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Bovine mastitis caused by Klebsiella pneumoniae is usually treated with antibiotics, thereby potentially increasing antimicrobial resistance. The objective of this study was to evaluate efficacy of a bacteriophage, isolated from dairy farm wastewater, as a treatment for a murine model of K. pneumoniae mastitis. A lytic bacteriophage CM8-1 was isolated, morphological and biological characteristics were assessed with transmission electron microscopy and double-layer plate, and its genome was sequenced and analyzed. Furthermore, effectiveness of this bacteriophage for treatment of a murine model of K. pneumoniae mastitis was evaluated based on the following mammary gland characteristics: morphological changes; number of K. pneumoniae; and mRNA and protein expression of pro-inflammatory factors TNF-α, IL-1β, IL-6, and IL-8. Bacteriophage CM8-1 had an incubation period of 30 min and a burst time of 20 min. Its viability and adsorption were stable at 30 to 50°C, but decreased significantly at >60°C, with no significant change in viability or infectivity at pH 6 to 10. In a murine model of K. pneumoniae mastitis, injecting bacteriophage CM8-1 into the mammary gland 2 h after inoculation with K. pneumoniae resulted in reductions in bacterial counts in the murine mammary gland, improvements in mammary gland tissue morphology, and reductions in mRNA and protein expression of pro-inflammatory factors. Bacteriophage CM8-1 had stable biological characteristics and suppressed K. pneumoniae mastitis when injected into the mammary gland 2 h latera in mice bacterial inoculation., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Klebsiella pneumoniae, an environmental pathogen causing mastitis in dairy cattle, is often resistant to antibiotics. K. pneumoniae was used as the host bacteria to support bacteriophage replication; 2 bacteriophages, CM8-1 and SJT-2 were isolated and considered to have therapeutic potential. In the present study, we determined the ability of these 2 bacteriophages to mitigate cytotoxicity, pathomorphological changes, inflammatory responses and apoptosis induced by K. pneumoniae (bacteriophage to K. pneumoniae MOI 1:10) in bovine mammary epithelial cells (bMECs) cultured in vitro., Results: Bacteriophages reduced bacterial adhesion and invasion and cytotoxicity (lactate dehydrogenase release). Morphological changes in bMECs, including swelling, shrinkage, necrosis and hematoxylin and eosin staining of cytoplasm, were apparent 4 to 8 h after infection with K. pneumoniae, but each bacteriophage significantly suppressed damage and decreased TNF-α and IL-1β concentrations. K. pneumoniae enhanced mRNA expression of TLR4, NF-κB, TNF-α, IL-1β, IL-6, IL-8, caspase-3, caspase-9 and cyt-c in bMECs and increased apoptosis of bMECs, although these effects were mitigated by treatment with either bacteriophage for 8 h., Conclusions: Bacteriophages CM8-1 and SJT-2 mitigated K. pneumoniae-induced inflammation in bMECs cultured in vitro. Therefore, the potential of these bacteriophages for treating mastitis in cows should be determined in clinical trials.

Phage therapy has potential to combat antibiotic-resistant bacteria causing bovine mastitis. Our objective was to use 3 Klebsiella lytic phages to create a phage cocktail, and to compare bactericidal activity of this phage cocktail versus an individual phage, both in vitro and in vivo. Based on transmission electron microscopy, phage CM_Kpn_HB154724 belonged to Podoviridae and on double agar plates, it formed translucent plaques on the bacterial lawn of Klebsiella pneumoniae KPHB154724. In one-step growth curves, this phage had a latent period of 40 min, an outbreak period of 40 min, a burst size of 1.2 × 107 PFU/mL, and an optimal multiplicity of infection (MOI) of 1. Furthermore, it was inactivated under extreme conditions (pH ≤ 3.0 or ≥ 12.0 and temperatures of 60 or 70 °C). It had a host range of 90% and had 146 predicted genes (Illumine NovaSeq). Based on histopathology and expression of inflammatory factors interleukin-1β, tumor necrosis factor-α, interleukin-6, and prostaglandin, phage cocktail therapy had better efficiency than an individual phage in K. pneumoniae -infected murine mammary glands. In conclusion, we used 3 Klebsiella lytic phages to create a phage cocktail and confirmed its effectiveness against K. pneumoniae both in vitro (bacterial lawn) and in vivo (infected murine mammary glands). • Phage cocktail composed of 3 Klebsiella lytic phages had good bactericidal activity. • Phage cocktail reduced the bacterial burden in K. pneumoniae -infected murine mammary gland. • Phage cocktail decreased the expression of inflammatory factors in K. pneumoniae -infected murine mammary gland. • Phage cocktail therapy had better efficiency for K. pneumoniae -infected murine mastitis. [ABSTRACT FROM AUTHOR]