Peptide synthesis
The synthetic melittin peptide was obtained from a yeast expression system as previously described [15]. Briefly, the sequence of melittin was cloned in the pPIC9 vector and then transformed into the Pichia pastoris GS115. Finally, a product yield of 105 µg melittin/L was achieved. During the peptide synthesis and purification, a standard melittin peptide obtained from Sigma-Aldrich (St. Louis, MO, USA) was used.
Cytotoxicity assay
The Human primary fibroblast cell line (C654, Pasteur Institute of Iran, Iran) was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) (Biosera Co. France). Once established, fibroblast cultures were trypsinized (Gibco, Canada) and transferred into a polystyrene 96-well plate with 3 × 103 cells per well in 200-µL medium and incubated at 37 °C in 5 % CO2 for 12-h or when they reached 80 % confluence. Afterward, the medium was replaced with PBS (phosphate-buffered saline) overnight for cell starving, and then cells were exposed to melittin at the two-fold concentration (0.625-10 µg/mL) for 24-h at 37 °C in 5 % CO2. The control wells were maintained with PBS. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) dye was used for cytotoxicity evaluation. The cells were rinsed with PBS and incubated with 0.5 mg/mL MTT diluted in complete DMEM for 4-h. Then, supernatants were removed, and 150 mL of dimethyl sulfoxide was added, and the plate was incubated for 10 min. The absorbance was read at 570 nm using a 96-well ELISA plate reader (BioTek, Vermont, USA). Five replicates for all concentrations were performed. The percentage of cell viability was calculated as follows: percentage of cell viability = [(A treatment – A blank)/(A control – A blank)] × 100 (where, A = absorbance)[16].
Hemolysis assay
The hemolysis assay was carried out as previously reported by Evans et al. [17]. Briefly, heparinized blood samples were collected from healthy volunteers. Red blood cells (RBCs) were extracted from the whole human blood sample by removing white blood cells and platelets through centrifugation (500 rpm, 5 min). The remained RBCs were washed in PBS. The melittin was dissolved in PBS and added to the suspension of RBCs (2 % final in v/v in PBS) at a concentration range from 0.061 to 15.6 µg/mL and incubated at 37 °C for 1-h. The samples were centrifuged (500 rpm, 5 min), supernatants collected, and 100 µL of each sample’s supernatant was transferred into a 96-well plate. The hemoglobin release was evaluated by measuring the absorbance (A-sample) of the samples at 540 nm. For negative and positive controls, PBS (A-blank) and Triton X-100 (A-Triton) (0.2 % v/v) were used, respectively. The percentage of hemolysis was calculated according to the equation.
Percentage of hemolysis = [(A-sample – A-blank)/ (A-Triton – A-blank)] × 100.
In vivo toxicity assessment
Animals
Male adult BALB/c mice (8-week old) weighing 20–25 g were used in the present study. All animals were housed in polypropylene cages in a temperature-controlled room (24 ± 2ºC) with 30–35 % relative humidity and a 12-h light/dark cycle. Mice were given free access to water and standard chow during the study period. All procedures involving animals were in accordance with the national guides in care and use of Laboratory Animals in Scientific Affairs provided by the Iranian Ministry of Health and Medical Education (2020). The guideline complies with the ARRIVE guidelines [18]. Moreover, all the study protocols, including the animal experiments, were approved by the Mashhad University of Medical Sciences Ethics Committee (permit code: IR.MUMS.MEDICAL.REC.1399.151).
In the present study, two main types of experiments were used to assess melittin toxicity in mice:
-
(I)
Acute toxicity assessment upon single-dose administration of melittin.
-
(II)
Sub-acute toxicity assessment upon multi-dose administration of melittin with different intervals.
Acute toxicity experiment
A two-fold concentration gradient test was carried out to determine the in vivo LD50 (median lethal dose) value for melittin. Accordingly, four different doses (1.2 mg/kg, 2.4 mg/kg, 4.8 mg/kg, and 9.6 mg/kg) were tested. For each concentration, five mice were injected intraperitoneally (i.p.) once and monitored for five days [19].
Sub-acute toxicity
Evaluation of sub-acute toxicity was conducted with two-time intervals (12-h vs. 8-h) to assess the probable cumulative toxic effect. Firstly, melittin at the dose of 2.4 mg/kg (sub-lethal dose determined in acute assay) was injected (i.p.) into mice (n = 10 each), every 12-h for five consecutive days (totally 11 injections). During the study period, animals were monitored daily for mortality, changes in their fur, eyes, mucous membranes, and behavioral signs (salivation, tremors, convulsions, diarrhea, and lethargy). In order to determine the safe dosing interval, additional multi-dose treatments were performed by injection of 2.4 mg/kg of melittin into other mice (n = 10 each), every 8-h for five consecutive days (totally 16 injections) [19]. Finally, animals were anesthetized with ketamine-xylazine (100:10 mg/kg i.p.), and blood samples were collected via cardiac puncture. Blood samples of half of the mice from each group were collected into tubes containing no anticoagulants. Samples allowed to clot, centrifuged (3000 g for 15 min), and sera were obtained for blood chemistry. Biochemical evaluation of creatinine, urea, aspartate transaminase (AST), and alanine transaminase (ALT) were performed. The blood samples of another half of the mice from each group were collected in ethylene diamine tetra-acetic acid (EDTA) tubes for hematological evaluation. Immediately after the blood collection, liver and kidney (left kidney) were dissected out, weighed, and fixed in 4 % paraformaldehyde solution for histological evaluation.
In vitro and in vivo antibacterial efficacies
Bacterial strains and reagents
In the present study, different strains of the three most common pathogens responsible for nosocomial infections were used. The pathogens were including XDR A. baumannii, MRSA, and KPC-KP. DNA sequencing analysis was performed for confirming A. baumannii using 16 S rRNA specific primer and PCR method. S. aureus strains were also confirmed using standard culture and biochemical tests. Multiplex PCR was used to detect XDR (blaIMP, blaVIM, and blaNDM genes) and MRSA (mceA gene) strains as previously reported [20, 21]. In addition, a clinical isolate of KPC-producing Klebsiella pneumonia (KPC-KP-C11) with positive KPC, NDM, and OXA-48 genes was chosen from previous work [22]. All media, including Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA), Brain heart infusion Broth (BHIB), Blood-agar (BA), and Trypticase soy broth (TSB), were purchased from Merck (Germany). Antibiotics including colistin sulfate (CAS No. 1264-72-8) and vancomycin hydrochloride (CAS No. 1404-93-9) were purchased from Sigma (USA).
In vitro antibacterial evaluation
The minimum inhibitory concentrations (MICs) of melittin, vancomycin and colistin were determined by the broth microdilution method proposed by the Clinical and Laboratory Standards Institute [23]. Briefly, the numbers of 1.5 × 105 (CFU/mL) bacteria were added to each well of a polystyrene 96-well plate containing different concentrations of melittin (2-100 µg/mL), vancomycin (0.125-128 µg/mL), and colistin (0.25–128 µg/mL) and incubated at 37° for 16–20 h. All experiments were performed in duplicate. The MIC was defined as the lowest concentration of the agent (peptide or antibiotic) to produce complete inhibition of visible growth.
In the next step, to determine the minimum bactericidal concentrations (MBCs) of melittin, vancomycin, and colistin, two 10µL samples from each well containing no visible growth were sub-cultured on MHA medium and incubated at 37° for 12–18. The MBC was defined as the concentration that killed all the tested bacteria (99.9 % killing) [9].
In order to observe the dynamic picture of the bactericidal activity of melittin, the time-killing curve (TKC) assay was carried out as previously described [9]. Briefly, a strain from each of the studied organisms was cultured in MHB medium at 37 °C for 24-h. Afterward, 107 CFU/mL of each strain was added to every well of a polystyrene 96-well plate in the presence of melittin at 1–4× MICs. Eventually, 10µL from each well was sampled at different time points (0, 0.5. 2, 3, 5, and 24-h) after incubation and sub-cultured on MHA medium at 37 °C for 24-h. Experiments were performed in duplicate. TKCs were constructed by plotting mean colony count (log 10 CFU/mL) [19].
In vivo antibacterial evaluation
After in vivo toxicity assessment of melittin and determination of sub-lethal dose, this phase of the study was performed. The in vivo antibacterial efficacy of melittin was evaluated in three mouse models of intraperitoneal infection induced by the three most common nosocomial pathogens.
Mouse peritoneal model of A. baumannii
A mouse model of XDR A. baumannii sepsis was developed as previously described [24, 25]. Briefly, all mice were rendered neurotropic by i.p. injection of cyclophosphamide at 150 mg/kg (200 µL), 4 and 1 days before infection. Firstly, the minimal lethal dose of A. baumannii was determined by pilot experiments in which different doses of A. baumannii (XDR-CI66) (103-109 CFU/mouse) were administrated (single i.p.) to the neurotropic animals. The minimal lethal dose was considered a dose able to kill 100 % of the infected neurotropic animals (LD100) over 72-h. The minimal lethal dose of A. baumannii (XDR-CI66) was determined as 107 CFU/mouse.
For survival analysis, the neurotropic mice were infected by the LD100 of A. baumannii (107 CFU/mouse dissolved in 500µL PBS) and then randomly were divided into three main groups (n = 10 each), including untreated, melittin treated, and colistin treated. PBS, melittin (2.4 mg/kg), and colistin (1.5 mg/kg) were administrated (i.p.) to control untreated, melittin treated and colistin treated groups respectively started 1-h post-infection and repeated every 12-h up to 36-h (totally four injections). The animals were monitored for five days.
In order to estimate the peritoneal load of A. baumannii, mice were infected by a sub LD100 dose of A. baumannii (106 CFU/mouse). Then, the infected animals were randomly divided into three groups (n = 15 each), including untreated (PBS), melittin treated (2.4 mg/kg), and colistin treated (1.5 mg/kg). Treatments were started 1-h post-infection and repeated every 12-h up to 36-h (totally four injections). Every 12-h post-infection, five mice from each group were euthanized, their peritoneal cavities were lavaged with 5mL sterile chilled saline, and their blood samples were also collected. Blood samples were used for bacterial culture, and lavage fluids were firstly aliquoted into 10-fold serial dilutions and then cultured on the BA medium to quantify the number of viable A. baumannii in the respective samples.
Mouse peritoneal model of Staphylococcus aureus (MRSA)
Like the A. baumannii peritoneal model, mice were immunosuppressed and then subjected to LD100 and a sub-lethal dose of MRSA for survival and antibacterial evaluations. The LD100 of MRSA was determined through pilot experiments in which different doses (104-109 CFU/mouse) of S. aureus (MRSA-CI66) were administrated. The LD100 was considered a dose able to kill 100 % of the infected neurotropic animals over 36-h [26]. The LD100 for the tested S. aureus was determined as107 CFU/mouse.
Mice were infected by a single i.p. injection of the LD100 dose of MRSA (107 CFU/mouse). The infected mice were randomly allocated into three main groups (n = 10 each) including, untreated (PBS), melittin treated (2.4 mg/kg), and vancomycin treated (200 mg/kg). The dose of vancomycin was optimized according to previous works and pilot experiments in which three doses of this drug (25, 150, and 200 mg/kg ) were tested. Vancomycin (200 mg/kg subcutaneously) was only administrated once at 0.5-h after infection [27]. The untreated and melittin treated groups were treated with PBS, and melittin (2.4 mg/kg i.p.) respectively initiated 1-h post-infection and every 12-h up to 36-h (totally four injections). Similar to A. baumannii sepsis, survival analysis was performed for MRSA.
In order to estimate the peritoneal load of MRSA, mice were infected by a sub LD100 dose of MRSA (106 CFU/mouse). Then, the infected animals were randomly divided into three groups (n = 15 each) including, untreated (PBS), melittin treated (2.4 mg/kg), and vancomycin treated (200 mg/kg). Treatments were similar to those performed in survival assessment. Every 12-h post-infection, five mice from each group were euthanized, their blood and lavage samples were collected and used for bacterial culture and quantifying the number of viable MRSA in the respective samples.
Mouse peritoneal model of Klebsiella pneumonia (KPC-KP)
Mice were immunosuppressed as described above. Like the other studied bacteria strains, the LD100 of KPC-KP was determined. Briefly, different doses (106-109 CFU/mouse) of KPC-KP were administrated, and the minimum dose that was able to kill 100 % of the infected neurotropic animals over 72-h was considered as LD100 [28]. The minimal lethal dose of K pneumonia (CI1 KPC-KP) was determined as 108 CFU/mouse.
The immunosuppressed mice were infected by a single i.p. injection of the LD100 of KPC-KP (108 CFU/mouse). The infected mice were randomly allocated into three main groups (n = 10 each), including untreated (PBS), melittin treated (2.4 mg/kg), and colistin treated (12.5 mg/kg). The colistin dose was optimized according to previous works and pilot experiments in which three doses of this drug (2.5, 12.5, and 25 mg/kg) were tested. Treatments with PBS, melittin (2.4 mg/kg), and colistin (12.5 mg/kg) were initiated at 1-h post-infection and repeated every 12-h up to 72-h (totally seven injections). For survival analysis, the animals were monitored for five days.
In order to estimate the peritoneal load of KPC-KP, the immunosuppressed mice were infected by a sub LD100 dose of KPC-KP (107 CFU/mouse). Then, the infected animals were randomly divided into three groups (n = 15 each). Grouping and treatments were similar to those performed in the survival assessment. Blood and lavage samples were collected at 24, 36, and 48-h post-infection and used for bacterial culture and quantifying the number of viable KPC-KP in the respective samples.
Statistical analysis
Data are presented as mean ± standard deviation (SD) for each group unless otherwise specified. Differences in quantitative measurements were assessed by one-way analysis of variance followed by Dunnett T3 post-hoc multi-comparison test, Kruskal Wallis, and Student’s t-tests, when appropriate. A log-rank test was run to determine differences in the survival distribution for the different types of intervention. The number of repetitions is given in the method. Differences were considered significant when P < 0.05.