1. Materials
Methicillin-resistant Staphylococcus epidermidis (MRSE) was previously cultured and maintained in our laboratory. Male C57BL/6J mice, aged 6–8 weeks, were procured from Changsha Tianqin Biotechnology Co., Ltd.. The ethanol extract of emilia sonchifolia (10g crude drug/ 1g extract ) was purchased from Shanxi Xibolan Biotechnology Co., Ltd. (20220315).. The assay kits for succinate dehydrogenase, NADP-Malate dehydrogenase, catalase, and superoxide dismutase activities were acquired from Beijing Solarbio Science & Technology Co., Ltd. Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) were procured from Oxoid.
2.3.1 Morphological characterizations
Morphological characterizations of MRSE treated with emilia sonchifolia were explored using Scanning electron microscopy (SEM) and Transmission Electron Microscopy (TEM).
The scanning electron microscopy (SEM) was conducted using a modified method that had been previously employed. In summary, emilia sonchifolia (1 MIC) was introduced to a logarithmic phase bacterial suspension. The control group received an equal volume of phosphate buffer saline (PBS). All groups were incubated at 37℃ for a duration of 4 hours. Subsequently, the treated bacterial cells were harvested, washed, and subsequently fixed in a 3% glutaraldehyde solution overnight at 4℃. Next, the bacterial cells underwent dehydration through the application of ethanol with varying concentrations, followed by replacement with tertiary butyl alcohol. Subsequently, the samples were subjected to freeze-drying, sputter coated with gold, and examined through microscopic observations utilizing a scanning electron microscope. For the transmission electron microscopy (TEM) analysis, the MRSE were treated in a manner consistent with the SEM procedure. As per the report, the specimens were affixed to a fibrous carbon film and observed using TEM [21].
2.3.2 Effect of Emilia Sonchifolia on Energy Metabolism
In order to assess the impact of Emilia sonchifolia on the activity of SDH and NADP-MDH in MRSE, the SDH activity in bacterial cells was measured. A bacterial suspension in logarithmic phase was treated with Emilia sonchifolia (1 MIC), while the control group received an equivalent volume of PBS. Subsequently, all groups were incubated at 37℃ for a duration of 4 hours. Following the incubation period, the cultures were individually centrifuged at 12000r/min for 5 minutes at 4℃.The bacterial cells that had been collected were resuspended in 1 mL of phosphate-buffered saline (PBS) and subjected to ultrasonication at a strength of 30% for a duration of 30 minutes with an interval of 5 seconds. Subsequently, the supernatant was obtained through centrifugation at a speed of 12,000 revolutions per minute for 5 minutes at a temperature of 4 ºC. The activity of succinate dehydrogenase (SDH) and NADP-dependent malate dehydrogenase (NADP-MDH) was determined using the SDH and NADP-MDH assay kit.
2.3.3 Effect of Emilia Sonchifolia on Antioxidant system
The activities of CAT and SOD were evaluated in MRSE under emilia sonchifolia stress. MRSE in the logarithmic growth phase was introduced to emilia sonchifolia at a concentration of 1 minimum inhibitory concentration (MIC). Subsequently, the mixtures were incubated at 37°C for 4 hours. A negative control was also prepared, consisting of phosphate-buffered saline (PBS) instead of emilia sonchifolia. The mixtures were then subjected to centrifugation at 12000 revolutions per minute for 5 minutes at 4°C, and the resulting supernatant was discarded. The cells were subsequently washed and suspended in PBS. Subsequently, the samples were subjected to ultrasonication at a concentration of 30% for a duration of 30 minutes (with an interval of 5 seconds) and then centrifuged at a speed of 12000 revolutions per minute for 5 minutes at a temperature of 4℃. Ultimately,the supernatant was divided into aliquots and used for the CAT and SOD assays by kits from Beijing Solarbio Science & Technology Co.,Ltd..
2.4 The Antibacterial Activity of Emilia Sonchifolia In Vivo
2.4.1 Animals conditioning
The mice were assigned to individual cages in a pathogen-free facility, with a maximum occupancy of six mice per cage. Prior to their utilization, the mice were subjected to a 12-hour light-dark cycle at a temperature of 22 ± 2°C for a duration of one week. Additionally, they were provided unrestricted access to both food and water.
2.4.2 Establishment of mouse immunosuppressive models
Based on the existing literature, a mouse immunosuppressive model was established through intraperitoneal injection of CP doses at 120 mg/kg of body weight for a duration of 4 days. Control mice were administered PBS. Subsequently, the thymus and spleen were extracted from both the immunosuppressive and control mice, and the thymus and spleen index was computed.
2.4.3 Treatment of Emilia Sonchifolia against MRSE-induced BSIs
The animals were randomly assigned to different groups prior to the experiment, including the immunosuppressive group, bloodstream infection group, Emilia sonchifolia group, and blank group. In the Emilia sonchifolia group, Emilia sonchifolia was administered orally at a dosage of 8g/kg, 7 days prior to infection. The other three groups received an equal volume of PBS.
The immunosuppression model was implemented three days prior to infection, following the aforementioned method, in the immunosuppressive group, bloodstream infection group, and Patrinia scabiosaefolia group, and the blank group were dealt with PBS. Furthermore, bloodstream infection group, Emilia sonchifolia group were infected at 7 d after Emilia sonchifoli oral administration by tail vein injection with 1× 108cfu/mL MRSE, and the immunosuppressive group,blank group were dealt with PBS. The way of tail veins were consistent with the procedure outlined in Liu et al.[24] After that, at 12 h post infection (pi), blood samples were collected from tail veins in all groups. Meanwhile the above mouse were euthanized by 100mg/kg Pentobarbitol Sodium (IP). Subsequently, the aforementioned samples underwent serial dilution in phosphate-buffered saline (PBS) and were subsequently plated on tryptic soy broth (TSB) agar plates for a duration of 24 hours at a temperature of 37◦C. Ultimately, the enumeration of clones was conducted to assess the efficacy of the mouse bloodstream infection (BSI) models.[25]
The aforementioned mouse experiments were authorized by the University Committee on Use and Care of Animals at the Guizhou University of Traditional Chinese Medicine, in accordance with the Laboratory Animal – Guideline for Ethical Review of Animal Welfare (GB/T 35892—2018).
2.5 Statistical analysis
The values were reported as means ± standard deviations (SDs). To assess the statistical differences among the various groups, a one-way analysis of variance (ANOVA) was conducted. Significant differences between means were determined using Tukey's Honest Significant Difference test, with a significance level set at p < 0.05.