DOI: https://doi.org/10.21203/rs.3.rs-1549550/v1
With the recent rise of multidrug-resistant bacteria (MDRB) in most communities, it has become one of the most important concerns confronting health professionals globally. Today, Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for a great deal of hospital-acquired (nosocomial) and community-acquired infections. MRSA is difficult to treat with limited options for treatment such as vancomycin, linezolid, or clindamycin. Thus, an innovative methodology is required to compact infections with resistant organisms. In this study we synthesis a novel ultra-short antimicrobial peptide consists of three unit of tryptophan and three unit of lysine. We examined the effect of this peptide against MRSA and biofilm forming MRSA. Promising result showed that the peptide was effective in killing against both MRSA and biofilm forming MRSA with low toxicity to human red blood cells. Moreover, our novel peptide also show excellent synergistic effect when combined with vancomycin against MRSA. Furthermore, combination of our peptide with levofloxacin and clarithromycin revealed synergistic effect with biofilm forming MRSA. In conclusion, the result of this study presented a novel USAMPs which potentially can be utilized as a new generation of antibiotics to compact resistant bacteria globally.
According to a recent world health organization (WHO), Staphylococcus aureus is the major bacterial species causing nosocomial infections globally. S. aureus is a Gram-positive bacterium that causes a wide range of human diseases responsible for many critical hospital-acquired (nosocomial) and community-acquired infections1. The strain has become a serious problem internationally with the development of methicillin resistant causes invasive infection which led to a 20% increase in mortality rate. Methicillin resistant Staphylococcus aureus (MRSA) was induced with a transfer of mecA gene from an ancestral Staphylococcus species, with the gene being mediated by a special mobile genetic element2. The risk of developing staphylococcal infections rises from the pathogen’s ability to develop drug resistance to multiple antibiotics complicated by its ability to form biofilm3. According to the central of disease control (CDC) initiative (National Nosocomial Infections Surveillance System), the incidence of MRSA infection has risen 60% in intensive care units4. Treating these infections is challenging due to resistance to most traditional antibiotic, including second-and third-line drugs5.
Some strains of Staphylococcus aureus not only display resistance to antibiotics such as methicillin but also are prolific producers of biofilm materials [Ref]. Biofilm is formed when cells stick to each other’s, after adhering to solid surfaces, the process is expatiated with the production of extracellular polymeric substance6. This matrix composes of DNA, proteins, polysaccharides, water; and microorganism cell. Various microorganism has the ability to form biofilm such as bacteria, fungi and protists7. The biofilm formation ensures good strategy to microorganism to survive and adapt to living environmental and nutritional conditions8. The biofilm formation process undergoes important multi-steps. The first step is the attachment of floating microorganisms to a surface. Attachment is followed by a period of growth and formation of micro-colony, creating a complex 3D structure. Followed by development of a small biofilm, maturation and detachment. In fact, when compared to planktonic, those growing as a biofilm can be up to 1,500 times more resistant to antibiotics and other biological and chemical agents9. To control the biofilm formation, several treatment strategies have been proposed. The intensive and aggressive antibiotic treatment are used to retard their spreading but not to eradicate the whole biofilm community. Due to the great problem caused by these types of bacteria and to increase their immunity to the types of antibiotics currently available10. A new type of drug was needed to combat it, and one of these drugs is antimicrobial peptides (AMPs). These peptides are characterized by their ability to attack the cell by binding to the cell wall and causing cell lysis11. In this study, we synthesized an ultra-short antimicrobial peptide (USAMPs) and combined it with a number of antibiotics and measured its effectiveness against planktonic and biofilm forming Staphylococcus aureus12.
When we synthesis the peptide we use a lysin amino acid to charge the peptide (+ 3) which has been deemed as a suitable charge that presented effective antimicrobial effect 20. We also use Tryptophan because it’s have a good lipophilic characteristic and good interaction with bacteria membrane. We also combined the peptide with ferulic acid to increase the peptide hydrophobicity. The structure of the peptide shown in Fig. 1.
The result of the MIC and MBC value of the conventional antibiotics shown in Table 1
| Antibiotics | MIC / MRSA (ATCC BAA-41) | MBC / MRSA (ATCC BAA-41) |
|---|---|---|
| Levofloxacin | 10 | 10 |
| Chloramphenicol | 25 | 40 |
| Rifampicin | 0.005 | 0.005 |
| Amoxicillin | 40 | 40 |
| Clarithromycin | 125 | 150 |
| Doxycycline | 10 | 20 |
| Vancomycin | 2 | 2 |
| cefixime | 30 | 30 |
The result of the viable cell count was shown in Fig. 2, the result indicates that the combination of the peptide with antibiotics lead to dramatically decrease of the viable cell.
We also investigated the effect of the peptide alone against MRSA and in combination with the antibiotics panel, then we determined the synergistic effect using FIC equation (Table 2) .
| Bacterial strains | Antibiotic | Antibiotic MIC before combination | Antibiotic MIC after combination | Peptide MIC before combination | Peptide MIC after combination | FIC results |
|---|---|---|---|---|---|---|
| MRSA (ATCC BAA-41) | Levofloxacin | 10 | 8 | 15 | 6 | 1.2 |
| Chloramphenicol | 40 | 20 | 15 | 6 | 0.9 | |
| Rifampicin | 0.005 | 0.0025 | 15 | 10 | 1.17 | |
| Amoxicillin | 40 | 25 | 15 | 10 | 1.3 | |
| Clarithromycin | 200 | 80 | 15 | 15 | 1.4 | |
| Doxycycline | 20 | 10 | 15 | 6 | 0.9 | |
| Vancomycin | 2 | 0.5 | 15 | 4 | 0.5 | |
| Cefixime | 30 | 15 | 15 | 8 | 1.03 |
Results in Table 3 show that the conjugation at concentration of 100 µM only causes 1% hemolysis on Human erythrocytes after 30 minutes’ incubation.
| Hemolysis % | Concentration (µM) |
|---|---|
| 0 | 5 |
| 0 | 10 |
| 0 | 20 |
| 0 | 40 |
| 2 | 60 |
| 2 | 80 |
| 1 | 100 |
Biofilm forming pathogens such as S. aureus are one of the most difficult to treat, and are considered a major problem to human in medicine, particularly after biofilm is established. In this study, we used MRSA as an example for biofilm producing bacteria that is also resistant to antibiotic and involved in hospital-acquired infections 21. The SARS2 pandemic resulted in a great number of patients with pulmonary destress, great number of elderly patients were put on ventilators. Studies have showen that a large number of corona patients die as a result of pneumonia after using ventilators rather than the viral infection caused by the coronavirus. The major bacterial pathogen associated with corona patients were MRSA 22.
In this study, we synthesized an ultra-short anti-microbial peptide to study its effect on the biofilm producing Staphylococcus aureus bacteria and MRSA. The results proved a good efficacy of this compound to resist this type of bacteria after measuring the counting the percentage of viable cells. This peptide mechanism of action involved the ability of the peptide to attack the cell wall and make pores in it, which causes cell lysis and death23. We also studied the degree of toxicity of our synthesized peptide to red blood cells, as toxicity seems to be one of the biggest issues in developing peptide therapeutics in the future, our experiment showed that our synthesized peptide had very low toxicity to red blood cells.
In order to increase the effectiveness of the peptide, we combined it with eight antibiotics to see the effect against biofilm, and the results showed a significant increase in effectiveness24 with Levofloxacin, Chloramphenicol, Rifampicin, Clarithromycin and Doxycycline. This can be explained by the fact that these antibiotics work inside the cell and the peptide facilitates their entry into the cell because it makes holes in the cell wall, which promote its entry into the cell and do its work easily25.
MIC values of eight conventional antibiotics (levofloxacin, chloramphenicol, rifampicin, amoxicillin, clarithromycin, vancomycin, cefixime, and doxycycline) against MRSA (ATCC BAA-41). demonstrated that rifampicin is the most potent antibiotic against Gram-positive bacteria namely S. aureus (ATCC: 29215 and BAA-41) with MICs of 0.025 and 0.005 µM, respectively26. Vancomycin displayed a synergistic effect against Gram-positive bacteria. This might be explained based on vancomycin function against the cell wall thus facilitating the ease entry of the peptides to their target sites in the cell membrane these led finally cause rapid cell lysis and decrease the effective concentrations needed to inhibit bacterial growth displayed by peptide and vancomycin27.
In conclusion, the results proved that the peptide that we synthesized has a good efficacy against MRSA, with low toxicity to red blood cells. The results of the study indicate that the combination of peptides with available antibiotics could be one of the most significant methods to enhance the efficacy of existing antibiotics today. The peptide mode of action delivers a high concentration of antibiotics with peptides to bacterial species and ensure bacterial killing (bactericidal activities) regardless of bacterial resistance status.
We use methicillin resistance Staphylococcus aureus (ATCC 33591) as a biofilm forming bacteria and MRSA (ATCC BAA-41) as a planktonic strain was obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA, USA).
The synthesized peptide is made up of three tryptophan (w) subunits and three lysine (K) amino acids, and ferulic acid was used to conjugate the peptide. The peptide was created using the solid-phase Fmoc chemistry. Purity was determined using reverse phase high performance liquid chromatography (RPHPLC), and validated using mass spectrometry and electrospray ionization mass spectrometry (ESIMS)13
For biofilm formation we used the Calgary biofilm device, as previously reported14 with Staphylococcus aureus (ATCC 33591).
We used sterile 96-well microtiter plates to determine the minimum inhibition and bactericidal concentrations (MIC/MBC), we used the microbroth dilution method for the later determination. Muller–Hinton broth (MHB) was used as a revival growth medium for the staphylococcus aureus, it was also used as the main broth media in determining the MIC. Briefly, S. aureus were revived from glycerol stock at -70C using MUB, S. aureus cells were grown overnight and diluted to 106 CFU/mL in MHB. We also diluted our peptide in different concentrations and in separate 96-well microtiter plates, we mixed 50 µL of peptide with 50 µL of diluted bacterial suspension, we performed six replica for each peptide concentration. Plates were incubated for 18 h at 37 ◦C. Bacterial growth were determined via measuring the optical density at λ = 570 nm. MIC was defined as the lowest concentration of peptide which inhibited the growth of S. aureus. We included positive and negative controls with each plate to ensure bacterial growth and MHB sterility. For MBC determination we streaked 10 µL from the clear negative wells on nutrient agar and incubated the plates overnight at 37 ◦C. MBC value we defined as the lowest concentration that killed 99.9% of S. aureus (< 0.1% viable cells). Experiments were performed in triplicate15.
Levofloxacin, Chloramphenicol, Rifampicin Amoxicillin, Clarithromycin,Doxycycline,Vancomycin and and Cefixime was obtained from sigma Aldrich .
MICs and MBCs determined against planktonic type 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 broth16.
According to the broth microdilution checkerboard technique, MICs of peptide-antibiotics combinations against planktonic type MRSA was tested However, in this assay, each microtiter well contained a mixture of one peptide and one antibiotic in different concentrations. 25 µl of the 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 triplicate17.
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 ≤ 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 technique18.
Hemolytic assay of red blood cells was determined according to previous study 19 where we used the equation below to determine the RBCs hemolysis due to the use of peptide.
% Hemolysis = \(\frac{(A - AO) }{(AX -AO)}\) × 100
Where A: is Optical density 450 with the peptide solution
A0: is Optical density 450 of the blank.
And AX: is Optical density 450 of control (0.1% triton X-100).
Competing interests
The authors declare no competing interests
Acknowledgments
The authors are thankful to both Middle East University and the deanship of research at Jordan University of Science and Technology for the financial support granted to cover the publication fee of this research article.
Availability of Data and Materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].