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Research article
Evaluating the Antimicrobial Efficacy of a Designed Synthetic Peptide against Pathogenic Bacteria
1Protein Purification Laboratory and its Biological Functions; Faculty of Pharmaceutical Sciences, Food and Nutrition; Faculty of Pharmacy, Food and Nutrition; Federal University of Mato Grosso do Sul (UFMS), Campo Grande, Mato Grosso do Sul, Brazil
2Center for Natural and Human Sciences of the Federal University of ABC (UFABC), São Paulo, SP, Brazil
J. Microbiol. Biotechnol. 2024; 34(11): 2231-2244
Published November 28, 2024 https://doi.org/10.4014/jmb.2405.05011
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
The recent surge in multidrug-resistant bacteria, a pressing issue in public health, directly results from the overuse of antibiotics and the sluggish pace of antimicrobial drug development [1]. While discovering antimicrobials has historically been a life-saving breakthrough, the persistent challenge of multi-resistance infections underscores the urgent need for new, less toxic, and more effective antimicrobials [2-4].
A new proposal for new drug development is antimicrobial peptides (AMPs). These molecules act as effective antibiotics, whose antimicrobial activity results from disrupting the cell membrane integrity. Unlike conventional antimicrobial agents, AMPS acts on multiple targets within the cell [5]. Most antibiotics interact with and inhibit specific biochemical processes in bacteria, such as cell wall synthesis (peptidoglycan), protein synthesis (translation), DNA replication, RNA synthesis (transcription), and folic acid biosynthesis. However, some antibiotics interfere with ion channels and cause bacteriolysis [6]. AMPs exert their activity mainly through the rupture of the bacterial cell membrane, with consequent cell death or immunomodulatory actions [7].
AMPs have garnered significant interest as potential antibiotics for the future. Cellular organisms internally produce these small molecules, encompassing 2-50 amino acids and are essential components of the host's natural immune response. Their small size and diverse mechanisms of action, which impede the development of bacterial resistance, make them a promising alternative to conventional therapeutics. AMPs also display rapid bactericidal action, independent of phenotypic resistance, and good solubility in water with thermal stability [8, 9].
Their cationic and amphiphilic properties drive AMP antibacterial activity. It presents a variable net positive charge and about 50% hydrophobic amino acids. These peptides establish electrostatic interactions with anionic bacterial membranes, rupturing them. Their hydrophobic and hydrophilic properties facilitate interaction with the lipid tails of lipopolysaccharide (LPS) and the hydrophilic heads of phospholipids in the bacterial membrane. For this reason, it compromises the membrane permeability and leads to bacterial cell death [9]. There is significant interest in researching natural products despite their limitations. Applying synthetic biology can overcome production challenges and enhance chemical diversity. Computational tools assist in peptide sequence design and structure with bactericidal action. The broad-spectrum in vitro, in vivo and silico antibacterial activity of AMPs offers a promising alternative to conventional therapeutics [10].
Our research involved the development of the RK8 peptide using computer-aided methods to leverage its physicochemical properties in investigating its antibacterial activity and cell selectivity. The peptide sequence comprises eight amino acid residues and has a molecular mass of 1,273.54 Da, net charge +5, and hydrophobic ratio of 38%. Its amino acid residues do not fold into regular secondary structure elements. Therefore, we investigated its antimicrobial, antibiofilm, and toxicity properties in the present study. In addition, our study has illustrated the synergistic interaction of RK8 in combination with ciprofloxacin, along with its non-toxic attributes in both in vitro and in vivo settings. Finally, we also determined that its mechanism of action involves membrane damage. However, it acts differently on Gram-negative and Gram-positive membranes.
Material and Methods
Peptide Design and Synthesis
Based on the structural characteristics shared by arginine- and tryptophan-rich peptides, our researchers created the RK8 peptide sequence. As our aims involved the synthesis of an ultrashort peptide, we started with a sequence of eight amino acid residues containing arginines, lysines, and tryptophan. We defined a net electrical charge of +5 to secure the sequence distributed between two arginines and three lysines. Three tryptophan residues influence 37.5% of the hydrophobic amino acid composition. The molecular structure was carefully devised to incorporate specific amino acid residues, allowing it to exhibit concurrently hydrophilic and hydrophobic properties. The Antimicrobial Peptide Database (APD3) and the PepDraw server (http://pepdraw.com/) had the resulting RKWRKWWK sequence submitted and analysed. Physicochemical parameters such as isoelectric point, molecular weight, and side chain arrangement were also evaluated [11].
Aminotech, in Diadema-SP, carried out the peptide synthesis. A synthesis > 95% was requested. Mass spectrometry (ESI-TOF) and high-performance liquid chromatography (HPLC) evaluated molecular mass and purity parameters. The concentration of the RK8 peptide in solution was determined by determining the absorbance at 280 nm, based on the extinction coefficient of the tryptophan residue (ε = 5.560 mol/ml). RK8 was solubilised in ultrapure water, and its absorbance was measured using an Evolution 201 spectrophotometer (Thermo Fisher Scientific, USA) at 280 nm (A280). To determine its concentration (C), the following formula was used:
C (mg/ml) = (A280 × FD × PM) / ε
In Daltons, FD is the dilution factor, and PM is the molecular weight of the peptide.
Modelling and Validation
The peptide's secondary structure was determined using the APPTEST server (https://research.timmons.eu/apptest), which interactive simulations complete atomic models [12]. After modelling, we performed validation using PROCHECK software (https://www.ebi.ac.uk/thorntonsrv/software/PROCHECK/) to verify the stereochemistry and atoms' spatial arrangement of the peptide.
Circular Dichroism
CD analyses were performed with a Jasco J-1100 spectropolarimeter (Jasco Inc., Japan) using a quartz cuvette with a 0.1 cm optical path. RK8 peptide was synthesised at a concentration of 30 μM in an aqueous solution and subjected to analysis in ultra-pure water in the presence of SDS (sodium dodecyl sulfate). The experiment involved six scans at 25°C, covering wavelengths from 190 to 280 nm. As part of the peptide preparation process, we gathered spectra of the used solutions, referring to them as blank solutions. We collect the resulting data after subtracting the implemented spectra from the blank solution. Afterwards, the data was transformed into residual molar ellipticity [θ] using the following equation:
[θ] =
Where θ is the ellipticity measured in milliseconds, C is the concentration in molar units, l is the path length in cm, and nr is the number of amino acid residues.
Microorganisms
To determine the antimicrobial activity of the RK8 peptide, bacteria purchased commercially and catalogued in the American Type Culture Collection (ATCC, USA) were used: Gram-negative:
Strains of multidrug-resistant clinical isolates were tested: Gram-negative
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays
Clinical and Laboratory Standards Institute (CLSI) guidelines performed the peptide MIC assays. Bacterial colonies were isolated on the Muller Hinton Agar (MHA) and plated at a final concentration of 1.5 × 105 CFU/ml. The peptides were tested at concentrations ranging from 0.5 μM to 32 μM. Antibiotic control, positive control, was carried out using 10 μl of ciprofloxacin (for targeting Gram-negative bacteria) or vancomycin (for targeting Gram-positive bacteria) at a concentration of 1 μM, in combination with 90 μl of MH culture medium.
The term "MIC" refers to the minimum inhibitory concentration of a peptide, which is the lowest concentration capable of reducing bacterial growth by 90% or more. For the results, bacterial growth was determined by visual inspection and confirmed by absorbance measurement at 595 nm after 24 h of incubation using a Multiskan GO microplate reader (Thermo Fisher Scientific, USA). For assays involving resistant strains such as MRSA strains and clinical isolates of
The MBC was determined based on the MIC results. Three replicates of 10 μl were extracted from the microplate wells, placed on MHA, and then kept in an incubator at 37°C for 24 h. The MBC was determined to have the lowest peptide concentration, and no bacterial growth was detected. All experiments were performed in triplicate.
RK8 and Ciprofloxacin Synergistic Effect
An investigation of the potential synergistic effect of combining the RK8 peptide with the quinolone ciprofloxacin antibiotic was conducted. The assay was performed in a 96-well microplate, where the two drugs, RK8 and ciprofloxacin, were present. Serial dilution was undertaken to establish a range of concentrations aligned with their corresponding minimum inhibitory concentration (MIC) values. Only the multidrug-resistant strains
An assessment of synergistic effects was conducted using synergy scoring models on the SynergyFinder platform (https://synergyfinder.org/). The models included Highest Single Agent (HSA), Loewe Additivity, Bliss Independence, and Zero Interaction Potency (ZIP). Loewe Additivity is a model that assumes that drugs with similar modes of action produce an additive effect, identifying synergy if the observed effect exceeds this additive expectation. These models compare the observed combination effect to expected outcomes, identifying synergy when the observed effect exceeds the predicted effect based on each model's assumptions. We used the Bliss model to calculate the synergistic impact of RK8 in combination with ciprofloxacin. This model assumes that the two drugs exert their effects independently, and the expected combination effect can be calculated based on the probability of independent events [13].
yB L ISS = y1+y2-y1·y2
RK8 concentrations ranging from 0 to 16 μM were combined to assemble the concentration matrix with ciprofloxacin concentrations ranging from 0 to 32 μM. For the analyses carried out with
Membrane Permeability
Membrane permeability was investigated as described by Mohanram & Bhattacharjya (2016), and Almeida
Biofilm Activity
The RK8 eradication effect on mature
The microplate was incubated with the bicarbonate plasma solution overnight at 4°C. The following day, the wells were aspirated and washed once with 500 μl of saline. A culture of
After biofilm formation, the wells were washed twice with a BHI medium containing 0.4% glucose to remove non-adherent bacteria. They received 500 μl of the medium used in the assay containing RK8 at the MIC and half this concentration (MIC /two). Biofilm control received only a BHI medium containing 0.4% glucose, and control with the antibiotic ciprofloxacin at MIC (128 μM) was also performed. The biofilm was incubated under constant agitation (100 rpm) at 37°C for 24 h to analyse the effect of RK8 on eradicating the mature form of the
The plate was incubated at 37°C for 30 min, the culture medium was aspirated, and the reduced formazan crystals were dissolved with Dimethyl sulfoxide (DMSO). Three aliquots from each of the wells were transferred to a microplate (
Cytotoxic Effects
Hemolytic activity. As hemolysis is an effect widely observed among antimicrobial peptides, we investigated the RK8 peptide hemolytic activity based on the protocol proposed by Uggerhøj
Cellular viability. RAW 264.7 murine macrophages' cellular viability was evaluated in the presence of the RK8 peptide, according to the method proposed by Mosmann [18], using the enzymatic reduction of the reagent 3-(4,5-dimethylthiazol-2-yl bromide) -2,5-diphenyltetrazolium (MTT). The cells were provided by Octávio Franco (Dom Bosco Catholic University - UCDB) and cultivated in Dulbeccós Modified Eagle Medium - high glucose (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Gibco, Brazil), at 37°C in an incubator at 5% CO2.
After detaching from the culture bottle, their density was determined, and they were seeded in a 96-well microplate at a density of 6 × 103 cells per well. After being placed in microplates, the cells were incubated for 24 h. The culture medium was replaced with a DMEM culture medium lacking SFB supplementation. Instead, it contained a serial dilution of RK8 ranging from 1 to 64 μM. After 24 h, the medium containing RK8 was removed and replaced with 100 μl of DMEM medium containing MTT 0.5 mg/ml. After four hours of incubation, the medium was removed again, and the reduced formazan crystals were resuspended with the addition of 100 μl of DMSO. Then, the absorbance of the wells was determined at 630 nm in a Varioskan microplate reader (Thermo Fisher Scientific). Three independent experiments were performed in triplicate. Cell viability was calculated from the following formula:
Cell viability (%) = (AbsSample÷AbsNegative control) × 100
Peptide Stability
The RK8 peptide stability was evaluated using a 25% FBS solution prepared in ultrapure water. This assay was performed by adding 40 μl of the peptide solution (10 mg/ml) to 1 ml of 25% FBS solution. The mixture was maintained at 30°C. Aliquots of 150 μl were collected after 0, 15, 30, 45, 60, and 120 min, mixed with ten μl of Trifluoroacetic Acid (TFA) and centrifuged at 14 000 rpm at 4°C for 10 min [19]. High-Performance Liquid Chromatography (HPLC) monitored the stability of the peptide using the C-18 reversed-phase hydrophobic column (μ Bondapak C18, 3.9 × 300 mm, Waters, USA) at 25°C. The peptide's remaining percentage during degradation kinetics was calculated by integrating the peak area corresponding to the RK8 peptide, injecting 40 μl of the incubation product in triplicate. The assay was conducted in an Ultimate 3000 chromatograph (Thermo Fisher Scientific). A separation program of 60 min at 1 ml/min was implemented, with monitoring of the 220 and 280 nm wavelengths. Thermo Scientific's ChromeleonTM Chromatography Data System (CDS) 7 software determined the area under the curve.
Statistical Analysis
The experimental results showed that statistical significance was determined by a one-way Student's
Results
RK8 Peptide Racional Design
RK8 sequence (RKWRKWWK) has eight amino acid residues, +5 net charge, and hydrophobicity of 38%, with the first residue being arginine (R) and the eighth being lysine (K) (Table 1). Peptide sequence was created from a two sequences combination: 2 repetitions of a three amino acids sequence: Arg–Lys–Trp, occupying positions 1 to 6, and an inverted repeat of residues 5 and 6 (Lys–Trp), giving rise to amino acids at positions 7 and 8 (Trp–Lys). The structural design was produced to ensure positively charged residues at the amino and carboxyl termini. The physicochemical properties and primary structure of RK8 were collected from the APD3 database and the PepDraw platform. To validate this, PROCHECK software was used to confirm the chiral centres and ensure no steric hindrances in the correct orientation (Fig. 1).
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Table 1 . Physicochemical characteristics of the RK8 peptide attained from the APD3 server.
Sequence Total net charge Boman Index (Kcal/mol) pI Molecular weight (Da) Hydrophobic reason (%) RKWRKWWK +5 4.93 12.51 1.273,54 38%
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Fig. 1. Primary structure of the RK8 peptide, collected through the PepDraw server.
Circular Dichroism
After synthesising, we characterised the RK8 secondary structure through Circular Dichroism (CD) studies. The results exhibit RK8 dissolution in water and 30 mM SDS. RK8 exhibits a random structure in both solutions, with low-magnitude negative bands at 200 nm (Fig. 2), which suggests that the peptide does not adopt an alpha-helical conformation. Therefore, we speculate that the peptide has an extended conformation.
-
Fig. 2. Circular dichroism spectra of the RK8 peptide at 30 μM, in both water and SDS at 25°C.
Antibacterial Activity
The MIC was determined using the broth microdilution method (CLSI, 2012), using different concentrations of RK8 against susceptible and resistant bacteria. For Gram-positive bacteria, RK8 exhibited MIC values ranging from 8 μM to > 64 μM and MBC values from 8 μM to 64 μM. Ciprofloxacin showed MIC values between 1 μM and four μM, with corresponding MBC values of 1 μM to 8 μM. Vancomycin had consistent MIC and MBC values of 1μM for
-
Table 2 . MIC and MBC values, in μM, of Gram-positive and Gram-negative strains were evaluated with an initial density of bacteria corresponding to 1.5 × 105 CFU/ml.
Microorganisms (1.5 × 105 CFU/ ml) Bacteria RK8 Ciprofloxacin Vancomycin MIC (μM) MBC (μM) MIC (μM) MBC (μM) MIC (μM) MBC (μM Gram-positive Staphylococcus aureus (MRSA) ATCC 335918 8 4 8 1 1 Staphylococcus aureus (MRSA) ATCC 4330016 8 2 2 1 0.5 Staphylococcus saprophyticus ATCC 29970>64 64 1 1 0.5 1 Gram-negative Escherichia coli KPC+ 00181244616 8 32 1 0.5 0.5 Escherichia coli ATCC 35218>64 2 1 2 8 1 Acinetobacter baumannii IC 00332121664 4 128 1 4 1 Acinetobacter baumannii ATCC 19906>64 4 1 1 4 1 Pseudomonas aeruginosa ATCC 27853>64 64 0.5 8 0.5 1 Klebsiella pneumoniae ATCC 700603>64 64 12 1 8 1 MIC, Minimum Inhibitory Concentration; MBC, minimum Bactericidal Concentration
Following the detection of antibacterial activity against susceptible strains, we evaluated the effectiveness of RK8 against multi-resistant bacterial strains. A heat map was used to improve the clarity of RK8's antibacterial activity data, making it easier to interpret and present the peptide's efficacy against different bacterial strains and concentrations (Fig. 3A and 3B).
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Fig. 3. Heatmap graph showing the MICs of RK8 (A) and ciprofloxacin (B) for resistant bacteria, evaluated with an initial density of bacteria corresponding to 1.5 × 105 CFU/ml. Darker red represents growth inhibition and was considered to establish the MIC; lighter shades reveal partial growth inhibition, while dark blue shades show microbial growth.
The heat map primarily evaluates the effectiveness of RK8. In this context, it is possible to denote that for
In summary, RK8 is more effective against
RK8 and Ciprofloxacin Synergistic Effect
The RK8 and ciprofloxacin combination showed a Bliss Synergy Score 3.99 against
-
Fig. 4. Dose-response inhibition map and Synergy Score for the combination of RK8 and ciprofloxacin against the
E. coli strain KPC+ IC 001812446.
-
Fig. 5. Inhibition dose-response map and Synergy Score for the combination of RK8 and ciprofloxacin against the
A. baumannii strain IC 003321216.
Antibiofilm Activity
The bacterial eradication biofilm assay was performed with the
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Fig. 6. Effect of RK8 on mature biofilm of
A. baumannii IC 003321216. Viable cells are stained in green, and non-viable cells are stained in red. Image of the control slide without treatment with RK8 in (A) and slide with biofilm treated with RK8 in (B).P < 0.0001 compared with cells treated with control.
-
Fig. 7. Viability of bacterial cells in mature biofilm of
A. baumannii IC 003321216, in the presence of RK8 (A) and ciprofloxacin (B).P < 0.0001 compared with cells treated with control.
The fluorescence microscopy images (Fig. 6) represent the biofilm viability assay. After exposure to RK8 at the minimum inhibitory concentration (MIC), the biofilm displayed red areas revealing cell death and green areas signifying cell viability. Image analysis using Image J revealed an 82.5% reduction in cell viability. The image shows structural damage to the biofilm, with an irregular distribution of bacteria suggesting significant damage.
Membrane Permeability
To verify whether the RK8 mechanism against bacterial cells involves damage to the cell membrane, we performed the assay with the fluorophore Sytox green, a bacterial membrane damage indicator. Fig. 8 shows that the peptide has an action mechanism that involves membrane damage. However, it acts differently on Gram-negative and Gram-positive membranes. For
-
Fig. 8. Absorption of Sytox Green by
E. coli (ATCC 35218) and MRSA (ATCC 43300) treated with RK8 in MIC. Sytox green uptake was measured during 120 min of incubation with RK8.
Hemolytic Activity
The RK8 hemolytic activity was evaluated against human erythrocytes, where the peptide maximum concentration was 100 μM, performing serial dilution up to 0.78 μM. The RK8 peptide induced approximately 4.6% hemolysis at the initial concentration, having even lower percentages in the MIC bacterial strains tested values (Fig. 9).
-
Fig. 9. Percentage of hemolysis in the presence of the RK8 peptide at concentrations collected from serial dilution starting at 100 μM.
P < 0.0001 compared with cells treated with control.
Cell Viability
The cytotoxic effects of RK8 against mammalian cells were evaluated using RAW 264.7 murine macrophages in serial dilution starting at a concentration of 64 μM. At the initial peptide concentration, macrophages maintained their cell viability at 90.1% after 24 h of incubation (Fig. 10). At the concentrations tested, the toxicity index (IC50), which is the concentration capable of inducing the death of 50% of cells, was not reached. These results denote that RK8 exhibits low cytotoxicity.
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Fig. 10. Cell viability test of RAW 264.7 murine macrophages in the presence of the RK8 peptide. Values are means ± SD of three repetitions.
P > 0.0001 compared with cells treated with the control vehicle.
Acute in vivo Toxicity in G. mellonella Assessment
As shown in Fig. 11, during the 72 h of the experiment, none of the RK8 concentrations promoted the death of
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Fig. 11. The number of dead
G. mellonella larvae from 0 h to 72 h was treated with samples at concentrations of 16 μM, 160 μM, and 960 μM (n = 10).
Peptide Stability Test
After incubating with fetal bovine serum (FBS) at various time intervals, we observed the stability effect of RK8 (Fig. 12). At time zero, 100% of the area under the RK8 peak curve was determined, and this area was used as a parameter to assess the other time intervals. During the first 45-min incubation with 25% FBS, the total area analysed, corresponding to the intact structure of RK8, was unaffected. After 60 min of incubation, we calculated a 12.2% reduction in the area under the curve. Over 120 min, there was a 40% decrease in the overall area, demonstrating RK8's ability to sustain a high level of stability against FBS.
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Fig. 12. Resistance to degradation of the RK8 peptide incubated in a solution containing 25% FBS. The data represent an assay performed in triplicate, and the percentage of area over the curve was calculated using the Chromeleon Chromatography Data System (CDS) 7 software (Thermo Fisher Scientific).
P > 0.0001 compared with cells treated with the control vehicle.
Discussion
The peptide RK8 comprises 8 amino acid residues, classifying it as an ultrashort peptide belonging to the subfamily of antimicrobial peptides. This specific group offers multiple economic benefits because of its ease of synthesis and purification, resulting in resource and time savings for the synthesis process [20]. The mode of action of the ultrashort cationic peptides is not entirely understood. Recent studies mention that they penetrate phospholipid bilayers, disrupting essential processes such as respiration and cell wall biosynthesis [21]. As mentioned, shorter peptides adopt an extended conformation, which may be rich in one or more specific amino acids. Their mechanism of action is associated with efficiency in internalisation and membrane composition [22].
Boman index calculates the potential interaction of the peptide with membranes (protein-membrane) based on the amino acid sequence. RK8 peptide exhibits a Boman index of 4.93 kcal/mol, with values greater than 2.48 kcal/mol, demonstrating a high potential for binding to biological membranes [23]. The RK8 sequence repeats three amino acids: arginine and lysine, which possess a positive charge, and tryptophan, a nonpolar aromatic amino acid. This combination creates regions with both hydrophobic and hydrophilic properties, facilitating interactions with bacterial membranes. Including mostly positive residues increases the peptide's net electrical charge, enhancing the likelihood of electrostatic attraction between the AMP and the predominantly anionic microbial membrane [24]. Research suggests that in AMPs, there is an approximate 50% increase in the presence of essential amino acids like Arginine and Lysine compared to the overall genomic content. Conversely, acidic amino acids exhibit a notable reduction, approximately 75% less than expected. AMPs commonly contain hydrophobic amino acids, demonstrating their prevalence in these peptides. These findings are consistent because they are designed using simplified sets of amino acids, predominantly comprising essential and hydrophobic residues. Specifically, straightforward cationic/hydrophobic peptides, such as those containing Arginine and Tryptophan, demonstrate notable antimicrobial efficacy [25].
CD analysis reveals that the RK8 peptide lacks a structured conformation, meaning its amino acid residues do not form regular secondary structures. These findings categorise it as an extended AMP, a characteristic often observed in peptides rich in amino acids such as Arginine (Arg), Tryptophan (Trp), or Proline (Pro). Notably, Trp and Arg residues are prevalent in peptides with fewer than 15 residues, exemplified by the active antimicrobial fragments RRWQWR and RAWVAWR from bovine lactoferricin and human lysozyme, respectively. Screening of combinatorial peptide libraries uncovered potent broad-spectrum activity in the hexameric sequence RRWWRF [26].
In Gram-negative bacterial strains, RK8 demonstrated consistent MIC and MBC values, exhibiting a bactericidal effect within concentrations ranging from 0.5 to 4 μM against
In clinical isolates exhibiting resistance, RK8 displayed its most favourable MIC value against
The treatment of resistant pathogens often involves multiple antibiotic combinations to achieve a synergistic effect. However, this approach remains contentious because of the heightened toxicity risk, organ damage, and the potential for selecting resistant strains. In vitro synergy evaluations typically utilise checkerboard titration, although some studies also employ animal models. According to Pletzer
RK8 disrupted approximately 40% of mature
The outer membrane of Gram-negative bacteria involves an asymmetric bilayer comprising phospholipids and lipopolysaccharides (LPS). The initial interaction between AMPs and the membrane of Gram-negative bacteria involves electrostatic attractions between the positively charged peptide and the negatively charged LPS of the outer membrane, ultimately resulting in membrane disruption [37, 38]. To investigate the potential mechanism of action of RK8, we assessed whether it induces damage to bacterial membranes using the Sytox green reagent. Sytox green is a DNA intercalator that cannot penetrate intact membranes. By conducting fluorescence microscopy studies, we aimed to detect the leakage of intracellular contents into the extracellular environment [39]. In this study, we aimed to observe an increase in fluorescence when bacteria were exposed to the peptide, illustrating potential damage to microbial membranes. Our results revealed that membrane permeation occurred more rapidly in Gram-negative strains than in Gram-positive ones. The time required for Sytox green permeation was shorter than the interval needed to observe a reduced area under the stability test curve. This suggests that the peptide's action on bacterial membranes theoretically precedes its degradation by peptidases.
When designing a peptide, it is crucial to consider the potential cytotoxic effects on eukaryotic cells. The relationship between structure and activity plays an essential role in determining the selectivity of the peptide towards its target cells [40]. Eukaryotic plasma membranes primarily contain neutrally charged or zwitterionic lipids, unlike bacterial membranes containing anionic components. Therefore, the interaction of AMPs with other cells is generally considered selective [41]. The concentrations tested proved entirely safe, with negligible hemolysis observed even at concentrations much higher than the MIC and MBC. The presence of RK8 did not significantly affect the cell viability of murine macrophages if its cytotoxicity index (IC50), signalling the concentration capable of inducing cell death in 50% of the cells, could not be determined in the assay, thus suggesting potential pharmacological safety.
There is a growing trend towards minimising the use of mammals in vivo testing, leading to increased reliance on in vitro toxicity data derived from cell cultures. While in vitro models are useful for studying certain aspects of compound metabolism, such as absorption rates, biotransformation, distribution, and excretion, they may not fully replicate the complexities of these processes in the human body.
The peptide's stability in blood is critical for its in vivo antimicrobial effectiveness, demanding prolonged circulation at high blood concentrations. Peptides' in vivo stability in blood can be simulated by assessing their stability in serum or plasma in vitro [19]. In this study, the RK8 peptide was tested in the presence of fetal bovine serum to determine its half-life against enzymatic degradation. Nguyen
Conclusion
The findings from this study underscore the efficacy of the design approach employed in creating RK8, yielding a novel ultrashort and extended AMP. RK8 exhibited significant efficacy against Gram-positive and Gram-negative bacteria, including multidrug-resistant clinical isolates. These results suggest that the bacterial plasma membrane may serve as a target for RK8, acting before its degradation by peptidases found in blood plasma. In vitro toxicity assessments against erythrocytes and macrophages and in vivo experiments with
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant numbers 305679/2016-3, 430694/2016-4, 426912/2018-7, 302175/2020-2), financiadora de Estudos e Projetos (FINEP), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado do Mato Grosso do Sul (Fundect grant numbers 009/2015, 047/2018, 040/2020, and 132/2020). This study was financed in part by the Fundação Universidade Federal de Mato Grosso do Sul – UFMS/MEC – Brazil.
Author Contributions
Cavalheiro, M.C.M.: Conceptualization, Methodology; De Oliveira, C.F. R: Conceptualization, Methodology; Boleti, A.P.B.: Original Draft Preparation; Rocha, L. S: Original Draft Preparation; Jacobowski, A.C.: Original Draft Preparation; Macedo, M.L.R.: Conceptualization, Visualization, Supervision.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Muteeb G, Rehman MT, Shahwan M, Aatif M. 2023. Origin of antibiotics and antibiotic resistance, and their impacts on drug development: A narrative review.
Pharmaceuticals 16 : 1-54. - Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH,
et al . 2018. Antibiotic resistance: a rundown of a global crisis.Infect. Drug Resist. 11 : 1645-1658. - Chinemerem Nwobodo D, Ugwu MC, Oliseloke Anie C, Al-Ouqaili MTS, Chinedu Ikem J, Victor Chigozie U,
et al . 2022. Antibiotic resistance: the challenges and emerging strategies for tackling a global menace.J. Clin. Lab. Anal. 36 : e24655. - Terreni M, Taccani M, Pregnolato M. 2021. New antibiotics for multidrug-resistant bacterial strains: latest research developments and future perspectives.
Molecules 26 : 2671. - Zhang QY, Yan Z Bin, Meng YM, Hong XY, Shao G, Ma JJ,
et al . 2021. Antimicrobial peptides: mechanism of action, activity and clinical potential.Mil. Med. Res. 8 : 1-25. - Lade H, Kim JS. 2021. Bacterial targets of antibiotics in methicillin-resistant
Staphylococcus aureus .Antibiotics 10 : 398. - Elibe Mba I, Innocent Nweze E. 2022. Antimicrobial peptides therapy: an emerging alternative for treating drug-resistant bacteria.
Yale J. Biol. Med. 95 : 445-463. - Pirtskhalava M, Vishnepolsky B, Grigolava M, Managadze G. 2021. Physicochemical features and peculiarities of interaction of amp with the membrane.
Pharmaceuticals 14 : 471. - Bakare OO, Gokul A, Niekerk LA, Aina O, Abiona A, Barker AM,
et al . 2023. Recent progress in the characterization, Synthesis, delivery procedures, treatment strategies, and precision of antimicrobial peptides.Int. J. Mol. Sci. 24 : 11864. - Almeida CV, de Oliveira CFR, Almeida LH de O, Ramalho SR, Gutierrez C de O, Sardi J de CO,
et al . 2024. Computer-made peptide RQ18 acts as a dual antifungal and antibiofilm peptide though membrane-associated mechanisms of action.Arch. Biochem. Biophys. 753 : 109884. - Wang G, Li X, Wang Z. 2016. APD3: the antimicrobial peptide database as a tool for research and education.
Nucleic Acids Res. 44(D1) : D1087-1093. - Timmons PB, Hewage CM. 2021. APPTEST is a novel protocol for the automatic prediction of peptide tertiary structures.
Brief Bioinform. 22 : bbab308. - BLISS CI. 1939. The toxicity of poisons applied jointly.
Ann. Appl. Biol. 26 : 585-615. - Almeida CV, de Oliveira CFR, dos Santos EL, dos Santos HF, Júnior EC, Marchetto R,
et al . 2021. Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes.Biochim. Biophys. Acta Gen. Subj. 1865 : 129937. - Mohanram H, Bhattacharjya S. 2016. Salt-resistant short antimicrobial peptides.
Biopolymers 106 : 345-356. - Walker JN, Horswill AR. 2012. A coverslip-based technique for evaluating
Staphylococcus aureus biofilm formation on human plasma.Front. Cell. Infect. Microbiol. 2 : 39. - Uggerhøj LE, Poulsen TJ, Munk JK, Fredborg M, Sondergaard TE, Frimodt-Moller N,
et al . 2015. Rational design of alpha-helical antimicrobial peptides: Dós and don'ts.ChemBioChem. 16 : 242-253. - Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J. Immunol. Methods 65 : 55-63. - Powell M, Stewart S, Otvos L, Urge L, Gaeta FCA, Sette A. 1993. Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum.
Pharm. Res. 10 : 1268-1273. - Almaaytah A, Qaoud MT, Mohammed GK, Abualhaijaa A, Knappe D, Hoffmann R,
et al . 2018. Antimicrobial and antibiofilm activity of UP-5, an ultrashort antimicrobial peptide designed using only arginine and biphenylalanine.Pharmaceuticals 11 : 3. - Kraszewska J, Beckett MC, James TC, Bond U. 2016. Comparative analysis of the antimicrobial activities of plant defensin-like and ultrashort peptides against food-spoiling bacteria.
Appl. Environ. Microbiol. 82 : 4288-4298. - Kalafatovic D, Giralt E. 2017. Cell-penetrating peptides: design strategies beyond primary structure and amphipathicity.
Molecules 22 : 1929. - Kardani K, Bolhassani A. 2021. Exploring novel and potent cell penetrating peptides in the proteome of SARS-COV-2 using bioinformatics approaches.
PLoS One 16 : e0247396. - Ye H. 2018. Molecular design of antimicrobial peptides based on hemagglutinin fusion domain to combat antibiotic resistance in bacterial infection.
J. Pept. Sci. 24 . doi: 10.1002/psc.3068. - WC W. 2010. Describing the mechanism of antimicrobial peptide action with the interfacial activity model.
Acs Chem. Biol. 5 : 905-917. - Nguyen LT, Haney EF, Vogel HJ. 2011. The expanding scope of antimicrobial peptide structures and their modes of action.
Trends Biotechnol. 29 : 464-472. - Wei SY, Wu JM, Kuo YY, Chen HL, Yip BS, Tzeng SR,
et al . 2006. Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity.J. Bacteriol. 188 : 328-334. - Almeida LH de O, Oliveira CFR de, Rodrigues M de S, Neto SM, Boleti AP de A, Taveira GB,
et al . 2020. Adepamycin: design, synthesis and biological properties of a new peptide with antimicrobial properties.Arch. Biochem. Biophys. 691 : 108487. - McKenna M. 2013. Antibiotic resistance: the last resort.
Nature 499 : 394-396. - Menousek J, Mishra B, Hanke ML, Heim CE, Kielian T, Wang G. 2012. Database screening and in vivo efficacy of antimicrobial peptides against methicillin-resistant
Staphylococcus aureus USA300.Int. J. Antimicrob. Agents 39 : 402-406. - Das T, Nath C, Das P, Ghosh K, Logno TA, Debnath P,
et al . 2023. High prevalence of ciprofloxacin resistance inEscherichia coli isolated from chickens, humans and the environment: an emerging one health issue.PLoS One 18 : e0294043. - Shariati A, Arshadi M, Khosrojerdi MA, Abedinzadeh M, Ganjalishahi M, Maleki A,
et al . 2022. The resistance mechanisms of bacteria against ciprofloxacin and new approaches for enhancing the efficacy of this antibiotic.Front. Public Health 10 : 1025633. - Pletzer D, Mansour SC, Hancock REW. 2018. ynergy between conventional antibiotics and anti-biofilm peptides in a murine, subcutaneous abscess model caused by recalcitrant ESKAPE pathogens.
PLoS Pathog. 14 : e1007084. - Lin MF, Lin YY, Lan CY. 2019. A method to assess influence of different medical tubing on biofilm formation by
Acinetobacter baumannii .J. Microbiol. Methods 160 : 84-86. - Lin Y, Chang RYK, Britton WJ, Morales S, Kutter E, Chan HK. 2018. Synergy of nebulized phage PEV20 and ciprofloxacin combination against
Pseudomonas aeruginosa .Int. J. Pharm. 551 : 158-165. - Rishi P, Vashist T, Sharma A, Kaur A, Kaur A, Kaur N,
et al . 2018. Efficacy of designer K11 antimicrobial peptide (a hybrid of melittin, cecropin A1 and magainin 2) againstAcinetobacter baumannii -infected wounds.Pathog. Dis. 76 . doi: 10.1093/femspd/fty072. - Powers JPS, Hancock REW. 2003. The relationship between peptide structure and antibacterial activity.
Peptides 24 : 1681-1691. - Sharma P, Ayappa KG. A 2022. Molecular dynamics study of antimicrobial peptide interactions with the lipopolysaccharides of the outer bacterial membrane.
J. Membr. Biol. 255 : 665-675. - Thakur S, Cattoni DI, Nöllmann M. 2015. The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent.
Eur. Biophys. J. 44 : 337-348. - Shagaghi N, Palombo EA, Clayton AHA, Bhave M. 2018. Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality.
World J. Microbiol. Biotechnol. 34 : 62. - Travkova OG, Moehwald H, Brezesinski G. 2017. The interaction of antimicrobial peptides with membranes.
Adv. Colloid Interface Sci. 247 : 521-532. - Allegra E, Titball RW, Carter J, Champion OL. 2018.
Galleria mellonella larvae allow the discrimination of toxic and non-toxic chemicals.Chemosphere 198 : 469-472. - Browne N, Heelan M, Kavanagh K. 2013. An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes.
Virulence 4 : 597-603. - Wojda I. 2017. Immunity of the greater wax moth
Galleria mellonella .Insect Sci. 24 : 342-357. - Nguyen LT, Chau JK, Perry NA, de Boer L, Zaat SAJ, Vogel HJ. 2010. Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs.
PLoS One 5 : e12684. - Jia F, Zhang Y, Wang J, Peng J, Zhao P, Zhang L,
et al . 2019. The effect of halogenation on the antimicrobial activity, antibiofilm activity, cytotoxicity and proteolytic stability of the antimicrobial peptide Jelleine-I.Peptides 112 : 56-66.
Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2024; 34(11): 2231-2244
Published online November 28, 2024 https://doi.org/10.4014/jmb.2405.05011
Copyright © The Korean Society for Microbiology and Biotechnology.
Evaluating the Antimicrobial Efficacy of a Designed Synthetic Peptide against Pathogenic Bacteria
Maria Caroline de Moura Cavalheiro1, Caio Fernando Ramalho de Oliveira1, Ana Paula de Araújo Boleti1, Layza Sá Rocha1, Ana Cristina Jacobowski1, Cibele Nicolaski Pedron2, Vani Xavier de Oliveira Júnior2, and Maria Lígia Rodrigues Macedo1*
1Protein Purification Laboratory and its Biological Functions; Faculty of Pharmaceutical Sciences, Food and Nutrition; Faculty of Pharmacy, Food and Nutrition; Federal University of Mato Grosso do Sul (UFMS), Campo Grande, Mato Grosso do Sul, Brazil
2Center for Natural and Human Sciences of the Federal University of ABC (UFABC), São Paulo, SP, Brazil
Correspondence to:Maria Lígia Rodrigues Macedo, Ligiamacedo18@gmail.com
Abstract
Recent research has focused on discovering peptides that effectively target multidrug-resistant bacteria while leaving healthy cells unharmed. In this work, we describe the antimicrobial properties of RK8, a peptide composed of eight amino acid residues. Its activity was tested against multidrug-resistant Gram-negative and Gram-positive bacteria. RK8's efficacy in eradicating mature biofilm and increasing membrane permeability was assessed using Sytox Green. Cytotoxicity assays were conducted both in vitro and in vivo models. Circular dichroism analysis revealed that RK8 adopted an extended structure in water and sodium dodecyl sulfate (SDS). RK8 exhibited MICs of 8-64 μM and MBCs of 4-64 μM against various bacteria, with higher effectiveness observed in Methicillin-resistant Staphylococcus aureus (MRSA) and E. coli KPC+ strains than others. Ciprofloxacin and Vancomycin showed varying MIC and MBC values lower than RK8 for Gram-positive bacteria, but competitive for Gram-negative bacteria. The combination of RK8 and ciprofloxacin showed a synergistic effect. The RK8 peptides could reduce 38% of the mature Acinetobacter baumannii biofilm. Sytox Green reagent achieved 100% membrane permeation of Gram-positive and Gram-negative bacteria. The RK8 peptide did not show cytotoxic effects against murine macrophages (64 μM), erythrocytes (100 μM) or Galleria mellanella larvae (960 μM). In the stability test against peptidases, the RK8 peptide was stable, maintaining around 60% of the molecule intact after 120 min of incubation. These results highlight the potential of RK8 to be a promising strategy for developing a new antimicrobial and antibiofilm agent, inspiring and motivating further research in antimicrobial peptides.
Keywords: Bacterial resistance, biofilm, drug design
Introduction
The recent surge in multidrug-resistant bacteria, a pressing issue in public health, directly results from the overuse of antibiotics and the sluggish pace of antimicrobial drug development [1]. While discovering antimicrobials has historically been a life-saving breakthrough, the persistent challenge of multi-resistance infections underscores the urgent need for new, less toxic, and more effective antimicrobials [2-4].
A new proposal for new drug development is antimicrobial peptides (AMPs). These molecules act as effective antibiotics, whose antimicrobial activity results from disrupting the cell membrane integrity. Unlike conventional antimicrobial agents, AMPS acts on multiple targets within the cell [5]. Most antibiotics interact with and inhibit specific biochemical processes in bacteria, such as cell wall synthesis (peptidoglycan), protein synthesis (translation), DNA replication, RNA synthesis (transcription), and folic acid biosynthesis. However, some antibiotics interfere with ion channels and cause bacteriolysis [6]. AMPs exert their activity mainly through the rupture of the bacterial cell membrane, with consequent cell death or immunomodulatory actions [7].
AMPs have garnered significant interest as potential antibiotics for the future. Cellular organisms internally produce these small molecules, encompassing 2-50 amino acids and are essential components of the host's natural immune response. Their small size and diverse mechanisms of action, which impede the development of bacterial resistance, make them a promising alternative to conventional therapeutics. AMPs also display rapid bactericidal action, independent of phenotypic resistance, and good solubility in water with thermal stability [8, 9].
Their cationic and amphiphilic properties drive AMP antibacterial activity. It presents a variable net positive charge and about 50% hydrophobic amino acids. These peptides establish electrostatic interactions with anionic bacterial membranes, rupturing them. Their hydrophobic and hydrophilic properties facilitate interaction with the lipid tails of lipopolysaccharide (LPS) and the hydrophilic heads of phospholipids in the bacterial membrane. For this reason, it compromises the membrane permeability and leads to bacterial cell death [9]. There is significant interest in researching natural products despite their limitations. Applying synthetic biology can overcome production challenges and enhance chemical diversity. Computational tools assist in peptide sequence design and structure with bactericidal action. The broad-spectrum in vitro, in vivo and silico antibacterial activity of AMPs offers a promising alternative to conventional therapeutics [10].
Our research involved the development of the RK8 peptide using computer-aided methods to leverage its physicochemical properties in investigating its antibacterial activity and cell selectivity. The peptide sequence comprises eight amino acid residues and has a molecular mass of 1,273.54 Da, net charge +5, and hydrophobic ratio of 38%. Its amino acid residues do not fold into regular secondary structure elements. Therefore, we investigated its antimicrobial, antibiofilm, and toxicity properties in the present study. In addition, our study has illustrated the synergistic interaction of RK8 in combination with ciprofloxacin, along with its non-toxic attributes in both in vitro and in vivo settings. Finally, we also determined that its mechanism of action involves membrane damage. However, it acts differently on Gram-negative and Gram-positive membranes.
Material and Methods
Peptide Design and Synthesis
Based on the structural characteristics shared by arginine- and tryptophan-rich peptides, our researchers created the RK8 peptide sequence. As our aims involved the synthesis of an ultrashort peptide, we started with a sequence of eight amino acid residues containing arginines, lysines, and tryptophan. We defined a net electrical charge of +5 to secure the sequence distributed between two arginines and three lysines. Three tryptophan residues influence 37.5% of the hydrophobic amino acid composition. The molecular structure was carefully devised to incorporate specific amino acid residues, allowing it to exhibit concurrently hydrophilic and hydrophobic properties. The Antimicrobial Peptide Database (APD3) and the PepDraw server (http://pepdraw.com/) had the resulting RKWRKWWK sequence submitted and analysed. Physicochemical parameters such as isoelectric point, molecular weight, and side chain arrangement were also evaluated [11].
Aminotech, in Diadema-SP, carried out the peptide synthesis. A synthesis > 95% was requested. Mass spectrometry (ESI-TOF) and high-performance liquid chromatography (HPLC) evaluated molecular mass and purity parameters. The concentration of the RK8 peptide in solution was determined by determining the absorbance at 280 nm, based on the extinction coefficient of the tryptophan residue (ε = 5.560 mol/ml). RK8 was solubilised in ultrapure water, and its absorbance was measured using an Evolution 201 spectrophotometer (Thermo Fisher Scientific, USA) at 280 nm (A280). To determine its concentration (C), the following formula was used:
C (mg/ml) = (A280 × FD × PM) / ε
In Daltons, FD is the dilution factor, and PM is the molecular weight of the peptide.
Modelling and Validation
The peptide's secondary structure was determined using the APPTEST server (https://research.timmons.eu/apptest), which interactive simulations complete atomic models [12]. After modelling, we performed validation using PROCHECK software (https://www.ebi.ac.uk/thorntonsrv/software/PROCHECK/) to verify the stereochemistry and atoms' spatial arrangement of the peptide.
Circular Dichroism
CD analyses were performed with a Jasco J-1100 spectropolarimeter (Jasco Inc., Japan) using a quartz cuvette with a 0.1 cm optical path. RK8 peptide was synthesised at a concentration of 30 μM in an aqueous solution and subjected to analysis in ultra-pure water in the presence of SDS (sodium dodecyl sulfate). The experiment involved six scans at 25°C, covering wavelengths from 190 to 280 nm. As part of the peptide preparation process, we gathered spectra of the used solutions, referring to them as blank solutions. We collect the resulting data after subtracting the implemented spectra from the blank solution. Afterwards, the data was transformed into residual molar ellipticity [θ] using the following equation:
[θ] =
Where θ is the ellipticity measured in milliseconds, C is the concentration in molar units, l is the path length in cm, and nr is the number of amino acid residues.
Microorganisms
To determine the antimicrobial activity of the RK8 peptide, bacteria purchased commercially and catalogued in the American Type Culture Collection (ATCC, USA) were used: Gram-negative:
Strains of multidrug-resistant clinical isolates were tested: Gram-negative
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Assays
Clinical and Laboratory Standards Institute (CLSI) guidelines performed the peptide MIC assays. Bacterial colonies were isolated on the Muller Hinton Agar (MHA) and plated at a final concentration of 1.5 × 105 CFU/ml. The peptides were tested at concentrations ranging from 0.5 μM to 32 μM. Antibiotic control, positive control, was carried out using 10 μl of ciprofloxacin (for targeting Gram-negative bacteria) or vancomycin (for targeting Gram-positive bacteria) at a concentration of 1 μM, in combination with 90 μl of MH culture medium.
The term "MIC" refers to the minimum inhibitory concentration of a peptide, which is the lowest concentration capable of reducing bacterial growth by 90% or more. For the results, bacterial growth was determined by visual inspection and confirmed by absorbance measurement at 595 nm after 24 h of incubation using a Multiskan GO microplate reader (Thermo Fisher Scientific, USA). For assays involving resistant strains such as MRSA strains and clinical isolates of
The MBC was determined based on the MIC results. Three replicates of 10 μl were extracted from the microplate wells, placed on MHA, and then kept in an incubator at 37°C for 24 h. The MBC was determined to have the lowest peptide concentration, and no bacterial growth was detected. All experiments were performed in triplicate.
RK8 and Ciprofloxacin Synergistic Effect
An investigation of the potential synergistic effect of combining the RK8 peptide with the quinolone ciprofloxacin antibiotic was conducted. The assay was performed in a 96-well microplate, where the two drugs, RK8 and ciprofloxacin, were present. Serial dilution was undertaken to establish a range of concentrations aligned with their corresponding minimum inhibitory concentration (MIC) values. Only the multidrug-resistant strains
An assessment of synergistic effects was conducted using synergy scoring models on the SynergyFinder platform (https://synergyfinder.org/). The models included Highest Single Agent (HSA), Loewe Additivity, Bliss Independence, and Zero Interaction Potency (ZIP). Loewe Additivity is a model that assumes that drugs with similar modes of action produce an additive effect, identifying synergy if the observed effect exceeds this additive expectation. These models compare the observed combination effect to expected outcomes, identifying synergy when the observed effect exceeds the predicted effect based on each model's assumptions. We used the Bliss model to calculate the synergistic impact of RK8 in combination with ciprofloxacin. This model assumes that the two drugs exert their effects independently, and the expected combination effect can be calculated based on the probability of independent events [13].
yB L ISS = y1+y2-y1·y2
RK8 concentrations ranging from 0 to 16 μM were combined to assemble the concentration matrix with ciprofloxacin concentrations ranging from 0 to 32 μM. For the analyses carried out with
Membrane Permeability
Membrane permeability was investigated as described by Mohanram & Bhattacharjya (2016), and Almeida
Biofilm Activity
The RK8 eradication effect on mature
The microplate was incubated with the bicarbonate plasma solution overnight at 4°C. The following day, the wells were aspirated and washed once with 500 μl of saline. A culture of
After biofilm formation, the wells were washed twice with a BHI medium containing 0.4% glucose to remove non-adherent bacteria. They received 500 μl of the medium used in the assay containing RK8 at the MIC and half this concentration (MIC /two). Biofilm control received only a BHI medium containing 0.4% glucose, and control with the antibiotic ciprofloxacin at MIC (128 μM) was also performed. The biofilm was incubated under constant agitation (100 rpm) at 37°C for 24 h to analyse the effect of RK8 on eradicating the mature form of the
The plate was incubated at 37°C for 30 min, the culture medium was aspirated, and the reduced formazan crystals were dissolved with Dimethyl sulfoxide (DMSO). Three aliquots from each of the wells were transferred to a microplate (
Cytotoxic Effects
Hemolytic activity. As hemolysis is an effect widely observed among antimicrobial peptides, we investigated the RK8 peptide hemolytic activity based on the protocol proposed by Uggerhøj
Cellular viability. RAW 264.7 murine macrophages' cellular viability was evaluated in the presence of the RK8 peptide, according to the method proposed by Mosmann [18], using the enzymatic reduction of the reagent 3-(4,5-dimethylthiazol-2-yl bromide) -2,5-diphenyltetrazolium (MTT). The cells were provided by Octávio Franco (Dom Bosco Catholic University - UCDB) and cultivated in Dulbeccós Modified Eagle Medium - high glucose (DMEM), supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml of penicillin and 100 μg/ml of streptomycin (Gibco, Brazil), at 37°C in an incubator at 5% CO2.
After detaching from the culture bottle, their density was determined, and they were seeded in a 96-well microplate at a density of 6 × 103 cells per well. After being placed in microplates, the cells were incubated for 24 h. The culture medium was replaced with a DMEM culture medium lacking SFB supplementation. Instead, it contained a serial dilution of RK8 ranging from 1 to 64 μM. After 24 h, the medium containing RK8 was removed and replaced with 100 μl of DMEM medium containing MTT 0.5 mg/ml. After four hours of incubation, the medium was removed again, and the reduced formazan crystals were resuspended with the addition of 100 μl of DMSO. Then, the absorbance of the wells was determined at 630 nm in a Varioskan microplate reader (Thermo Fisher Scientific). Three independent experiments were performed in triplicate. Cell viability was calculated from the following formula:
Cell viability (%) = (AbsSample÷AbsNegative control) × 100
Peptide Stability
The RK8 peptide stability was evaluated using a 25% FBS solution prepared in ultrapure water. This assay was performed by adding 40 μl of the peptide solution (10 mg/ml) to 1 ml of 25% FBS solution. The mixture was maintained at 30°C. Aliquots of 150 μl were collected after 0, 15, 30, 45, 60, and 120 min, mixed with ten μl of Trifluoroacetic Acid (TFA) and centrifuged at 14 000 rpm at 4°C for 10 min [19]. High-Performance Liquid Chromatography (HPLC) monitored the stability of the peptide using the C-18 reversed-phase hydrophobic column (μ Bondapak C18, 3.9 × 300 mm, Waters, USA) at 25°C. The peptide's remaining percentage during degradation kinetics was calculated by integrating the peak area corresponding to the RK8 peptide, injecting 40 μl of the incubation product in triplicate. The assay was conducted in an Ultimate 3000 chromatograph (Thermo Fisher Scientific). A separation program of 60 min at 1 ml/min was implemented, with monitoring of the 220 and 280 nm wavelengths. Thermo Scientific's ChromeleonTM Chromatography Data System (CDS) 7 software determined the area under the curve.
Statistical Analysis
The experimental results showed that statistical significance was determined by a one-way Student's
Results
RK8 Peptide Racional Design
RK8 sequence (RKWRKWWK) has eight amino acid residues, +5 net charge, and hydrophobicity of 38%, with the first residue being arginine (R) and the eighth being lysine (K) (Table 1). Peptide sequence was created from a two sequences combination: 2 repetitions of a three amino acids sequence: Arg–Lys–Trp, occupying positions 1 to 6, and an inverted repeat of residues 5 and 6 (Lys–Trp), giving rise to amino acids at positions 7 and 8 (Trp–Lys). The structural design was produced to ensure positively charged residues at the amino and carboxyl termini. The physicochemical properties and primary structure of RK8 were collected from the APD3 database and the PepDraw platform. To validate this, PROCHECK software was used to confirm the chiral centres and ensure no steric hindrances in the correct orientation (Fig. 1).
-
Table 1 . Physicochemical characteristics of the RK8 peptide attained from the APD3 server..
Sequence Total net charge Boman Index (Kcal/mol) pI Molecular weight (Da) Hydrophobic reason (%) RKWRKWWK +5 4.93 12.51 1.273,54 38%
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Figure 1. Primary structure of the RK8 peptide, collected through the PepDraw server.
Circular Dichroism
After synthesising, we characterised the RK8 secondary structure through Circular Dichroism (CD) studies. The results exhibit RK8 dissolution in water and 30 mM SDS. RK8 exhibits a random structure in both solutions, with low-magnitude negative bands at 200 nm (Fig. 2), which suggests that the peptide does not adopt an alpha-helical conformation. Therefore, we speculate that the peptide has an extended conformation.
-
Figure 2. Circular dichroism spectra of the RK8 peptide at 30 μM, in both water and SDS at 25°C.
Antibacterial Activity
The MIC was determined using the broth microdilution method (CLSI, 2012), using different concentrations of RK8 against susceptible and resistant bacteria. For Gram-positive bacteria, RK8 exhibited MIC values ranging from 8 μM to > 64 μM and MBC values from 8 μM to 64 μM. Ciprofloxacin showed MIC values between 1 μM and four μM, with corresponding MBC values of 1 μM to 8 μM. Vancomycin had consistent MIC and MBC values of 1μM for
-
Table 2 . MIC and MBC values, in μM, of Gram-positive and Gram-negative strains were evaluated with an initial density of bacteria corresponding to 1.5 × 105 CFU/ml..
Microorganisms (1.5 × 105 CFU/ ml) Bacteria RK8 Ciprofloxacin Vancomycin MIC (μM) MBC (μM) MIC (μM) MBC (μM) MIC (μM) MBC (μM Gram-positive Staphylococcus aureus (MRSA) ATCC 335918 8 4 8 1 1 Staphylococcus aureus (MRSA) ATCC 4330016 8 2 2 1 0.5 Staphylococcus saprophyticus ATCC 29970>64 64 1 1 0.5 1 Gram-negative Escherichia coli KPC+ 00181244616 8 32 1 0.5 0.5 Escherichia coli ATCC 35218>64 2 1 2 8 1 Acinetobacter baumannii IC 00332121664 4 128 1 4 1 Acinetobacter baumannii ATCC 19906>64 4 1 1 4 1 Pseudomonas aeruginosa ATCC 27853>64 64 0.5 8 0.5 1 Klebsiella pneumoniae ATCC 700603>64 64 12 1 8 1 MIC, Minimum Inhibitory Concentration; MBC, minimum Bactericidal Concentration.
Following the detection of antibacterial activity against susceptible strains, we evaluated the effectiveness of RK8 against multi-resistant bacterial strains. A heat map was used to improve the clarity of RK8's antibacterial activity data, making it easier to interpret and present the peptide's efficacy against different bacterial strains and concentrations (Fig. 3A and 3B).
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Figure 3. Heatmap graph showing the MICs of RK8 (A) and ciprofloxacin (B) for resistant bacteria, evaluated with an initial density of bacteria corresponding to 1.5 × 105 CFU/ml. Darker red represents growth inhibition and was considered to establish the MIC; lighter shades reveal partial growth inhibition, while dark blue shades show microbial growth.
The heat map primarily evaluates the effectiveness of RK8. In this context, it is possible to denote that for
In summary, RK8 is more effective against
RK8 and Ciprofloxacin Synergistic Effect
The RK8 and ciprofloxacin combination showed a Bliss Synergy Score 3.99 against
-
Figure 4. Dose-response inhibition map and Synergy Score for the combination of RK8 and ciprofloxacin against the
E. coli strain KPC+ IC 001812446.
-
Figure 5. Inhibition dose-response map and Synergy Score for the combination of RK8 and ciprofloxacin against the
A. baumannii strain IC 003321216.
Antibiofilm Activity
The bacterial eradication biofilm assay was performed with the
-
Figure 6. Effect of RK8 on mature biofilm of
A. baumannii IC 003321216. Viable cells are stained in green, and non-viable cells are stained in red. Image of the control slide without treatment with RK8 in (A) and slide with biofilm treated with RK8 in (B).P < 0.0001 compared with cells treated with control.
-
Figure 7. Viability of bacterial cells in mature biofilm of
A. baumannii IC 003321216, in the presence of RK8 (A) and ciprofloxacin (B).P < 0.0001 compared with cells treated with control.
The fluorescence microscopy images (Fig. 6) represent the biofilm viability assay. After exposure to RK8 at the minimum inhibitory concentration (MIC), the biofilm displayed red areas revealing cell death and green areas signifying cell viability. Image analysis using Image J revealed an 82.5% reduction in cell viability. The image shows structural damage to the biofilm, with an irregular distribution of bacteria suggesting significant damage.
Membrane Permeability
To verify whether the RK8 mechanism against bacterial cells involves damage to the cell membrane, we performed the assay with the fluorophore Sytox green, a bacterial membrane damage indicator. Fig. 8 shows that the peptide has an action mechanism that involves membrane damage. However, it acts differently on Gram-negative and Gram-positive membranes. For
-
Figure 8. Absorption of Sytox Green by
E. coli (ATCC 35218) and MRSA (ATCC 43300) treated with RK8 in MIC. Sytox green uptake was measured during 120 min of incubation with RK8.
Hemolytic Activity
The RK8 hemolytic activity was evaluated against human erythrocytes, where the peptide maximum concentration was 100 μM, performing serial dilution up to 0.78 μM. The RK8 peptide induced approximately 4.6% hemolysis at the initial concentration, having even lower percentages in the MIC bacterial strains tested values (Fig. 9).
-
Figure 9. Percentage of hemolysis in the presence of the RK8 peptide at concentrations collected from serial dilution starting at 100 μM.
P < 0.0001 compared with cells treated with control.
Cell Viability
The cytotoxic effects of RK8 against mammalian cells were evaluated using RAW 264.7 murine macrophages in serial dilution starting at a concentration of 64 μM. At the initial peptide concentration, macrophages maintained their cell viability at 90.1% after 24 h of incubation (Fig. 10). At the concentrations tested, the toxicity index (IC50), which is the concentration capable of inducing the death of 50% of cells, was not reached. These results denote that RK8 exhibits low cytotoxicity.
-
Figure 10. Cell viability test of RAW 264.7 murine macrophages in the presence of the RK8 peptide. Values are means ± SD of three repetitions.
P > 0.0001 compared with cells treated with the control vehicle.
Acute in vivo Toxicity in G. mellonella Assessment
As shown in Fig. 11, during the 72 h of the experiment, none of the RK8 concentrations promoted the death of
-
Figure 11. The number of dead
G. mellonella larvae from 0 h to 72 h was treated with samples at concentrations of 16 μM, 160 μM, and 960 μM (n = 10).
Peptide Stability Test
After incubating with fetal bovine serum (FBS) at various time intervals, we observed the stability effect of RK8 (Fig. 12). At time zero, 100% of the area under the RK8 peak curve was determined, and this area was used as a parameter to assess the other time intervals. During the first 45-min incubation with 25% FBS, the total area analysed, corresponding to the intact structure of RK8, was unaffected. After 60 min of incubation, we calculated a 12.2% reduction in the area under the curve. Over 120 min, there was a 40% decrease in the overall area, demonstrating RK8's ability to sustain a high level of stability against FBS.
-
Figure 12. Resistance to degradation of the RK8 peptide incubated in a solution containing 25% FBS. The data represent an assay performed in triplicate, and the percentage of area over the curve was calculated using the Chromeleon Chromatography Data System (CDS) 7 software (Thermo Fisher Scientific).
P > 0.0001 compared with cells treated with the control vehicle.
Discussion
The peptide RK8 comprises 8 amino acid residues, classifying it as an ultrashort peptide belonging to the subfamily of antimicrobial peptides. This specific group offers multiple economic benefits because of its ease of synthesis and purification, resulting in resource and time savings for the synthesis process [20]. The mode of action of the ultrashort cationic peptides is not entirely understood. Recent studies mention that they penetrate phospholipid bilayers, disrupting essential processes such as respiration and cell wall biosynthesis [21]. As mentioned, shorter peptides adopt an extended conformation, which may be rich in one or more specific amino acids. Their mechanism of action is associated with efficiency in internalisation and membrane composition [22].
Boman index calculates the potential interaction of the peptide with membranes (protein-membrane) based on the amino acid sequence. RK8 peptide exhibits a Boman index of 4.93 kcal/mol, with values greater than 2.48 kcal/mol, demonstrating a high potential for binding to biological membranes [23]. The RK8 sequence repeats three amino acids: arginine and lysine, which possess a positive charge, and tryptophan, a nonpolar aromatic amino acid. This combination creates regions with both hydrophobic and hydrophilic properties, facilitating interactions with bacterial membranes. Including mostly positive residues increases the peptide's net electrical charge, enhancing the likelihood of electrostatic attraction between the AMP and the predominantly anionic microbial membrane [24]. Research suggests that in AMPs, there is an approximate 50% increase in the presence of essential amino acids like Arginine and Lysine compared to the overall genomic content. Conversely, acidic amino acids exhibit a notable reduction, approximately 75% less than expected. AMPs commonly contain hydrophobic amino acids, demonstrating their prevalence in these peptides. These findings are consistent because they are designed using simplified sets of amino acids, predominantly comprising essential and hydrophobic residues. Specifically, straightforward cationic/hydrophobic peptides, such as those containing Arginine and Tryptophan, demonstrate notable antimicrobial efficacy [25].
CD analysis reveals that the RK8 peptide lacks a structured conformation, meaning its amino acid residues do not form regular secondary structures. These findings categorise it as an extended AMP, a characteristic often observed in peptides rich in amino acids such as Arginine (Arg), Tryptophan (Trp), or Proline (Pro). Notably, Trp and Arg residues are prevalent in peptides with fewer than 15 residues, exemplified by the active antimicrobial fragments RRWQWR and RAWVAWR from bovine lactoferricin and human lysozyme, respectively. Screening of combinatorial peptide libraries uncovered potent broad-spectrum activity in the hexameric sequence RRWWRF [26].
In Gram-negative bacterial strains, RK8 demonstrated consistent MIC and MBC values, exhibiting a bactericidal effect within concentrations ranging from 0.5 to 4 μM against
In clinical isolates exhibiting resistance, RK8 displayed its most favourable MIC value against
The treatment of resistant pathogens often involves multiple antibiotic combinations to achieve a synergistic effect. However, this approach remains contentious because of the heightened toxicity risk, organ damage, and the potential for selecting resistant strains. In vitro synergy evaluations typically utilise checkerboard titration, although some studies also employ animal models. According to Pletzer
RK8 disrupted approximately 40% of mature
The outer membrane of Gram-negative bacteria involves an asymmetric bilayer comprising phospholipids and lipopolysaccharides (LPS). The initial interaction between AMPs and the membrane of Gram-negative bacteria involves electrostatic attractions between the positively charged peptide and the negatively charged LPS of the outer membrane, ultimately resulting in membrane disruption [37, 38]. To investigate the potential mechanism of action of RK8, we assessed whether it induces damage to bacterial membranes using the Sytox green reagent. Sytox green is a DNA intercalator that cannot penetrate intact membranes. By conducting fluorescence microscopy studies, we aimed to detect the leakage of intracellular contents into the extracellular environment [39]. In this study, we aimed to observe an increase in fluorescence when bacteria were exposed to the peptide, illustrating potential damage to microbial membranes. Our results revealed that membrane permeation occurred more rapidly in Gram-negative strains than in Gram-positive ones. The time required for Sytox green permeation was shorter than the interval needed to observe a reduced area under the stability test curve. This suggests that the peptide's action on bacterial membranes theoretically precedes its degradation by peptidases.
When designing a peptide, it is crucial to consider the potential cytotoxic effects on eukaryotic cells. The relationship between structure and activity plays an essential role in determining the selectivity of the peptide towards its target cells [40]. Eukaryotic plasma membranes primarily contain neutrally charged or zwitterionic lipids, unlike bacterial membranes containing anionic components. Therefore, the interaction of AMPs with other cells is generally considered selective [41]. The concentrations tested proved entirely safe, with negligible hemolysis observed even at concentrations much higher than the MIC and MBC. The presence of RK8 did not significantly affect the cell viability of murine macrophages if its cytotoxicity index (IC50), signalling the concentration capable of inducing cell death in 50% of the cells, could not be determined in the assay, thus suggesting potential pharmacological safety.
There is a growing trend towards minimising the use of mammals in vivo testing, leading to increased reliance on in vitro toxicity data derived from cell cultures. While in vitro models are useful for studying certain aspects of compound metabolism, such as absorption rates, biotransformation, distribution, and excretion, they may not fully replicate the complexities of these processes in the human body.
The peptide's stability in blood is critical for its in vivo antimicrobial effectiveness, demanding prolonged circulation at high blood concentrations. Peptides' in vivo stability in blood can be simulated by assessing their stability in serum or plasma in vitro [19]. In this study, the RK8 peptide was tested in the presence of fetal bovine serum to determine its half-life against enzymatic degradation. Nguyen
Conclusion
The findings from this study underscore the efficacy of the design approach employed in creating RK8, yielding a novel ultrashort and extended AMP. RK8 exhibited significant efficacy against Gram-positive and Gram-negative bacteria, including multidrug-resistant clinical isolates. These results suggest that the bacterial plasma membrane may serve as a target for RK8, acting before its degradation by peptidases found in blood plasma. In vitro toxicity assessments against erythrocytes and macrophages and in vivo experiments with
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grant numbers 305679/2016-3, 430694/2016-4, 426912/2018-7, 302175/2020-2), financiadora de Estudos e Projetos (FINEP), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado do Mato Grosso do Sul (Fundect grant numbers 009/2015, 047/2018, 040/2020, and 132/2020). This study was financed in part by the Fundação Universidade Federal de Mato Grosso do Sul – UFMS/MEC – Brazil.
Author Contributions
Cavalheiro, M.C.M.: Conceptualization, Methodology; De Oliveira, C.F. R: Conceptualization, Methodology; Boleti, A.P.B.: Original Draft Preparation; Rocha, L. S: Original Draft Preparation; Jacobowski, A.C.: Original Draft Preparation; Macedo, M.L.R.: Conceptualization, Visualization, Supervision.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Table 1 . Physicochemical characteristics of the RK8 peptide attained from the APD3 server..
Sequence Total net charge Boman Index (Kcal/mol) pI Molecular weight (Da) Hydrophobic reason (%) RKWRKWWK +5 4.93 12.51 1.273,54 38%
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Table 2 . MIC and MBC values, in μM, of Gram-positive and Gram-negative strains were evaluated with an initial density of bacteria corresponding to 1.5 × 105 CFU/ml..
Microorganisms (1.5 × 105 CFU/ ml) Bacteria RK8 Ciprofloxacin Vancomycin MIC (μM) MBC (μM) MIC (μM) MBC (μM) MIC (μM) MBC (μM Gram-positive Staphylococcus aureus (MRSA) ATCC 335918 8 4 8 1 1 Staphylococcus aureus (MRSA) ATCC 4330016 8 2 2 1 0.5 Staphylococcus saprophyticus ATCC 29970>64 64 1 1 0.5 1 Gram-negative Escherichia coli KPC+ 00181244616 8 32 1 0.5 0.5 Escherichia coli ATCC 35218>64 2 1 2 8 1 Acinetobacter baumannii IC 00332121664 4 128 1 4 1 Acinetobacter baumannii ATCC 19906>64 4 1 1 4 1 Pseudomonas aeruginosa ATCC 27853>64 64 0.5 8 0.5 1 Klebsiella pneumoniae ATCC 700603>64 64 12 1 8 1 MIC, Minimum Inhibitory Concentration; MBC, minimum Bactericidal Concentration.
References
- Muteeb G, Rehman MT, Shahwan M, Aatif M. 2023. Origin of antibiotics and antibiotic resistance, and their impacts on drug development: A narrative review.
Pharmaceuticals 16 : 1-54. - Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH,
et al . 2018. Antibiotic resistance: a rundown of a global crisis.Infect. Drug Resist. 11 : 1645-1658. - Chinemerem Nwobodo D, Ugwu MC, Oliseloke Anie C, Al-Ouqaili MTS, Chinedu Ikem J, Victor Chigozie U,
et al . 2022. Antibiotic resistance: the challenges and emerging strategies for tackling a global menace.J. Clin. Lab. Anal. 36 : e24655. - Terreni M, Taccani M, Pregnolato M. 2021. New antibiotics for multidrug-resistant bacterial strains: latest research developments and future perspectives.
Molecules 26 : 2671. - Zhang QY, Yan Z Bin, Meng YM, Hong XY, Shao G, Ma JJ,
et al . 2021. Antimicrobial peptides: mechanism of action, activity and clinical potential.Mil. Med. Res. 8 : 1-25. - Lade H, Kim JS. 2021. Bacterial targets of antibiotics in methicillin-resistant
Staphylococcus aureus .Antibiotics 10 : 398. - Elibe Mba I, Innocent Nweze E. 2022. Antimicrobial peptides therapy: an emerging alternative for treating drug-resistant bacteria.
Yale J. Biol. Med. 95 : 445-463. - Pirtskhalava M, Vishnepolsky B, Grigolava M, Managadze G. 2021. Physicochemical features and peculiarities of interaction of amp with the membrane.
Pharmaceuticals 14 : 471. - Bakare OO, Gokul A, Niekerk LA, Aina O, Abiona A, Barker AM,
et al . 2023. Recent progress in the characterization, Synthesis, delivery procedures, treatment strategies, and precision of antimicrobial peptides.Int. J. Mol. Sci. 24 : 11864. - Almeida CV, de Oliveira CFR, Almeida LH de O, Ramalho SR, Gutierrez C de O, Sardi J de CO,
et al . 2024. Computer-made peptide RQ18 acts as a dual antifungal and antibiofilm peptide though membrane-associated mechanisms of action.Arch. Biochem. Biophys. 753 : 109884. - Wang G, Li X, Wang Z. 2016. APD3: the antimicrobial peptide database as a tool for research and education.
Nucleic Acids Res. 44(D1) : D1087-1093. - Timmons PB, Hewage CM. 2021. APPTEST is a novel protocol for the automatic prediction of peptide tertiary structures.
Brief Bioinform. 22 : bbab308. - BLISS CI. 1939. The toxicity of poisons applied jointly.
Ann. Appl. Biol. 26 : 585-615. - Almeida CV, de Oliveira CFR, dos Santos EL, dos Santos HF, Júnior EC, Marchetto R,
et al . 2021. Differential interactions of the antimicrobial peptide, RQ18, with phospholipids and cholesterol modulate its selectivity for microorganism membranes.Biochim. Biophys. Acta Gen. Subj. 1865 : 129937. - Mohanram H, Bhattacharjya S. 2016. Salt-resistant short antimicrobial peptides.
Biopolymers 106 : 345-356. - Walker JN, Horswill AR. 2012. A coverslip-based technique for evaluating
Staphylococcus aureus biofilm formation on human plasma.Front. Cell. Infect. Microbiol. 2 : 39. - Uggerhøj LE, Poulsen TJ, Munk JK, Fredborg M, Sondergaard TE, Frimodt-Moller N,
et al . 2015. Rational design of alpha-helical antimicrobial peptides: Dós and don'ts.ChemBioChem. 16 : 242-253. - Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.
J. Immunol. Methods 65 : 55-63. - Powell M, Stewart S, Otvos L, Urge L, Gaeta FCA, Sette A. 1993. Peptide stability in drug development. II. Effect of single amino acid substitution and glycosylation on peptide reactivity in human serum.
Pharm. Res. 10 : 1268-1273. - Almaaytah A, Qaoud MT, Mohammed GK, Abualhaijaa A, Knappe D, Hoffmann R,
et al . 2018. Antimicrobial and antibiofilm activity of UP-5, an ultrashort antimicrobial peptide designed using only arginine and biphenylalanine.Pharmaceuticals 11 : 3. - Kraszewska J, Beckett MC, James TC, Bond U. 2016. Comparative analysis of the antimicrobial activities of plant defensin-like and ultrashort peptides against food-spoiling bacteria.
Appl. Environ. Microbiol. 82 : 4288-4298. - Kalafatovic D, Giralt E. 2017. Cell-penetrating peptides: design strategies beyond primary structure and amphipathicity.
Molecules 22 : 1929. - Kardani K, Bolhassani A. 2021. Exploring novel and potent cell penetrating peptides in the proteome of SARS-COV-2 using bioinformatics approaches.
PLoS One 16 : e0247396. - Ye H. 2018. Molecular design of antimicrobial peptides based on hemagglutinin fusion domain to combat antibiotic resistance in bacterial infection.
J. Pept. Sci. 24 . doi: 10.1002/psc.3068. - WC W. 2010. Describing the mechanism of antimicrobial peptide action with the interfacial activity model.
Acs Chem. Biol. 5 : 905-917. - Nguyen LT, Haney EF, Vogel HJ. 2011. The expanding scope of antimicrobial peptide structures and their modes of action.
Trends Biotechnol. 29 : 464-472. - Wei SY, Wu JM, Kuo YY, Chen HL, Yip BS, Tzeng SR,
et al . 2006. Solution structure of a novel tryptophan-rich peptide with bidirectional antimicrobial activity.J. Bacteriol. 188 : 328-334. - Almeida LH de O, Oliveira CFR de, Rodrigues M de S, Neto SM, Boleti AP de A, Taveira GB,
et al . 2020. Adepamycin: design, synthesis and biological properties of a new peptide with antimicrobial properties.Arch. Biochem. Biophys. 691 : 108487. - McKenna M. 2013. Antibiotic resistance: the last resort.
Nature 499 : 394-396. - Menousek J, Mishra B, Hanke ML, Heim CE, Kielian T, Wang G. 2012. Database screening and in vivo efficacy of antimicrobial peptides against methicillin-resistant
Staphylococcus aureus USA300.Int. J. Antimicrob. Agents 39 : 402-406. - Das T, Nath C, Das P, Ghosh K, Logno TA, Debnath P,
et al . 2023. High prevalence of ciprofloxacin resistance inEscherichia coli isolated from chickens, humans and the environment: an emerging one health issue.PLoS One 18 : e0294043. - Shariati A, Arshadi M, Khosrojerdi MA, Abedinzadeh M, Ganjalishahi M, Maleki A,
et al . 2022. The resistance mechanisms of bacteria against ciprofloxacin and new approaches for enhancing the efficacy of this antibiotic.Front. Public Health 10 : 1025633. - Pletzer D, Mansour SC, Hancock REW. 2018. ynergy between conventional antibiotics and anti-biofilm peptides in a murine, subcutaneous abscess model caused by recalcitrant ESKAPE pathogens.
PLoS Pathog. 14 : e1007084. - Lin MF, Lin YY, Lan CY. 2019. A method to assess influence of different medical tubing on biofilm formation by
Acinetobacter baumannii .J. Microbiol. Methods 160 : 84-86. - Lin Y, Chang RYK, Britton WJ, Morales S, Kutter E, Chan HK. 2018. Synergy of nebulized phage PEV20 and ciprofloxacin combination against
Pseudomonas aeruginosa .Int. J. Pharm. 551 : 158-165. - Rishi P, Vashist T, Sharma A, Kaur A, Kaur A, Kaur N,
et al . 2018. Efficacy of designer K11 antimicrobial peptide (a hybrid of melittin, cecropin A1 and magainin 2) againstAcinetobacter baumannii -infected wounds.Pathog. Dis. 76 . doi: 10.1093/femspd/fty072. - Powers JPS, Hancock REW. 2003. The relationship between peptide structure and antibacterial activity.
Peptides 24 : 1681-1691. - Sharma P, Ayappa KG. A 2022. Molecular dynamics study of antimicrobial peptide interactions with the lipopolysaccharides of the outer bacterial membrane.
J. Membr. Biol. 255 : 665-675. - Thakur S, Cattoni DI, Nöllmann M. 2015. The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent.
Eur. Biophys. J. 44 : 337-348. - Shagaghi N, Palombo EA, Clayton AHA, Bhave M. 2018. Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality.
World J. Microbiol. Biotechnol. 34 : 62. - Travkova OG, Moehwald H, Brezesinski G. 2017. The interaction of antimicrobial peptides with membranes.
Adv. Colloid Interface Sci. 247 : 521-532. - Allegra E, Titball RW, Carter J, Champion OL. 2018.
Galleria mellonella larvae allow the discrimination of toxic and non-toxic chemicals.Chemosphere 198 : 469-472. - Browne N, Heelan M, Kavanagh K. 2013. An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes.
Virulence 4 : 597-603. - Wojda I. 2017. Immunity of the greater wax moth
Galleria mellonella .Insect Sci. 24 : 342-357. - Nguyen LT, Chau JK, Perry NA, de Boer L, Zaat SAJ, Vogel HJ. 2010. Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs.
PLoS One 5 : e12684. - Jia F, Zhang Y, Wang J, Peng J, Zhao P, Zhang L,
et al . 2019. The effect of halogenation on the antimicrobial activity, antibiofilm activity, cytotoxicity and proteolytic stability of the antimicrobial peptide Jelleine-I.Peptides 112 : 56-66.