase genes in the sequenced phages. Conclusions: Phages against the equine genital pathogens K. pneumoniae and P. aeruginosa were evaluated in vitro for potential clinical use. While the bactericidal activity of the isolated phages was demonstrated in liquid culture, none of the tested phages showed significant reduction in in vitro biofilm models. However, due to synergistic activities between phages and antibiotics, further studies on combinatorial approaches are needed to develop strategies for managing difficult infections.
© 2025. The Author(s).
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Publication Date: 2025-10-03 PubMed ID: 41044673PubMed Central: PMC12492686DOI: 10.1186/s12917-025-04989-1Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigates newly isolated bacteriophages that target the equine genital pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa.
  • These phages demonstrated bacterial killing ability in liquid cultures and showed synergy when combined with antibiotics, though they were less effective against biofilm-associated bacteria in vitro.

Background and Importance

  • Bacterial infections in the equine genital tract cause significant reproductive issues.
  • Klebsiella pneumoniae and Pseudomonas aeruginosa are key pathogens in these infections, often forming biofilms that protect bacteria from conventional antibiotic treatments.
  • Biofilms are complex, surface-attached bacterial communities embedded in a protective matrix that make infections more difficult to eradicate.
  • Reducing antibiotic use is important to combat resistance, motivating the search for alternative therapies such as bacteriophages (viruses that infect bacteria).

Research Objectives

  • To isolate and characterize bacteriophages specific to K. pneumoniae and P. aeruginosa associated with equine genital infections.
  • To assess the bacterial killing efficacy of these phages against planktonic (free-floating) bacteria and biofilms in vitro.
  • To evaluate potential synergy between these phages and common antibiotics used in veterinary medicine.

Methods

  • Fifteen lytic phages were initially isolated: 6 specific to K. pneumoniae and 9 to P. aeruginosa.
  • Host range testing identified four broad-spectrum phages for further characterization: three targeting K. pneumoniae and one targeting P. aeruginosa.
  • Characterization included electron microscopy to visualize phage morphology and whole genome sequencing to analyze genetic content.
  • Planktonic killing assays exposed bacteria to phages at multiplicities of infection (MOI) of 1, 10, and 100 to measure antibacterial effects in liquid culture.
  • Synergy tests combined phages with antibiotics gentamicin, enrofloxacin, and trimethoprim-sulfadiazine to determine whether combined treatments enhanced bacterial killing compared to either alone.
  • Biofilm models were used to assess phage efficacy against bacteria embedded in biofilms under laboratory conditions.

Key Results

  • Phages vB_KpnS_LmqsRe28-1, vB_KpnS_LmqsRe28-2, vB_KpnM_LmqsRe27-1 (against K. pneumoniae), and vB_PaeS-LmqsRe25-1 (against P. aeruginosa) showed significant bacterial growth reduction in planktonic killing assays at all tested MOIs.
  • When combined with antibiotics, these phages showed synergistic activity, meaning the combination was more effective at killing bacteria than the sum of their individual effects.
  • No significant bacterial reduction was observed in biofilm models after phage treatment, likely because these phages lack depolymerase enzymes which can degrade the biofilm matrix and improve phage penetration.

Conclusions and Implications

  • This study presents novel bacteriophages with broad activity against relevant equine genital pathogens.
  • Phages alone effectively reduced planktonic bacterial growth but were limited against biofilms in vitro.
  • Phage-antibiotic combinations showed promising synergistic effects, suggesting potential for improved treatment strategies that could reduce antibiotic doses and combat resistant infections.
  • Further research is needed to enhance phage activity against biofilms, potentially by identifying or engineering phages with depolymerase enzymes.
  • Ultimately, such combinatorial approaches could lead to better management of difficult equine genital infections while minimizing antibiotic use.

Cite This Article

APA
Köhne M, Hüsch R, Peh E, Hirnet J, Tönissen A, Müsken M, Plötz M, Kittler S, Sieme H. (2025). Newly isolated bacteriophages show efficacy and phage-antibiotic synergy in vitro against the equine genital pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa. BMC Vet Res, 21(1), 568. https://doi.org/10.1186/s12917-025-04989-1

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 21
Issue: 1
Pages: 568
PII: 568

Researcher Affiliations

Köhne, Martin
  • Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine, Foundation, Bünteweg 15, 30559, Hannover, Germany. martin.koehne@tiho-hannover.de.
Hüsch, Ronja
  • Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine, Foundation, Bünteweg 15, 30559, Hannover, Germany.
Peh, Elisa
  • Institute of Food Quality and Food Safety, University of Veterinary Medicine, Bischofsholer Damm 115, 30173, Foundation, Hannover, Germany.
Hirnet, Juliane
  • Institute of Food Quality and Food Safety, University of Veterinary Medicine, Bischofsholer Damm 115, 30173, Foundation, Hannover, Germany.
Tönissen, Anna
  • Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine, Foundation, Bünteweg 15, 30559, Hannover, Germany.
Müsken, Mathias
  • Central Facility for Microscopy, Helmholtz Centre for Infection Research - HZI, Inhoffenstraße 7, 38124, Braunschweig, Germany.
Plötz, Madeleine
  • Institute of Food Quality and Food Safety, University of Veterinary Medicine, Bischofsholer Damm 115, 30173, Foundation, Hannover, Germany.
Kittler, Sophie
  • Institute of Food Quality and Food Safety, University of Veterinary Medicine, Bischofsholer Damm 115, 30173, Foundation, Hannover, Germany.
Sieme, Harald
  • Unit for Reproductive Medicine, Clinic for Horses, University of Veterinary Medicine, Foundation, Bünteweg 15, 30559, Hannover, Germany.

MeSH Terms

  • Animals
  • Pseudomonas aeruginosa / virology
  • Pseudomonas aeruginosa / drug effects
  • Horses
  • Klebsiella pneumoniae / virology
  • Klebsiella pneumoniae / drug effects
  • Biofilms / drug effects
  • Anti-Bacterial Agents / pharmacology
  • Bacteriophages / physiology
  • Bacteriophages / isolation & purification
  • Horse Diseases / microbiology
  • Horse Diseases / therapy
  • Phage Therapy / veterinary
  • Pseudomonas Infections / veterinary
  • Pseudomonas Infections / therapy
  • Pseudomonas Infections / microbiology
  • Klebsiella Infections / veterinary
  • Klebsiella Infections / therapy
  • Klebsiella Infections / microbiology
  • Reproductive Tract Infections / veterinary
  • Reproductive Tract Infections / microbiology
  • Reproductive Tract Infections / therapy

Conflict of Interest Statement

Declarations. Ethics approval and consent to participate: The animal study was approved by Animal Welfare Officer University of Veterinary Medicine Hannover. The study was conducted in accordance with the local legislation and institutional requirements. All authors agreed to participate in this study. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.

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Citations

This article has been cited 1 times.
  1. Bajrak O, Cieślik M, Górski A, Jończyk-Matysiak E. A comprehensive analysis of the kinetics of infection of lytic bacteriophages specific to the ESKAPE and critical pathogens. World J Microbiol Biotechnol 2026 Feb 28;42(3).
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