Research Article |
Corresponding author: Ali H. Salama ( belarabi99@hotmail.com ) Academic editor: Georgi Momekov
© 2023 Ali H. Salama.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Salama AH (2023) Study the activity of conjugated antimicrobial peptide WW-185 against clinically important bacteria. Pharmacia 70(2): 331-336. https://doi.org/10.3897/pharmacia.70.e103133
|
Multidrug-resistance bacteria are a serious problem for health specialists and all the people in the world. The main reasons for this problem are the misuse of antibiotics and the limited number of antibiotics as compared to the different human diseases. Important antibiotic-resistant bacteria include Methicillin-resistant Staphylococcus aureus (MRSA) and Extended-Spectrum β-Lactamases E. coli (ESBL E. coli), These two types of bacteria can cause life-threatening diseases and poses a big problem in choosing suitable antibiotics for infections caused by them.
Antimicrobial peptides (AMPs) are considered promising antimicrobial agents that meet the required criteria for novel antimicrobial drugs. This study aims to design novel and safe AMP to be used as antimicrobial agents. In this study, an unique modified AMPs called WW-158 was designed to have a hydrophilic and hydrophobic balance using arginine to represent the cationic part and tryptophan to show the hydrophobic part. It showed good activity against MRSA with a MIC value of 35 μM. These effective concentrations were associated with negligible toxicity toward human red blood cells. Furthermore, our results showed that most of the combined groups of peptides with eight conventional antibiotics displayed synergistic modes of action or additive effects.
antimicrobial peptides, hemolytic activity, MRSA, antibiotics resistance and peptides
The growing threat of antibiotic resistance has become a significant concern worldwide. The Interagency Task Force on Antimicrobial Resistance released an action plan in 2001 to combat the issue in the United States, which contained 84 action elements, 13 of which were designated as “top priorities” and fell into 4 overarching activity areas: surveillance, prevention and control, research, and product development (
Antimicrobial peptides (AMPs) are natural antibiotics that are obtained from various living organisms such as plants, frogs, insects, fungi, bacteria, and other organisms. The increasing bacterial drug resistance has made AMPs a critical solution to combat bacterial infection (
In silico designing of a series of novel, improved, and conjugated antimicrobial peptide of tripeptides was performed taking into account the balance between the hydrophobic and charged moieties
The synthesized peptide were analyzed using RP-HPLC for purification using an acetonitrile / H2O gradient. The identification of the synthesized peptides was confirmed by ESI-MS.
Using sterile 96-well polypropylene microtiter plates, the micro broth dilution method outlined by the Clinical and Laboratory Standards Institute (CLSI) guidelines was adopted to determine the MIC and MBC of the peptide (
MBC was determined by taking 10 μL was taken from clear negative wells, and turbid positive control wells and they were streaked on sterile labeled nutrient media agar and incubated for 24 hours at 37 °C. The lowest concentration that led to having < 0.1% viable cells (killing 99.9%) was referred to as the MBC value (
MICs and MBCs determined against stander bacterial strains of S. aureus and E. coli and resistance ESBL E. coli and MRSA via preparing different concentrations of each antibiotic (the concentration range was from 0.25 to 250 μM). Every antibiotic solution was prepared by dissolving it in water then diluted in the sterile broth (
According to the broth microdilution checkerboard technique (Sueke et al. 2010), MICs of peptide-antibiotics combinations against stander bacterial strains of S. aureus and E. coli and resistance ESBL E. coli and MRSA were tested and determined as described. However, in this assay, each microtiter well contained a mixture of one peptide and one antibiotic in different concentrations. 25 µl of each peptide concentration and 25 µl of each antibiotic concentration (from 0.25 to 200 μM) were added to six wells of a sterile flat–bottomed 96 well-plate that contained 50 µl of the diluted bacterial suspension. MICs determination made in triplicate (Mishra et al. 2017).
The fractional inhibitory concentration (FIC) is the summation of the inhibitory concentration values of each component resulted in the antimicrobial combination divided by the inhibitory concentration alone (
The FIC indices were interpreted as follows:
≤ 0.5: synergistic activity, 0.5–1: additive activity, 1–4 indifferent, >4: antagonistic. Interpretation and assessment of the FIC index and antimicrobial activity of peptides-antibiotics combinations were conducted according to the broth microdilution checkerboard technique (
Determination of the ability of the designed peptide to cause hemolysis to human erythrocyte, hemolytic assays was performed. Two ml of human blood was placed into a 50-ml centrifuge tube, centrifuged at 3000× g for 5 min. The supernatant was discarded and the cell pellet was suspended in 48 ml of PBS and centrifuged at 3000× g for 5 min; this step was repeated three times. Finally, the cell pellet was re-suspended in a sterile tube containing 50 ml PBS to reach a final concentration of 4% RBC and PBS containing different concentrations of the peptides. Then 1 ml of each concentration was added to 1 ml of erythrocyte suspension.
Controls were prepared by the addition of 5 µl of Triton X-100 to 1 ml of RBC suspension (positive control). The blank (negative control) was prepared by adding 1 ml of RBC suspension with PBS. The suspension was incubated for 60 min at 37 °C. Tubes were gently vortexed and 1 ml of each sample was aspirated and placed into sterilized Eppendorf tubes and then centrifuged for 5 min at 3000× g. From each supernatant 100 µl were placed into the wells of a 96-well plate. Absorbance was measured at λ= 570 μM with the aid of an Absorbance Microplate Reader The percentage of hemolysis was calculated according to the following equation (
Where A: is OD 450 with the peptide solution,
A0: is OD 450 of the blank.
And AX: is OD 450 of control (0.1% Triton X-100).
A novel improved tri- AMP contins two subunits of tryptophan and one ornithine amino acid. Ornithine was used to give the charge for the peptide; it has the advantage of being an unnatural amino acid and a non-coded amino acid, thus its stability against proteases is excellent. Tryptophan was integrated as hydrophobic moieties because of its membrane interface interaction. it exhibits a strong preference when compared to the other hydrophobic amino acids. our designed peptide was conjugated to Para-hydroxycinnamic acid (PHCA) which was used to increase the hydrophobic properties of the peptide. Also, PHCA has antimicrobial activity on its own. So it is expected to increase the activity of the peptide. The structure of the peptide is shown in Fig.
The peptide was tested against different strains of Gram-positive and Gram-negative bacteria, including resistant strains. The bacteria used were standard strains of S. aureus (ATCC 29215), MRSA (ATCC BAA-41) and standard strains of E. coli (ATCC 25922); in addition to ESBL – E. coli. To determine the MIC and MBC.
As shown in (Table
According to the checkerboard dilution method, WW-158 was combined with the eight conventional antibiotics to determine the outcomes of these combinations on antibacterial potency. The shift in antibacterial potency is reported by calculating the MIC values of the ultrashort peptides and the antibiotic combinations against standard types of bacterial strains of Gram-positive and Gram-negative bacteria. Also, it is out righted by calculating the FIC indices, so these combinations can be classified as synergistic (FIC ≤ 0.5), additive (FIC 0.5< FIC≤ 1), indifferent (1<FIC≤4), or antagonist (FIC>4).
The only combination that showed a synergistic effect against Gram-positive strains was that with vancomycin against S. aureus and MRSA. with FIC values of 0.21 and 0.25 respectively, On the other hand, combinations of WW-158 with rifampicin showed a synergistic effect with a FIC index less than 0.5 against the control strain E. coli, while no combination showed synergistic effect against the resistance gram-negative strain ESBL E. coli isolated strain (BAA-3054). The results are summarized in Table
Minimal inhibitory concentrations (MIC) and the FIC index of combinations of WW-158 & the antibiotics against all the tested bacterial strains.
MIC in combination with bacterial strains | FIC* Index | |||||
---|---|---|---|---|---|---|
MIC in combination and MIC alone | ||||||
Bacterial strains | Antibiotics | Antibiotic MIC before combination | Antibiotic MIC after combination | WW-158 MIC before combination | WW-158 MIC after combination | |
S. aureus (ATCC 6538) | Levofloxacin | .5 | .25 | 25 | 10 | .9 |
Chloramphenicol | 30 | 15 | 25 | 15 | 1.1 | |
Rifampicin | .025 | .0125 | 25 | 10 | 0.9 | |
Amoxicillin | 5 | .5 | 25 | 15 | 0.7 | |
Clarithromycin | 1.5 | .5 | 25 | 20 | 1.13 | |
Doxycycline | 10 | 2 | 25 | 8 | 0.52 | |
Vancomycin | .5 | 0.00125 | 25 | 5 | 0.21 | |
Cefixime | 4 | 2 | 25 | 15 | 1.1 | |
MRSA (ATCC BAA-41) | Levofloxacin | 10 | 6 | 35 | 20 | 1.2 |
Chloramphenicol | 40 | 15 | 35 | 15 | 0.8 | |
Rifampicin | .005 | .0025 | 35 | 20 | 1.1 | |
Amoxicillin | 40 | 40 | 35 | 30 | 1.9 | |
Clarithromycin | 100 | 100 | 35 | 25 | 1.7 | |
Doxycycline | 20 | 10 | 35 | 15 | 0.9 | |
Vancomycin | 2 | .05 | 35 | 8 | 0.25 | |
Cefixime | 30 | 15 | 35 | 20 | 1.1 | |
E. coli (ATCC 8739) | Levofloxacin | 2 | .0025 | 100 | 60 | 0.6 |
Chloramphenicol | 100 | 20 | 100 | 80 | 1 | |
Rifampicin | 15 | 1 | 100 | 40 | 0.47 | |
Amoxicillin | 25 | 10 | 100 | 80 | 1.2 | |
Clarithromycin | 150 | 100 | 100 | 60 | 1.3 | |
Doxycycline | 15 | 2 | 100 | 40 | 0.53 | |
Vancomycin | 150 | 60 | 100 | 50 | 0.9 | |
Cefixime | 6 | 1 | 100 | 40 | 0.57 | |
ESBL E. coli (BAA-3054) | Levofloxacin | 12 | 6 | 100 | 60 | 1.1 |
Chloramphenicol | 200 | 40 | 100 | 80 | 1 | |
Rifampicin | 50 | 15 | 100 | 40 | .7 | |
Amoxicillin | 250 | 150 | 100 | 80 | 1.4 | |
Clarithromycin | 200 | 100 | 100 | 40 | 0.9 | |
Doxycycline | 25 | 6 | 100 | 60 | 0.84 | |
Vancomycin | 200 | 175 | 100 | 50 | 1.4 | |
Cefixime | 80 | 40 | 100 | 60 | 1.1 |
In the present study, novel antimicrobial peptide (WW-158) was designed based on rational design. The mode of antimicrobial activity of AMPs was proposed to occur as a result of targeting the bacterial cell membranes. The proposed mechanism of action for AMPs indicates that peptide entry and membrane targeting are facilitated by the hydrophilic positively charged moieties of AMPs, which are driven electrostatically towards bacterial negatively charged surfaces, leading to pore formation and later cell membrane lysis and death. Previous work on AMPs has shown that only those peptides that carried a cationic net charge (≥ +3) managed to possess antibacterial activities and inflict damage on bacterial membranes ; thus, our strategy relied on designing novel peptides displaying a net cationic charge around +3 (Porto et al. 2018). While balancing other physicochemical parameters within the primary sequence, such as hydrophobicity, the design hypothesis is in complete alignment with the proposed mechanism of action of AMPs. Our in-house designed peptide will be adequately able to create sufficient electrostatic attraction with the negative head groups of phospholipids in the bacterial cell membrane and consequently cause membrane perforation and cell death (Xia et al. 2018). The cationic charge of AMPs designed in our study is mainly attributed to the presence of arginine amino acids within the primary structure. Since the side chain of the amino acids interacts through the formation of hydrogen bonds and also through electrostatic interaction with the negatively charged surface of bacteria (Rodriguez et al. 2016). Our results are consistent with previous studies , which demonstrated that the membrane disruption is cationic residue-specific and hence the positive charges must be dependent on arginine and not lysine or ornithine, especially when the peptide is less than three residues (
MIC values of eight conventional antibiotics (levofloxacin, chloramphenicol, rifampicin, amoxicillin, clarithromycin, vancomycin, cefixime, and doxycycline) against various strains of Gram-positive (S. aureus (ATCC 6538) MRSA (ATCC BAA-41)) and Gram-negative (E. coli (ATCC 8739) and ESBL E. coli isolated strain (BAA-3054)) demonstrated that rifampicin is the most potent antibiotic against Gram-positive bacteria namely, S. aureus (ATCC: 6538 and BAA-41) with MICs of 0.025 and 0.005 μM, respectively. Regarding the Gram-negative bacterial strains E. coli (ATCC: 8739 and BAA-3054), levofloxacin exhibited the highest antimicrobial activity with a MIC value equal to 2 and 12 μM. The combination between WW-158 and rifampicin shows a synergistic effect. The mechanism of the synergistic effects of the peptide- rifampicin combinations is unclear yet, but one hypothesis for the synergistic effect proposes that the destruction and pore formation effects of AMPs in the bacterial membranes enhance the intracellular entry of antibiotics, which allows them to reach their targets and accomplish their function rapidly (
In conclusion, we report the design and antibacterial properties of a novel conjugated ultrashort antimicrobial peptide with potent activity against clinically resistant strains of Gram-positive and Gram-negative bacteria with negligible hemolytic activity. When used in combination with traditional antibiotics, the peptide showed multiple synergistic effects and may prove to be an important candidate for further development of antibacterial drugs.
The author is grateful to the Middle East University (MEU), Amman, Jordan, for the financial support granted to cover the publication fee of this research article.