Bacteria and reagents
Escherichia coli ATCC 25, was purchased from American Type Culture Collection (ATCC). Human clinical MCR-1-producing isolates K. pneumoniae ZJ02, E. coli ZJ40, and E. coli ZJ478 were collected in our previous study [7]. E. coli DH5α (pUC19-mcr-1) [20], which carries an mcr-1 gene originating from K. pneumoniae ZJ05, Salmonella sp. strain HYM2, Salmonella sp. strain ZZW20 and Salmonella sp. strain 15E464 were provided by Professor Jian Sun [25] in South China Agricultural University. Pingwei Pill was obtained from Bei Jing Tong Ren Tang Group (Beijing, China). Colistin sulfate was purchased from the National Institute for Food and Drug Control (Beijing, China). Penicillin, Cephalothin sodium, Meropenem, Streptomycin, Kanamycin sulfate, Gentamycin, Chloramphenicol, Achromycin, Ciprofloxacin and Erythromycin were obtained from Dalian Melian Biotechnology Co., Ltd. (Dalian, China). Stock solutions of Pingwei Pill dissolved in dimethyl sulfoxide or sodium carboxymethyl cellulose solution (Sigma-Aldrich, St. Louis, MO).
Antibacterial test
Checkerboard assay
The synergistic effects of antibiotics and Pingwei Pill were evaluated using checkerboard assays with two-fold serial dilutions according to the guidelines of the Clinical and Laboratory Standards Institute [26]. All compounds were diluted two-fold in LB (Luria–Bertani) broth and mixed with bacterial suspension equally in sterilized 96-well polypropylene microtiter plates. MIC values were defined as the lowest concentrations of compounds with no visible growth of bacteria between 18 and 24 h of incubation at 37 °C. The efficacies of the combinations were evaluated by calculating the fractional inhibitory concentration (FIC) index values [27] as follows: FIC index = FIC colistin + FIC Pingwei Pill = MIC combination /MIC colistin + MIC combination /MIC Pingwei Pill. (MIC colistin is the MIC of colistin alone; MIC combination is the MIC of colistin in combination with Pingwei Pill; MIC Pingwei Pill is the MIC of Pingwei Pill alone; Synergy is defined as an FIC index of P ≤ 0.5.
Growth curves
All the tested strains were cultured in LB broth with shaking at 37 °C until the absorbance value reached 0.3 at OD600nm. Each bacterial suspension was equally divided into five identical conical flasks with different concentrations of Pingwei Pill (0, 0.256, 0.512, 1.024 and 2.048 mg/mL). The growth of bacteria cultured at 37 °C with shaking was evaluated by measuring the optical density at OD600nm every 30 min.
Kill-curve kinetics analysis
A time-killing curve was used to estimate the potential synergistic effect of Pingwei Pill and colistin [28]. The test strains were incubated overnight at 37 °C with shaking, diluted 1/500 in LB and incubated for 4 h (exponential phase). The bacterial cells were diluted to 5 × 105 CFU/mL in LB broth treated with colistin (2 µg/mL), Pingwei Pill (1.024 mg/mL), or a combination of colistin and Pingwei Pill or DMSO (control). Following incubation at 37 °C for the indicated time points (0, 1, 3, 5, 7 and 9 h), the samples were removed, diluted appropriately, and plated on agar plates for the calculation of CFU after incubation at 37 °C overnight.
Combined disk tests
A combined disk test was performed according to a previous report [29, 30]. The tested strain suspension with an absorbance value of 0.1 was used for LB agar plates. The indicated concentrations of Pingwei Pill (10 µL) were added to the agar plates with 10 µg colistin disks (Oxoid Ltd., Basingstoke, United Kingdom). Subsequently, the agar plates were incubated for 24 h at 37 °C before the inhibition zone diameters were observed and recorded.
Western blot assay
The strain suspension with an absorbance value of 0.1 at OD600nm was cocultured with Pingwei Pill (0, 0.256, 0.512 and 1.024 mg/mL) at 37 °C for 4 or 6 h. The sample solution was removed via centrifugation, precipitated by boiling with loading buffer, separated using SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Following incubation with antibodies according to a previous report [9], the blots were observed using a Western blotting visualizer (Tanon 4200). The density of each band was evaluated using ImageJ software.
The strain suspensions with an absorbance value of 0.1 at OD600nm were cocultured with the indicated concentrations of Pingwei Pill (0.256 mg/mL, 0.512 mg/mL, or 1.024 mg/mL) plus colistin (2 μg/mL) at 37 °C for 6 h. Then the sample solutions were performed in the same process as described above.
Cytotoxicity assessment
The hemolytic activity of colistin with or without Pingwei Pill was evaluated. Briefly, sheep blood cells were treated with colistin (from 0 to 128 μg/mL), Pingwei Pill (from 0 to 1.024 mg/mL) or their combination for 1 h at 37 °C [31]. The absorbance of supernatants at 543 nm was measured, and hemolysis of each sample was calculated via comparison with the positive control. PBS and water were used as negative and positive controls, respectively.
Caco-2 and HeLa cells were plated on 96-well plates at 1 × 104 cells/well and treated with colistin (from 0 to 128 μg/mL), Pingwei Pill (from 0 to 1.024 mg/mL) or a combination at 37 °C for 24 h. Following centrifugation, the release of LDH into the supernatants was examined using an LDH assay kit by determining the absorbance of each sample at 450 nm.
Peritoneal macrophages were obtained from C57BL /6 mice by intraperitoneal injection DifcoTM Fluid Thioglycollate Medium. After 4 days raised, the mouse was killed by cervical dislocation. Then, the peritoneal macrophage was collected by washing the abdominal region with RPMI-1640 medium with a centrifugation at 1000 rpm for 10 min. Subsequently, the peritoneal macrophage was re-suspended with RPMI-1640 medium with 10% FBS and seeded into 96-well plates at 1 × 104 cells/well. The treatment process with Pingwei Pill and colistin to peritoneal macrophage was the same to Caco-2 and HeLa cells described above.
Analysis of bacteria membrane damage
Bacterial suspensions in the logarithmic growth phase were collected, washed with phosphoric acid buffer (0.02 mmol/L, pH 7.4), resuspended to 1.0 × 108 CFU/mL and equally divided into four groups. Pingwei Pill (1.024 mg/mL), colistin (2 μg/mL), the combination or solvent control was added to the strain suspensions, which were subsequently cultured on glass slides with 0.1% polylysine treatment in 24-well plates. Following incubation at 37 °C for 4–6 h, the glass slide was washed with phosphoric acid buffer three times and fixed with 2.5% glutaraldehyde for 12 h for SEM inspection (S-3400N, HITACHI).
Bacterial viability assay
Salmonella sp. strain HYM2 was cultured overnight, diluted (1/50) in 10 mL fresh THY and incubated at 37 °C for 3 h until the OD600nm value was 0.3. The cultures were treated with Pingwei Pill, colistin, the combination or solvent control at 37 °C for another 8 h. The bacterial pellet was collected via centrifugation at a speed of 10,000 rpm for 2 min, resuspended in 300 μL PBS and treated with 1 μL fluorescent reagent of the LIVE/DEAD® BacLight ™ Bacterial Viability Kit according to the kit’s instructions. Following incubation in the dark, 5 μL of the mixture was used for imaging with a fluorescence microscope (IX83P2ZF, Olympus).
Serial passage assay
For each tested strain, the absorbance value of the bacterial suspension was adjusted to 0.1 at OD600nm with the addition of subinhibitory concentrations of colistin, Pingwei Pill, the combination, or solvent control and further incubated for 24 h at 37 °C under shaking. The next day, the treated bacterial suspension in tubes was added appropriately to other tubes with fresh LB broth, and the OD600nm value was adjusted to 0.1. Various treatments of colistin, Pingwei Pill and their combination were added to the tubes in the same as described above. The checkerboard method was performed in 96-well plates to detect changes in colistin MIC every day. Processing continued in this manner until 20 serial passages or when the passage MIC exceeded the resistance breakpoint for 2–3 consecutive passages.
Molecular docking
The protein crystal models (5GRR, IL6, and DGKA) were obtained from the RCSB Protein Data Bank (https://www.rcsb.org/). The top 7 ingredients of Pingwei Pill were selected with OB > 30% in TCMSP, and the 2D structures of each compound were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The protein receptor and small molecule ligands were docked together in Sail Vina v1.0, and the results were entered into the protein–ligand interaction profiler (PLIP) website for online analysis (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) [32]. The visual images were exported from PyMol v2.4.0 software.
Animal experiments
According to the animal use guidelines of Jilin University, all animal (mouse and chick) experiments were reviewed and approved by the animal experimentation ethics committee of Jilin University. For animal infection, Salmonella sp. strain HYM2 was grown in LB broth to mid-logarithmic phase (OD 600 = 0.6 ~ 0.8) at 37 °C with shaking, centrifuged at 6000×g for 5 min, washed three times with PBS and resuspended in PBS.
Six-to-eight-week-old female BALB /c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and housed in groups under standard ventilated cages independently with standard food and water 3 days before the experiment. Water and food were withdrawn 4 h before intragastric administration of 10 mg streptomycin to each mouse [33]. Mice were supplied with water and food normally afterward. Water and food were withdrawn again 20 h after streptomycin treatment and 4 h before the mice were infected intragastrically with the above Salmonella sp. strain HYM2 suspension (1 × 106 CFU/mouse for survival analysis and 1 × 105 CFU/mouse for other analyses) or treated with sterile PBS as a negative control. Pingwei Pill and colistin were suspended with 0.5% CMC-Na (sodium carboxymethyl cellulose) that was used as vehicle control. Two hours later, the mice were intragastrically administered colistin (2 mg/kg), Pingwei Pill (0.8 g/kg), or Pingwei Pill (0.8 g/kg) in combination with colistin (2 mg/kg) or solution (0.5% CMC-Na) as a positive control. The dosing interval was 12 h, and the negative control mice without infection were administered PBS intragastrically on the same schedule. The survival of the mice in each group (n = 10) was determined for 1 week. The body weight of mice in each group (n = 5) was monitored for 7 days. The other mice in each group (n = 10) were sacrificed via cervical dislocation at day 3 post infection for the determination of bacterial burden in the cecum using homogenization, serial dilutions, and plating on LB agar plates under streptomycin selection. The levels of cytokines (IL-1β, IL-6, TNF-α and IFN-γ) in the supernatants of homogenized cecum tissue were detected using enzyme-linked immunosorbent assay (ELISA). For histological analysis, cecum tissue was fixed in formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and observed under light microscopy.
A total of 100 one-day-old chicks were used for intragastric intestinal infection with the above Salmonella sp. strain HYM2 suspension (1 × 109 CFU/chick for survival analysis and 1 × 107 CFU/chick for other analyses). Then after 2 h, the chicks were intragastrically administered colistin (4 mg/kg), Pingwei Pill (1.6 g/kg), or Pingwei Pill (1.6 g/kg) in combination with colistin (4 mg/kg) or solution (0.5% CMC-Na) as a positive control. The dosing interval was 12 h, and the negative control chicks without infection were administered PBS intragastrically on the same schedule. The analyses of survival (n = 10), body weight (n = 5), bacterial burden (n = 10), histological injury (n = 10) and inflammatory response (n = 10) were performed as described for mouse infection.
The ingredients of Pingwei Pill and potential target prediction
The ETCM (The Encyclopedia of Traditional Chinese Medicine) and TCMSP (Traditional Chinese Medicine Database and Analysis Platform) databases were used to obtain ingredients with OB (oral bioavailability) > 30% and DL (drug-likeness) > 0.18 and potential targets of Pingwei Pill (http://www.tcmip.cn/ETCM/, https://tcmsp-e.com/). Pingwei Pill related TCMs include CANG ZHU (English name: rhizoma atractylodis; Latin name: Atractylodes lancea), HOU PU (English name: Bark of officinal Magnolia; Latin name: Cortex Magnoliae Officinalis), CHEN PI (English name: Dried Tangerine Peel; Latinname: Pericarpium Citri Reticulatae) and GAN CAO (English name: Glycyrrhiza; Latinname: Radix Glycyrrhizae). The chemical-gene co-occurrences of colistin were collected from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The disease related genes of Salmonella infection were obtained from GEO (Gene Expression Omnibus, https://www.ncbi.nlm.nih.Gov/geo/) and KEGG databases (Kyoto Encyclopedia of Genes and Genomes, https://www.genome.Jp/kegg/mapper/color.html). The subnetworks associated with the main targets were drawn in Cytoscape 3.7.2 software with Human Protein Reference Database Protein–Protein Interactions (hprdPPI) as the background network. The related pathways of target genes were enriched using the STRING and Metascape databases (https:// www. string. db. org/,http://metascape.org/), and a bubble diagram was obtained form OmicShare online (http://www.omicshare.com).
Bacterial adhesions and invasion assay
HeLa cells were plated into 24‐well plates at a density of 5 × 105 cells/well and incubated at 37 °C for 12 h in a CO2 incubator. Salmonella sp. cultured overnight and diluted 1:20 in LB broth, and Pingwei Pill DMSO solution was added in 0 μg/mL, 256 μg/mL, 512 μg/mL, and 1024 μg/mL, bacteria added only as positive, respectively. Then cultured for 5 h at 37 °C with 200 rpm. Afterwards, the bacteria were added to HeLa cells at an MOI of 100, colistin added at same time with final concentration was 2 μg/mL. After 20 min, the HeLa cells were washed by PBS three times. Subsequently, the cells were lysed with 0.02% triton and diluted appropriately, then plated on LB agar plates.
The steps of invasion assay, cell treatment and bacteria treatment were same to above, before washed cells, the bacteria and cells were cultured for 1 h, and the culture medium was replaced with fresh DMEM containing 100 µg/mL gentamicin for 1 h at 37 °C, 5% CO2. After washing three times, the cells lysed with 0.02% triton and diluted appropriately, then plated on LB agar plates.
Data and statistical analysis
Statistical Program for Social Sciences (SPSS) version 19.0 (IBM Corp. Armonk, NY, USA) was used to analyze experimental data, which are presented as the means ± SD (n ≥ 3). Significant differences were analyzed using one-way ANOVA followed by a Dunnett t-test. *P < 0.05; **P < 0.01.