Bacteriophages isolated from dairy farm mitigated Klebsiella pneumoniae-induced inflammation in bovine mammary epithelial cells cultured in vitro

Abstract

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.

dCas9‐guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus

“…Based on the characteristics of S. aureus mastitis in cows, the cells were treated for 0, 3, and 24 h, followed by experiments that assessed milk protein indices, immune indices, and cell viability (Jaeger et al, 2015; Oskarsson et al, 2017; Sajjanar et al, 2019). TNF‐α, IL‐6, and IL‐8, the key inflammatory factors involved in dairy cow mastitis, were used to detect immune responses in BMMECs (Li et al, 2016; Ran, Yan et al, 2020; Shi et al, 2021; Wojdak‐Maksymiec et al, 2013). The milk protein synthesis ability of BMMECs is shown in Figure 1b,c.…”

Biological and genomic characteristics of two bacteriophages isolated from sewage, using one multidrug-resistant and one non-multidrug-resistant strain of Klebsiella pneumoniae

“…Phage CM_Kpn_HB132952 differed in tail length from previously isolated long-tailed K . pneumoniae phages CM8-1 and SJT-2 ( Shi et al, 2021 ; Zhao et al, 2021 ). Furthermore, CM_Kpn_HB143742 had different plaque characteristics than K .…”

“…The culture broth was centrifuged 13,813 × g for 10 min, the supernatant was filtered through a 0.22 μm microporous filter, and the purified bacteriophage stock solution was stored at 4°C (~ 2–3 days). Purification procedures were conducted as described ( Shi et al, 2021 ) and the titer of the phage inoculum was determined by a plaque assay ( Richards et al, 2021 ). The plaque was examined by a double-layer agar method ( Zhao et al, 2021 ).…”

Bacteriophage Therapy to Control Bovine Mastitis: A Review

“…To contextualise and provide insight into the therapeutic safety and efficacy of phages, relevant ex vivo models involving bovine cells lines were investigated. The studied cell lines for bacterial and phage interactions for this are the mammary alveolar cells-large T antigen (MAC-T) and bovine mammary epithelial (bMEC) cell lines [ 51 , 52 , 65 ]. A cocktail of two phages, CM8-1 and SJT-2 was shown to reduce K. pneumonia numbers and consequently reduce adhesion, invasion, and cytotoxicity in bMEC cells [ 65 ].…”

“…The studied cell lines for bacterial and phage interactions for this are the mammary alveolar cells-large T antigen (MAC-T) and bovine mammary epithelial (bMEC) cell lines [ 51 , 52 , 65 ]. A cocktail of two phages, CM8-1 and SJT-2 was shown to reduce K. pneumonia numbers and consequently reduce adhesion, invasion, and cytotoxicity in bMEC cells [ 65 ]. S. aureus phages were shown to migrate intercellularly and could reach the nucleus within 3 h after exposure to MAC-T cell lines and have an endocytotic activity of 12% in a bovine ex vivo model [ 51 , 52 ].…”