Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Alternatives to antibiotics for treatment of mastitis in dairy cows.

Mastitis is considered the costliest disease on dairy farms and also adversely affects animal welfare. As treatment (and to a lesser extent prevention) of mastitis rely heavily on antibiotics, there are increasing concerns in veterinary and human medicine regarding development of antimicrobial resistance. Furthermore, with genes conferring resistance being capable of transfer to heterologous strains, reducing resistance in strains of animal origin should have positive impacts on humans. This article briefly reviews potential roles of non-steroidal anti-inflammatory drugs (NSAIDs), herbal medicines, antimicrobial peptides (AMPs), bacteriophages and their lytic enzymes, vaccination and other emerging therapies for prevention and treatment of mastitis in dairy cows. Although many of these approaches currently lack proven therapeutic efficacy, at least some may gradually replace antibiotics, especially as drug-resistant bacteria are proliferating globally.

Effective of phage cocktail against Klebsiella pneumoniae infection of murine mammary glands.

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-1ß, 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).

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

Bovine mastitis caused by multi-drug resistant (MDR) Klebsiella pneumoniae is difficult to treat with antibiotics, whereas bacteriophages may be a viable alternative. Our objective was to use 2 K. pneumoniae strains, 1 MDR and the other non-MDR, to isolate phages from sewage samples and compare their biological and genomic characteristics. Additionally, phage infected mouse mammary gland was also analyzed by H&E staining and ELISA kits to compare morphology and inflammatory factors, respectively. Based on assessments with double agar plates and transmission electron microscopy, phage CM_Kpn_HB132952 had clear plaques surrounded by translucent halos on the bacterial lawn of K. pneumoniae KPHB132952 and belonged to Siphoviridae, whereas phage CM_Kpn_HB143742 formed a clear plaque on the bacterial lawn of K. pneumoniae KPHB143742 and belonged to Podoviridae. In 1-step growth curves, CM_Kpn_HB132952 and CM_Kpn_HB143742 had burst sizes of 0.34 and 0.73 log10 PFU/mL, respectively. The former had a latent period of 50 min and an optimal multiplicity of infection (MOI) of 0.01, whereas for the latter, the latent period was 30 min (MOI = 1). Phage CM_Kpn_HB132952 had better thermal and acid-base stability than phage CM_Kpn_HB143742. Additionally, both phages had the same host range rate but different host ranges. Based on Illumina NovaSeq, phages CM_Kpn_HB132952 and CM_Kpn_HB143742 had 140 and 145 predicted genes, respectively. Genomic sequencing and phylogenetic tree analysis indicated that both phages were novel phages belonging to the Klebsiella family. Additionally, the histopathological structure and inflammatory factors TNF-α and IL-1ß were not significantly different among phage groups and the control group. In conclusion, using 1 MDR and 1 non-MDR strain of K. pneumoniae, we successfully isolated two phages from the same sewage sample, and demonstrated that they had distinct biological and genomic characteristics.