Staphylococcus aureus E48, a clinical mastitis isolate, had been used to induce mouse mastitis model (Wang et al., 2017). Tetracycline is one of the most extensively used antibiotics for mastitis treatment because of its relative safety, low cost, and a broad-spectrum activity (Kuang et al., 2009). It was used as a positive control in this study and was purchased from Sigma–Aldrich. The AMPs were prepared according to a previously described protocol (Zhang et al., 2015). The minimum inhibitory concentrations (MICs) were used to evaluate the antibacterial activities of antimicrobial agents (Wiegand et al., 2008). Briefly, S. aureus were grown to the log-phase at 37°C in the Mueller–Hinton broth, and diluted to 5 × 10⁵ CFU/mL with the fresh medium. A total of 180 μL of cell suspension and 20 μL of serially twofold diluted peptide were added to each well, and the plates were incubated at 37°C for 16–20 h. The final concentration of antimicrobial agents was prepared with gradients of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/mL. Three replicates were examined for each concentration. The MICs refer to the lowest concentration where no growth was visible after 16–20 h of incubation.

Time-kill assays of NZ2114, MP1102, and tetracycline were performed to evaluate the in vitro bactericidal activities. S. aureus E48 at the concentration of 5 × 10⁵ CFU/mL in fresh TSB medium was added to a flask, together with antimicrobial agents at concentrations of 0.5, 1, or 5 × MIC. The mixture was cultured at 37°C with shaking for 24 h. At 0, 2, 4, 8, 12, and 24 h of incubation, 100 μL sample was taken from each flask, serially diluted in sterile phosphate-buffered saline (PBS), plated onto tryptic soy agar (TSA) plates to count colonies after incubated at 37°C for 24 h. The experiments were repeated three times.

The activities of antimicrobial agents on S. aureus in bovine milk were determined in commercial whole-fat ultra-high-temperature-sterilized (UHT) milk as described by Schmelcher et al. (2012). UHT milk (Inner Mongolia Yili Industrial Group Co., Ltd.) at 37°C was inoculated with 5 × 10⁵ CFU/mL of exponentially growing cultures of S. aureus E48 in the presence of antimicrobial agents (100 μg/mL) or PBS buffer (negative control). The mixture was incubated without shaking. Samples were taken at 0, 2, 3, and 4 h after inoculation, and serially diluted in sterile PBS, plated onto TSA plates to count colonies after incubated at 37°C for 24 h. The absence of S. aureus E48 in non-inoculated UHT milk was verified by direct plating. Three independent assays were carried out on separate days.

An established bovine mammary epithelial cell line, designated MAC-T, which has been used for S. aureus internalization assays (Hensen et al., 2000; Bouchard et al., 2013), was used for this experiment. The MAC-T cell was cultured in cell culture dishes in the Dulbecco’s modified eagle medium (DMEM) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal calf serum. MAC-T cells were incubated in a humidified incubator at 37°C with 5% CO₂. After the cells were cultured to a confluent monolayer, the trypsin + EDTA solution (0.1%/0.04%) was added. The MAC-T cells were suspended in fresh medium at a concentration of 2 × 10⁵ cells/mL and cells were then seeded in 24-well plates (1 × 10⁵ cells/well) and incubated overnight at 37°C in 5% CO₂ to obtain a confluent monolayer for the internalization assay.

The effect of peptides on the mammary epithelial cell viability was measured by using a cell counting kit-8 (CCK-8, Shanghai Bangyi Biotechnology) as described previously (Fung and Demple, 2005).

The intracellular activities of AMPs were performed as previously described (Peyrusson et al., 2015) with minor modifications. Bacteria (1 × 10⁷ CFU) were added to mammary epithelial cell cultures at a bacterium-to-epithelial cell ratio of 100:1. After 1 h, extracellular bacteria were removed by thorough washing and gentamicin. Washing was performed by rinsing a well with PBS 10 times. After removing the washing fluid, DMEM supplemented with gentamicin (100 μg/mL) was added and incubated for 2.5 h to kill the extracellular bacteria. Mammary epithelial cells with intracellular bacteria were then re-cultured in a standard culture medium. Six wells of cells were lysed immediately as described later to calculate total numbers of intracellular bacteria at 0 h. The remaining wells of cells were cultured for additional 24 h in the presence of 3 antimicrobial agents. Afterward, the cells were washed thoroughly 10 times. Then, the cells were trypsinized with 450 μL of trypsin + EDTA solution approximately 15 min at 37°C. After cells were detached, 50 μL 1% Triton X-100 at a final concentration of 0.1% (vol/vol) was added to lyse the cells during a 10-min incubation at 37°C. The number of intracellular bacteria was determined. All tests were repeated three times.

The animal protocols used in this work were approved by Institutional Animal Care and Use Committee of Northwest A&F University. The 6- to 8-week-old specific-pathogen-free BALB/c mice were purchased from the Experimental Animal Center of the Medical College of Xi’an Jiao Tong University. To evaluate the efficacy of plectasin-derived AMPs in a mouse model of bovine mastitis, the pregnant female mice were randomly divided into five groups: snipped only group, S. aureus + PBS group, S. aureus + NZ2114 group, S. aureus + MP1102 group, and S. aureus + tetracycline group. The lactating mice were challenged with S. aureus E48 as described earlier (Schmelcher et al., 2015). The pentobarbital sodium (1.4 mg/20 g bodyweight) was used for anesthesia of the lactating mice by intraperitoneal injection. The distal end of the teat L4 (on the left) and R4 (on the right) was removed (snipped) by a small cut. These L4 and R4 glands constitute the fourth pair found from head to tail. For all groups except for the snipped only group, glands were injected with approximately 10⁴ CFU S. aureus in 50 μL PBS. At 45 min after S. aureus inoculation, the challenged glands were injected with 100 μg AMPs in 20 μL or 20 μL of PBS, depending on the treatment. The mice were euthanized 24 h after the S. aureus inoculation, and mammary glands were aseptically collected and weighed. Five mice (10 mammary glands) were used for each group. Seven mammary glands from each treatment were sampled and homogenized in PBS (100 mg/200 μL). Serial dilution plating on Baird-Parker agar plates was done to determine intramammary S. aureus. A part of the homogenate was centrifuged, and the supernatant was used for tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) assays, using the mouse TNF-α and IL-6 ELISA kits (Shanghai Bangyi Biotechnology). The tissue from three mammary glands from each treatment was fixed with 10% buffered formalin for histopathological analyses (HE staining).

The statistical procedures, means, and standard deviation (SD) were computed with SPSS 16.0. The data are expressed as mean ± SD. Comparisons between the groups were performed with ANOVA with a Duncan’s test. The differences were considered to be significant at P < 0.05.

The MICs of MP1102, NZ2114, and tetracycline against S. aureus E48 were 1, 2, and 1 μg/mL, respectively. As shown in Figure 1, MP1102 and NZ2114 at the concentrations of 5×MIC most significantly decreased S. aureus at 12 h after inoculation. However, bacterial re-growth occurred at the 24 h time point. At 1×MIC, bacterial counts initially decreased, but increased after 8 h post-exposure. In the presence of 0.5×MIC of MP1102 or NZ2114, the S. aureus grew more slowly than those in the control. All concentrates of tetracycline inhibited bacterial re-growth during 24 h. The data indicate that the bactericidal activities of plectasin-derived AMPs were both dose- and time-dependent.

The bactericidal activity of plectasin-derived AMPs against S. aureus in sterile homogenized whole milk was evaluated with the presence or absence of 100 μg/mL antimicrobials to milk at 37°C inoculated with ∼10⁵ CFU/mL of exponentially growing S. aureus cells. As shown in Figure 2, both AMPs significantly reduced bacterial numbers over the course of the experiment (4 h) as compared to the buffer control and the tetracycline group. After 4 h, there were almost no colonies recovered in the NZ2114 and MP1102 groups.

The potential cytotoxicity of NZ2114 and MP1102 was first evaluated. Cell viability was not affected by tetracycline, NZ2114, and MP1102 at the concentrations of 100 μg/mL, in comparison with the negative control without any antimicrobial components (data not shown).

In order to determine the bactericidal activities of NZ2114 and MP1102, S. aureus-infected bovine mammary epithelial cells were exposed to 100 μg/mL of antimicrobials for 24 h. As shown in Figure 3, both NZ2114 and MP1102 were efficient to reduce the internalized S. aureus. NZ2114 and MP1102 were similarly efficient and better than tetracycline.

The in vivo efficacy of the AMPs against S. aureus in a model of mastitis was evaluated. The S. aureus-induced inflammatory cell infiltration was significantly reduced by both AMPs and tetracycline (Figure 4). The numbers of recovered S. aureus from the mammary tissue are shown in Figure 5. After 24 h, S. aureus numbers in the PBS control (S. aureus + PBS treatment) glands exceeded 3.5 × 10⁷ per mammary gland. Both peptides and tetracycline significantly (P < 0.05) reduced S. aureus numbers in the mammary glands compared to the PBS control, but the effect of tetracycline was considerably weaker than those of the AMPs (1.48, 2.9, and 3.15 log₁₀ units reduction for tetracycline, NZ2114, and MP1102, respectively).

The concentrations of TNF-α and IL-6, which can be used as indicators of inflammation in mammary gland tissue, were examined (Figure 6). At 24 h post-infection, S. aureus infection without antimicrobial treatments showed a significant (P < 0.05) increase in TNF-α and IL-6 compared to snipped only glands with no S. aureus inoculation. Both AMPs and tetracycline significantly reduced TNF-α and IL-6 concentrations in comparison with the buffer control (P < 0.05).