Abstract
Bovine mastitis is mainly caused by Staphylococcus aureus, which is difficult to eliminate, prone to escape from antibacterial agents, and may cause recurring infections due to the intracellular nature of its infection and multidrug resistance. In this study, the intracellular activities of the NZ2114 derivative peptide H18R (H2) against methicillin-resistant S. aureus (MRSA) and multidrug-resistant bovine S. aureus strains were investigated in bovine mammary epithelial MAC-T cells and mouse mammary glands. The minimum inhibitory concentrations of H2 against S. aureus were 0.5‒1 μg/ml; H2 displayed a lower cytotoxicity than its parental peptide NZ2114 (survival rates of MAC-T cells: 100% [H2 treatment] vs 60.7% [NZ2114 (256 μg/ml) treatment]). H2 was internalized into MAC-T cells mainly via clathrin-mediated endocytosis, and distributed in the cytoplasm. The intracellular inhibition rates against MRSA ATCC43300, the mastitis isolates S. aureus CVCC 3051 and E48 were above 99%, 99%, and 94%, respectively; these were higher than those in case of vancomycin (23–47%). In the mouse model of S. aureus E48-induced mastitis, after treatment with 100 μg of H2 and vancomycin, bacterial numbers in each mammary gland were reduced by 3.96- and 1.59-log CFU, respectively. Additionally, similar to NZ2114 and vancomycin, H2 alleviated the histopathological damage of the mammary tissue and polymorphonuclear neutrophil infiltration in the alveoli. These results suggest that H2 can be used as a safe and effective candidate for treating S. aureus-induced mastitis.
Highlights
As an opportunistic pathogen with a broad range of hosts, Staphylococcus aureus can lead to the acute and chronic infections in humans or farm animals worldwide[1]; bovine mastitis is one of the most serious concerns with regards to milk production[2]
The antimicrobial susceptibility and typing results showed that the bovine mastitis isolates of S. aureus CVCC3051 and E48 can resist multiple antibiotics such as bacitracin, sulfisoxazole, streptomycin, or ampicillin, and they are assigned to spa type t3297 and t3583 (Supplementary Tables S1 and S2)
To determine the intracellular activities of the peptides against S. aureus, MAC-T cells were infected with methicillin-resistant S. aureus (MRSA) ATCC43300 and bovine mastitis isolates (S. aureus CVCC3051 and E48); the results showed that the pathogens entered the MAC-T cells and were localized in the vacuoles (Fig. 1a) and the cytoplasm, respectively (Fig. 1a,d,g,h)
Summary
As an opportunistic pathogen with a broad range of hosts, Staphylococcus aureus can lead to the acute and chronic infections in humans or farm animals worldwide[1]; bovine mastitis is one of the most serious concerns with regards to milk production[2]. The first fungal defensin-plectasin, isolated from Pseudoplectania nigrella, displays a potent activity against Gram-positive bacteria including S. aureus by binding to the cell wall precursor-lipid II15,16. Both NZ2114 (D9N, M13L, and Q14R) and MP1102 (D9Q, M13V, and Q14R), which are derivatives of plectasin, have a stronger activity than their parent; this may be due to their more positive net charges and higher hydrophobicity[17,18,19,20]. The intracellular activity of H2 was evaluated against one multidrug-resistant S. aureus strain methicillin-resistant S. aureus (MRSA) ATCC43300, and two clinical mastitis isolates: S. aureus CVCC3051 and E48) (Supplementary Tables S1 and S2) in MAC-T cells; the therapeutic efficacy of H2 was investigated in an S. aureus-infected mouse mastitis model
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