All strains used in this study were obtained from American Type Tissue Culture Collection (ATCC; Manassas, VA, USA). The four strains used in this study are Pseudomonas aeruginosa (ATCC 27853 and BAA-2114), Escherichia coli (ATCC 25992 and BAA-2452). All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless stated otherwise.

Rational design of the new hybrid peptide was performed using online bioinformatics software to achieve the optimal range in physiochemical properties. The Hierarchical Neural Network software (HNN) from the Network Protein Sequence Analysis (NPS) server was used to predict the secondary structure and calculate the helicity percentages of the parent peptides and the hybrid peptide. The physicochemical properties were predicted using ProtParam/ExPASy server (Wilkins et al. 1999) and the Antimicrobial Peptide Database (APD3) (Wang and Wang 2016). The properties include the molecular weight and the number of amino acids, the instability index, the aliphatic index, and finally, the hydrophobic ratio. Moreover, the HHpred (Zimmermann et al. 2018) and the MODELLER software (Webb and Sali 2016) were used for homology modeling. The validation method, the RAMPAGE (Lovell et al. 2003) was used to assess the Ramachandran Plot, and the I-TASSER (Zhang 2008) was used to confirm the helical structure and the quality of the model.

BKR1, a hybrid peptide of 21 amino acids (NH₂-VSKLAKKIKNLKNVKKSWKRQ-COOH) was synthesized following standard Fmoc solid-phase protocols on Wang resin. Peptide elongation was performed using standard HBTU coupling chemistry in dimethylformamide (DMF) solvent with a fourfold molar excess of diisopropyl ethylamine (DIEA) in N-methyl-2-pyrrolidone (NMP) and a threefold molar excess of each Fmoc-protected amino acid or 2-(6-methoxynaphthalen-2-yl) propanoic acid. BKR! was cleaved from the resin, using 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% thioanisole (3 h, room temperature), and precipitated using cold (–20 °C) diethyl ether. Reverse phase high-performance liquid chromatography (RP-HPLC) was used for purification of BKR1 using a C18 internsil ODS-SP column, the column was eluted with acetonitrile / H₂O-TFA gradient at flow rate of 1.0 ml/minute. The identification of BKR! was confirmed by mass analysis and through the employment of electrospray ionization mass spectrometry (ESI-MS).

Antimicrobial activities of the peptide alone, the antibiotics alone and in combination were tested using the broth dilution method as described by the Clinical and Laboratory Standards Institute guidelines and as performed previously (Almaaytah et al. 2014).

Briefly, four bacterial strains including a control and resistant clinical isolate of P. aeruginosa (ATCC 27853 and ATCC BAA-2114) in addition to a control and resistant clinical isolate of E. coli (ATCC 25922 and ATCC BAA-2452) were grown in Muller Hinton broth (MHB) medium. The initial concentration of the bacterial culture (1.5 × 10⁸ CFU/mL) were adjusted using 0.5 McFarland turbidity standard, and spectrophotometry at λ = 600 nm. The used concentration was diluted 100-fold to reach a value of 10⁶ CFU/mL. The bacterial suspension of the diluted concentration was distributed over 96-well plates. Each plate had a range different concentration of one of the four tested antibiotics (LVX, CHL, RIF, and AMP) that were determined according to previous analysis of the MIC values of each antibiotic against the target strain ranging from (1–100 µM), or different concentrations of the hybrid peptide. The growth control consisted of bacterial suspension without any concentration of an antimicrobial agent, while the negative control consisted of MHB broth alone.

The plates were incubated overnight, and the bacterial growth was evaluated by measuring the absorbance at λ = 600 nm using an Enzyme-Linked Immunosorbent Assay (ELISA) microplate reader (Epoch^TM; BioTek, Winooski, VT, USA). The minimum inhibitory concentration (MIC) values of the antimicrobial agent (antibiotics or hybrid peptide) were assessed by the bacterial growth at this stage since the MIC value is defined as The minimum inhibitory concentration (MIC) is the lowest antimicrobial concentration at which no visible microbial growth can be detected with the naked eye or where there is no difference in absorbance between the negative control and the test concentration (Omran et al. 2018).

Additonally, the minimum bactericidal concentration (MBC) which is defined as “the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic free media” (Mohseni et al. 2014), was determined by taking samples of 10 µL from turbidity-free wells, and spreading them over agar plates, followed by incubation for 24–48 h. Colonies were then counted and compared with growth controls.

The MIC and the MBC values for the four antibiotics and the hybrid peptide were tested alone and in combination against different bacterial strains as mentioned above. All experiments were made in triplicates. The checkerboard assay was adopted to check the synergistic activities between the hybrid peptide and the antibiotics.

The checkerboard assay employs different concentrations of antimicrobial agents to check their synergistic activities against a specific bacterial strain. Herein, samples of 25 µL of eight different concentrations of BKR1 peptide were distributed horizontally on 96-well plates, and each column had the same volume and the same concentration of the peptide. On the other hand, 25 mL of six different concentrations of a single antibiotic was added to different rows. Each row had the same concentration of the antibiotic. Meanwhile, two columns were reserved for testing the growth and negative controls. On each well, a fresh aliquot of 50 µL of bacterial suspension (10⁶ CFU/mL) was added. The plates were then incubated overnight (18 h), at 37 °C, and the MIC values were then measured by reading the absorbance at λ = 600 nm throygh an ELISA plate reader (Epoch^TM; BioTek, Winooski, VT, USA).

The act of synergism was measured by calculating the Fractional Inhibitory Concentration Index (FICI) according to the following equation:

$\text{FICI}=\frac{\text{MIC of BKR1 in combination}}{\text{ MIC of BKR1 alone}}+\frac{\text{MIC of antibiotic in combination}}{\text{ MIC of antibiotic alone}}$

The results were then interpreted as synergistic if FIC value was £ 0.5, additive if FIC was in the range between 0.5–1, and indifferent if FIC was higher than 1 and lower than 4 (Sheikholeslami et al. 2016).

2 mL of human blood (Sigma Aldrich, St. Louis, MO, USA) was added to 48 mL of sterile phosphate buffer saline (PBS) at pH 7.4. The suspension was centrifuged three times at 2000 rpm for 5 min. Each time the supernatant was discarded and replaced by a fresh PBS buffer. The final concentration of human blood (RBCs) suspension was 4%. Eight tubes were then prepared, in which six of them contained 2 mL RBCs suspension and 2 mL of 6 different peptide concentration, one of them was for the positive control (2 mL of RBCs suspension mixed with 5µL of 0.1% Triton X-100), and the final tube represents the negative control (2 mL of RBCs suspension). Subsequently, all tubes were incubated at 37 °C for 60 min, then centrifuged at 2000 rpm for 5 min. After that, 1 mL was taken from each tube, and their absorbance was checked using (ELISA) microplate reader (Epoch^TM; BioTek, Winooski, VT, USA) at λ = 450 nm.

The cell lines used in the present study were Vero cells (ATCC CCL-­81). The Vero lineage was isolated from kidney epithelial cells extracted from an African green monkey. The cells were grown in an RPMI media that consist of 10% fetal bovine serum and 1% v/v antibiotics (ampicillin, streptomycin), and antifungal agent (Amphotericin B), which were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Cells were seeded in 75 cm² flasks with 24 mL of prepared media, incubated in 5% CO₂ incubator at 37 °C. The media was changed every 24 h until the confluence reached 70%. The media was then discarded, leaving the adherent cells in the flask. A volume of 5–7 mL of trypsin 1X was added, and the flask was then returned to the CO₂ incubator for 5–10 min. The trypsin was removed, and the detachment of the cells was confirmed through the use of an an inverted microscope. The trypsinization process was repeated until sufficient cell detachment. Finally, 5 mL media was added to neutralize the low pH of trypsin, followed by centrifugation at 2500 rpm for 5 min. The supernatant was discarded and replaced with 10 mL media and mixed by a vortex.

Cells were mixed with an equal amount of trypan blue (4%). The cells that were stained by trypan blue stain were considered dying cells, whereas live cells looked like stars. The cells were then counted using hemocytometer.

This assay was performed by seeding the cells at 5 × 10⁵ cells/well in a 96-well plate flat bottom. The plates were incubated in a CO₂ incubator at 37 °C for 18 h, to reattached cells to the bottom. The media was then discarded from the plates, and six different concentrations of peptide were dissolved in prepared RPMI media. The plates were incubated again in a CO₂ incubator at 37 °C for 18 -24 hours. A volume of 25 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) suspension (4 mg/mL) was added, and the plates were further incubated for 4–6 hours. The MTT-Peptide solution was then removed, and 100 µL of DMSO was placed in each well followed by continuous pipetting to dissolve the formed Formazan crystals. The absorbance was then measured using ELISA microplate (Epoch^TM; BioTek, Winooski, VT, USA) at λ = 540 nm (Almaaytah et al. 2019).

The sequence of the peptide was designed by fragmenting the helical parts of the parent peptides; Esculentin-1a and Melitten with some modifications as shown in Table 1; the bold underlined letters represent the parts which were taken from the parents, and valine was added at the beginning of the sequence to increase the half-life of the peptide as suggested by ProtParam/ExPasy. The design of BKR1 peptide was adopted after obtaining the highest helicity percentage and acquiring the most optimum in silico physicochemical properties as suggested by ProtParam/ExPASy server and the Antimicrobial Peptide Database (APD3). BKR1 is a 21 amino acid hybrid peptide with 85.71% helicity (Fig. 1), which is higher than both parents as shown in the table below.

Table 1.

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Prediction of secondary structure for the parent peptides and the hybrid peptide (BKR1) using Hierarchical Neural Network software (HNN).

Name of Peptide	Sequence	# of a.a1	α- helix %	b- sheet %	Random coils %
		46	45.00	19.00	34.78
		26	42.31	26.92	30.77
		21	85.71	0.00	14.29

¹ # of a.a.; the number of amino acids.

Figure 1. 

Three-dimensional structure of BKR1 generated by homology modelling using MODELLER and the figure was prepared using PyMol.

The physicochemical properties of BKR1 mentioned above were tested by ProtParam/ExPASy server and the Antimicrobial Peptide Database (APD3). The peptide is expected to display a stable form in the test tube as suggested by the instability index (>40), and the aliphatic index which provides an indication of the peptide’s thermostability. The peptide is considered hydrophilic (33% hydrophobic ratio) with high protein interaction ability (Boman index 2.81 kcal/mol). The charge of BKR1 peptide is +9 compared with +5 and +6 for Esculentin-1a and Melitten, respectively (Table 2). Finally, the I-TASSER results confirmed the helicity of BKR1 peptide with C-score -0.05, TM score 0.71 ± 0.12, and RMSD 1.2 ± 1.2 Å.

BKR1 displays a net positive charge of +3 and a molecular weight of 1144.34 Da. BKR1 was purified to >98% purity using RP-HPLC (Suppl. material 1) and its identity was confirmed by electrospray ionization mass spectrometry (ESI-MS) with the synthetic peptide displaying major peaks in the +3, +4 and +5 charge state of 842.6, 632.2 and 506.0 Daltons (Fig. 2).

Figure 2. 

Positive electrospray ionization mass spectrometric (ESI-MS) analysis of the BKR1. The peptide shows major peaks in the +2, +3 and +5 state of 842.6, 532.2 and 506.0 Daltons.

The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) for BKR1 displayed high antimicrobial activity against different Gram-negative bacteria. The average MIC value for all strains in this study was in the range of 25–30 mM, against the control strains and the resistant strains of the bacterial strains employed in the study (Table 3). For example, the MIC values were 25 mM for P. aeruginosa ATCC 27853 (control strain) and ATCC BAA-2114 (resistant strain), and the MIC values were also the same against E. coli ATCC 25992 (control strain) and BAA-2452 (resistant strain). On the other hand, the MBC values were equal to the MIC values in all experiments, which indicates the bactericidal behavior of the hybrid peptide.

BKR1 peptide displayed significant antimicrobial activity against different strains of Gram-negative bacteria as shown in the previous section. However, we tested its potential of BKR1 to act as an antimicrobial adjuvant with the four suggested antibiotics (levofloxacin, LVX; chloramphenicol, CHL; ampicillin, AMP; and rifampicin, RIF) against different strains of P. aeruginosa (control strain ATCC 27853 and a resistant strain ATCC BAA-2114) in addition to E. coli (control strain ATCC 25922 and resistant strain ATCC BAA-2452). The act of synergism was checked using Checkerboard assay, and the results are summarized in Table 4.

To calculate the percentage reduction in MIC, the following equation was used:

$\text{ Percentage of MIC reduction }=\frac{\text{MIC alone}-\text{MIC combination}}{\text{MIC alone}}\times 100\%$

Interestingly, the combination of LVX/BKR1 displayed additive activity against control strains of both P. aeruginosa and E. coli but expressed synergistic activities against the resistant strains. The synergism of this combination against the tested strains caused a huge reduction in the MIC values of the combined agents (LVX: BKR1 = > 75%: > 90%).

CHL/BKR1 combination, on the other hand, displayed synergism with all tested strains with ≥ 80% reduction in the CHL MIC values and ≥ 85% in the MIC values of BKR1 (except against the control strain of E. coli, which showed additive effect). Whereas the combination of RIF/BKR1 always displayed strong synergism with more than 70% reduction in the MIC of both antibiotics, and more than 90% reduction in the MIC of BKR1 in all cases. AMP/BKR1 combination showed no synergism, but the outputs showed that their combinations decreased the MIC values for both agents in all cases as shown in Table 4. The strongest synergism was found with CHL against the resistant strain of P. aeruginosa with a reduction percentage of 88% and 95% for CHL and BKR, respectively.

The toxicity of antimicrobial peptides against human red blood cells has been a significant obstacle against their development into clinically useful drugs (Yeung et al. 2011). Therefore, we tested different concentrations of BKR1 against human RBCs to evaluate its toxicity against mammalian cells. The concentrations used were 200, 160, 120, 80, 40, 20, and 10 μM), which were incubated with 4% human erythrocytes suspension. BKR1 peptide showed no hemolysis activity at its MIC values. Doubling the minimum inhibitory concentrations did not cause significant hemolysis against RBCs as the hemolysis did not exceed 8% as shown in Fig. 3.

Figure 3. 

The hemolysis effect of different concentration of BKR1. The data are representative of three independent experiments.

The MTT assay evaluated the toxicity profile of BKR1 peptide against Vero cell line. The selectivity of the new antimicrobial agent towards bacteria is a major issue when designing novel antimicrobial agents. Therefore, different concentrations of the hybrid peptide were tested, and the outputs are represented in Fig. 4. The IC₅₀ of BKR1 was 43.1 mM, and unfortunately, the MIC values of the peptide when used alone put 40% of the viable cells at risk of damage. Therefore, this peptide can be used safely in combination with other antibiotics rather than being used indivdiually.

Figure 4. 

The MTT analysis of BKR1 peptide alone against Vero cell line at different concentration range. Error bars represent the standard deviation (±SD). Values represent the means of six different experiments.