Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

Ali Salama

doi:10.12688/f1000research.136310.1

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Research Article

Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

[version 1; peer review: 2 not approved]

Ali Salama

PUBLISHED 18 Oct 2023

Author details Author details

Pharmacy, Middle East University, Amman, 11831, Jordan

Ali Salama
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell & Molecular Biology gateway.

Abstract

Background: The emergence of multidrug-resistant bacteria (MDRB) poses a significant global challenge for healthcare professionals. Methicillin-resistant Staphylococcus aureus (MRSA), a prominent pathogen responsible for both hospital-acquired (nosocomial) and community-acquired infections, is particularly difficult to treat. Existing treatment options, such as vancomycin, linezolid, or clindamycin, have limitations. Therefore, there is a need for innovative approaches to combat infections caused by drug-resistant organisms.
Methods: In this study, we synthesized a novel ultra-short antimicrobial peptide composed of three units of tryptophan and three units of lysine. We evaluated the effectiveness of this peptide against MRSA and MRSA that forms biofilms.
Results: Encouraging results demonstrated that the peptide effectively killed both MRSA and biofilm-forming MRSA, while exhibiting low toxicity to human red blood cells. Additionally, our novel peptide showed excellent synergistic effects when combined with vancomycin against MRSA. Furthermore, when combined with levofloxacin and clarithromycin, our peptide exhibited synergistic effects against biofilm-forming MRSA.
Conclusions: In conclusion, this study presents a novel ultra-short antimicrobial peptide (USAMP) that holds potential as a new generation of antibiotics to combat globally prevalent drug-resistant bacteria.

Keywords

multidrug-resistant bacteria , community-acquired infections , resistance , peptides and biofilm

Corresponding author: Ali Salama

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2023 Salama A. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions.

How to cite: Salama A. Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus [version 1; peer review: 2 not approved]. F1000Research 2023, 12:1369 (https://doi.org/10.12688/f1000research.136310.1) First published: 18 Oct 2023, 12:1369 (https://doi.org/10.12688/f1000research.136310.1) Latest published: 18 Oct 2023, 12:1369 (https://doi.org/10.12688/f1000research.136310.1)

Introduction

According to the 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 infections.¹ The strain has become a serious problem internationally with the development of methicillin resistant causes invasive infection which led to a 18% increase in mortality rate. For the study, which appears in the Journal of the American Board of Family Medicine, researchers analyzed data from the 2001-2004 National Health and Nutrition Examination Survey, a large, nationally representative study that combines survey questions with laboratory testing, including nasal swabs to test for the presence of MRSA. The researchers linked data on participants ages 40-85 with data from the National Death Index to track deaths over an 11-year period. Researchers adjusted for factors including gender, race and ethnicity, health insurance, poverty-income ratio, hospitalization in the previous 12 months, and doctor diagnosis of heart disease, diabetes and asthma. They found the mortality rate among participants without MRSA was about 18%, but among those with colonized MRSA, the mortality rate was 36%. Participants who carried staph bacteria on their skin, but not MRSA, did not have an increased risk for premature death.²

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 element.³ The incidence of staphylococcal infections has increased due to the pathogen's ability to develop resistance to multiple antibiotics and form biofilms.⁴ The Central of Disease Control (CDC) has reported a 60% rise in MRSA infections in intensive care units, according to the National Nosocomial Infections Surveillance System. Treating these infections has become challenging as they exhibit resistance to traditional antibiotics, including second- and third-line drugs.⁴^,⁵

Some strains of Staphylococcus aureus not only display resistance to antibiotics such as methicillin but also are prolific producers of biofilm materials. 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 substance.⁶ This matrix composes of DNA, proteins, polysaccharides, water; and microorganism cell. Various microorganism has the ability to form biofilm such as bacteria, fungi and protists.⁷ The biofilm formation ensures good strategy to microorganism to survive and adapt to living environmental and nutritional conditions.⁸ 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 agents.⁹ 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 available.¹⁰ 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 lysis.¹¹ 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 aureus.¹²

Methods

Bacterial cultures

We use methicillin resistance Staphylococcus aureus (ATCC 33591) as a biofilm forming bacteria and MRSA (ATCC BAA-41) as a planktonic strain which were obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA, USA).

Synthesis of ultra-short antimicrobial peptides (USAMPs)

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. The designed peptides used in the present study were synthesized by (GL Biochem Ltd., Shanghai, China) using the solid-phase method and Fmoc chemistry was finally obtained as a lyophilized state. Reverse phase highperformance liquid chromatography (RP-HPLC) was used for purification of the peptide using a C18 internsil® ODS-SP column, the column was eluted with acetonitrile/H2O-TFA gradient at a flow rate of 1.0 mL/min. and validated using mass spectrometry and electrospray ionization mass spectrometry (ESIMS)¹³ The absorbance was at λ = 214 nm, the solvent which was use is dichloromethane.

Biofilm activity assay

For biofilm formation we used the Calgary biofilm device, as previously reported¹⁴ with Staphylococcus aureus (ATCC 33591). Biofilm formation was performed employing the Calgary biofilm device (Innovotech, Edmonton, Canada). The bacteria was left to grow in TSB at 37°C for 20 hours. Then, a concentration of 107 CFU/mL was prepared by diluting the cultures in the same medium. Using 96-peg lids on which the cells of biofilm can build up, 150 μL of that bacteria culture was added to each peg lid to allow the formation of biofilm on the purposed designed pegs, followed by incubating the pegs for 20 hours at 37°C under 125 rpm rotation. Blank lanes were prepared by adding 150 μL TSB to six wells. To discard planktonic cells after biofilm formation, PBS was used to wash pegs three times.

Determination of the minimum inhibitory & bactericidal concentrations (MIC/MBCs)

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 triplicate.¹⁵

Antibiotic used in this study

Levofloxacin, Chloramphenicol, Rifampicin Amoxicillin, Clarithromycin, Doxycycline, Vancomycin and Cefixime were obtained from sigma Aldrich.

MIC and MBC determination of antibiotics alone

Various concentrations of each antibiotic (ranging from 0.25 to 250 μM) were prepared to determine the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against planktonic Methicillin-Resistant Staphylococcus aureus (MRSA). The antibiotics were dissolved in water and then further diluted in sterile broth for the preparation of each antibiotic solution.¹⁶

MIC determination of peptides-antibiotics combinations

The broth microdilution checkerboard technique was employed to test the MICs of peptide-antibiotic combinations against planktonic MRSA. In this assay, each well of a sterile flat-bottomed 96-well plate contained a mixture of one peptide and one antibiotic in varying concentrations. Specifically, 25 μl of the peptide concentration and 25 μl of each antibiotic concentration (ranging from 0.25 to 200 μM) were added to six wells. These wells also contained 50 μl of the diluted bacterial suspension. The MICs were determined in triplicate, ensuring robustness of the results.¹⁷

Determination of synergism using fractional inhibitory concentration

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 technique mention in the section above.¹⁸

Erythrocyte hemolytic assay

Hemolytic assay of red blood cells was determined according to previous study¹⁹ where we used the equation below to determine the RBCs hemolysis due to the use of peptide.

% Hemolysis = \frac{(A - AO)}{(AX - AO)} \times 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).

Stastical analysis

In this study, all data-generating experiments were conducted three times. The resulting values from these experiments were compared and analyzed using one-way analysis of variance (ANOVA) with least significant difference (LSD) multiple comparison tests on the means. Any variations observed were reported at a 95% confidence level (P<0.05). The analysis of the data was performed using SPSS software version 21.

Results

When we synthesised the peptide we used 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 used Tryptophan because it has 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 Figure 1.

Figure 1. The structure of the peptide.

The result of the viable cell count was shown in Figure 2, the result indicates that the combination of the peptide with antibiotics lead to dramatically decrease of the viable cell.²⁸

Figure 2. Percentage of viable cell after the treatment with KWKWKW-NH₂ peptide.

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 1 and 2).

Table 1. The minimum inhibitory concentration (MIC) (in μM) and MBC of the the antibiotics test panel.

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

Table 2. Minimum inhibitory concentration (MIC) in μM and the FIC index of combinations of peptide & the antibiotics against all the tested bacterial strains.

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.

Table 3. The hemolysis activity of the peptide in human erythrocytes in vitro.

Hemolysis %	Concentration (μM)
0	5
0	10
0	20
0	40
2	60
2	80
1	100

Discussion

Biofilm-producing pathogens, such as S. aureus, pose a significant challenge in medical treatment, especially once biofilm formation has occurred. These pathogens, exemplified by MRSA, are known for their ability to produce biofilms and their resistance to antibiotics. Furthermore, they are frequently associated with hospital-acquired infections, presenting a major concern for human health in the field of drug design.²¹ The coronavirus disease 2019 (COVID-19) pandemic resulted in a great number of patients with pulmonary destress, and great number of elderly patients were put on ventilators. Studies have shown that a large number of covid patients die as a result of pneumonia after using ventilators rather than the viral infection caused by the coronavirus.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 death.²³ 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 effectiveness 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 easily.²⁴^,²⁵

The minimum inhibitory concentration (MIC) values of eight conventional antibiotics, namely levofloxacin, chloramphenicol, rifampicin, amoxicillin, clarithromycin, vancomycin, cefixime, and doxycycline, were evaluated against Methicillin-Resistant Staphylococcus aureus (MRSA) strains, specifically ATCC BAA-41. The results showed that rifampicin exhibited the highest potency among these antibiotics against Gram-positive bacteria, including S. aureus (ATCC: 29215 and BAA-41), with MICs of 0.025 and 0.005 μM, respectively.²⁶ Furthermore, vancomycin demonstrated a synergistic effect against Gram-positive bacteria. This could be attributed to vancomycin's role in targeting the cell wall, facilitating the penetration of peptides to their target sites in the cell membrane. This ultimately led to rapid cell lysis and a decrease in the effective concentrations required to inhibit bacterial growth, as observed with the peptide and vancomycin combination.²⁷

Conclusions

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.

Data availability

Underlying data

Figshare: Peptide raw data. https://doi.org/10.6084/m9.figshare.23895903.v1.²⁸

This project contains the following underlying data:

- supplementry file (2).docx
- micS.xlsx
- combination peptide.xlsx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

An earlier version of this article can be found on Research Square (doi: https://doi.org/10.21203/rs.3.rs-1549550/v1).

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Version 1

VERSION 1 PUBLISHED 18 Oct 2023

Author details Author details

Pharmacy, Middle East University, Amman, 11831, Jordan

Ali Salama
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (1)

version 1

Published: 18 Oct 2023, 12:1369

https://doi.org/10.12688/f1000research.136310.1

Copyright

© 2023 Salama A. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions.

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Salama A. Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus [version 1; peer review: 2 not approved]. F1000Research 2023, 12:1369 (https://doi.org/10.12688/f1000research.136310.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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PUBLISHED 18 Oct 2023

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Reviewer Report 14 Mar 2024

Hui-min Neoh, Universiti Kebangsaan Malaysia Molecular Biology Institute, Kuala Lumpur, Malaysia

Not Approved

https://doi.org/10.5256/f1000research.149447.r250211

Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

According to the authors in the manuscript abstract, the manuscript was supposed to describe work performed in synthesizing USAMPS and testing ... Continue reading

Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

According to the authors in the manuscript abstract, the manuscript was supposed to describe work performed in synthesizing USAMPS and testing of the peptides' effectiveness in MRSA killing and biofilm eradication via determination of MIC, MBC and FIC. Synergism of the USAMPS and antibiotics in MRSA killing and also biofilm eradication was also supposed to be explored. Toxicity of the USAMPS towards red blood cells was to be investigated.

There are interesting data obtained by the authors especially on the apparent reduction in MIC and MBC readings of antibiotics when coupled with the synthesized USAMPs. However, no data about biofilm experiments can be found in the manuscript. There are also concerns in other areas of the manuscript, as stated in below comments:

1) Authors can expand the introduction and include more information about antimicrobial peptides and USAMPS. Why were tryptophan and lysine chosen for the synthesis - what was the basis? Reference for the methodology used should be provided. Figures depicting the synthesis will be good to help readers understand the synthesis process.

2) Methodology - biofilm activity assay: was the inoculum 107 CFU/mL? or was it 10*7 CFU/ml? Similar with 106 CFU/mL in MIC/MBC determination.

3) "We also diluted our peptide in different concentrations and in separate 96-well microtiter plates...". USAMPs concentration in MIC/MBC and checkerboard assay should be defined.

4) concentrations of antibiotics should preferably be in ug/ml or mg/L (as in EUCAST and CLSI) instead of uM.

5) will be good to give a brief description of the erythrocyte hemolytic assay, even though a reference of the method has been provided. Was the experiment carried out in exactly the same way as the reference?

6) Statistical analysis – Authors mentioned that “all data-generating experiments were conducted three times” and used ANOVA for analysis. This is not correct, as ANOVA is used to analyse difference between groups. I supposed the authors performed calculations to obtain the means from the experiment triplicates, and not ANOVA.

7) What is the KWKWKW-NH2 peptide? Was this the name given to the USAMPs? It should be properly defined in the manuscript.

8) Description about the experiment performed to obtain “viable cell counts” in Figure 2 should be added into the manuscript. What was the rationale of this experiment? What were the concentrations of the antibiotics and USAMP used? What was the condition of the experiment?

9) No results provided about biofilm assays though the methodology was described.

10) Will be good to include figures from the erythrocyte hemolytic assay in the manuscript.

11) Will be helpful for the authors to engage English editing for the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: antimicrobial resistance in MRSA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 25 Jan 2024

Yingxia Zhang, Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou,, China

Not Approved

https://doi.org/10.5256/f1000research.149447.r235412

In the manuscript "Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus", the authors created an ultra-short peptide that was characterized for its antibacterial activity. The peptide killed MRSA and exhibited low toxicity ... Continue reading

In the manuscript "Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus", the authors created an ultra-short peptide that was characterized for its antibacterial activity. The peptide killed MRSA and exhibited low toxicity to red blood cells. The manuscript is hard to follow at times and would benefit from a thorough review for typos and English grammar. And some issues seriously need to be addressed before it can be recommended for publication.

Introduction:

Some typos should be corrected, for example “Staphylococcus aureus” and “S. aureus” should be italic.
The full name of “MRSA” should be given when it mentioned at the first time. Then the abbreviation can be used. So does “Staphylococcus aureus”.

Methods:

Synthesis of ultra-short antimicrobial peptides (USAMPs): “the column was eluted with acetonitrile/H2O-TFA gradient at a flow rate of 1.0 mL/min. and validated using mass spectrometry and electrospray ionization mass spectrometry (ESIMS)13 The absorbance was at λ = 214 nm, the solvent which was use is dichloromethane.” Typos and English grammar errors were appeared.
Determination of the minimum inhibitory & bactericidal concentrations (MIC/MBCs): “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.” English grammar errors were appeared.

Results:
All the results were depicted too simply to understand.

Is there any evidence that ferulic acid can increase the peptide hydrophobicity?
In figure 2, what cells and the concentration of the peptide and antibiotics were used? The unit of Y-axis is CFU/ML, so is it MRSA? But why the peptide and antibiotics didn’t show antibacterial activity. And the method of cell viability was not mentioned in Methods.
The author mentioned in Methods that MRSA (ATCC 33591) as a biofilm forming bacteria and MRSA (ATCCBAA-41) as a planktonic strain were used. But in Results, only the antibacterial activity of MRSA (ATCCBAA-41) were described.
In Methods, Biofilm activity assay was mentioned. But I didn’t see any results.

Discussion:

The author mentioned “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 death”. There has no experimental evidence in the manuscript to support this conclusion.
The FIC≤ 0.5 was interpreted as synergistic activity. In table 2, the FIC of the peptide and vancomycin was the lowest (0.5), why the author claimed “the results showed a significant increase in effectiveness with Levofloxacin, Chloramphenicol, Rifampicin, Clarithromycin and Doxycycline”?

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: To find and design bioactive peptides with antimicrobial and anti-inflammatory activities.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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VERSION 1 PUBLISHED 18 Oct 2023

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Version 1 18 Oct 23	read	read

Yingxia Zhang, Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou,, China
Hui-min Neoh, Universiti Kebangsaan Malaysia Molecular Biology Institute, Kuala Lumpur, Malaysia

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Reviewer Report

6 Views

14 Mar 2024 | for Version 1

Hui-min Neoh, Universiti Kebangsaan Malaysia Molecular Biology Institute, Kuala Lumpur, Malaysia

6 Views Cite this report Responses(0)

Not Approved

Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

According to the authors in the manuscript abstract, the manuscript was supposed to describe work performed in synthesizing USAMPS and testing of the peptides' effectiveness in MRSA killing and biofilm eradication via determination of MIC, MBC and FIC. Synergism of the USAMPS and antibiotics in MRSA killing and also biofilm eradication was also supposed to be explored. Toxicity of the USAMPS towards red blood cells was to be investigated.

There are interesting data obtained by the authors especially on the apparent reduction in MIC and MBC readings of antibiotics when coupled with the synthesized USAMPs. However, no data about biofilm experiments can be found in the manuscript. There are also concerns in other areas of the manuscript, as stated in below comments:

1) Authors can expand the introduction and include more information about antimicrobial peptides and USAMPS. Why were tryptophan and lysine chosen for the synthesis - what was the basis? Reference for the methodology used should be provided. Figures depicting the synthesis will be good to help readers understand the synthesis process.

2) Methodology - biofilm activity assay: was the inoculum 107 CFU/mL? or was it 10*7 CFU/ml? Similar with 106 CFU/mL in MIC/MBC determination.

3) "We also diluted our peptide in different concentrations and in separate 96-well microtiter plates...". USAMPs concentration in MIC/MBC and checkerboard assay should be defined.

4) concentrations of antibiotics should preferably be in ug/ml or mg/L (as in EUCAST and CLSI) instead of uM.

5) will be good to give a brief description of the erythrocyte hemolytic assay, even though a reference of the method has been provided. Was the experiment carried out in exactly the same way as the reference?

6) Statistical analysis – Authors mentioned that “all data-generating experiments were conducted three times” and used ANOVA for analysis. This is not correct, as ANOVA is used to analyse difference between groups. I supposed the authors performed calculations to obtain the means from the experiment triplicates, and not ANOVA.

7) What is the KWKWKW-NH2 peptide? Was this the name given to the USAMPs? It should be properly defined in the manuscript.

8) Description about the experiment performed to obtain “viable cell counts” in Figure 2 should be added into the manuscript. What was the rationale of this experiment? What were the concentrations of the antibiotics and USAMP used? What was the condition of the experiment?

9) No results provided about biofilm assays though the methodology was described.

10) Will be good to include figures from the erythrocyte hemolytic assay in the manuscript.

11) Will be helpful for the authors to engage English editing for the manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

antimicrobial resistance in MRSA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report

9 Views

25 Jan 2024 | for Version 1

Yingxia Zhang, Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou,, China

9 Views Cite this report Responses(0)

Not Approved

In the manuscript "Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus", the authors created an ultra-short peptide that was characterized for its antibacterial activity. The peptide killed MRSA and exhibited low toxicity to red blood cells. The manuscript is hard to follow at times and would benefit from a thorough review for typos and English grammar. And some issues seriously need to be addressed before it can be recommended for publication.

Introduction:

Some typos should be corrected, for example “Staphylococcus aureus” and “S. aureus” should be italic.
The full name of “MRSA” should be given when it mentioned at the first time. Then the abbreviation can be used. So does “Staphylococcus aureus”.

Methods:

Synthesis of ultra-short antimicrobial peptides (USAMPs): “the column was eluted with acetonitrile/H2O-TFA gradient at a flow rate of 1.0 mL/min. and validated using mass spectrometry and electrospray ionization mass spectrometry (ESIMS)13 The absorbance was at λ = 214 nm, the solvent which was use is dichloromethane.” Typos and English grammar errors were appeared.
Determination of the minimum inhibitory & bactericidal concentrations (MIC/MBCs): “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.” English grammar errors were appeared.

Results:
All the results were depicted too simply to understand.

Is there any evidence that ferulic acid can increase the peptide hydrophobicity?
In figure 2, what cells and the concentration of the peptide and antibiotics were used? The unit of Y-axis is CFU/ML, so is it MRSA? But why the peptide and antibiotics didn’t show antibacterial activity. And the method of cell viability was not mentioned in Methods.
The author mentioned in Methods that MRSA (ATCC 33591) as a biofilm forming bacteria and MRSA (ATCCBAA-41) as a planktonic strain were used. But in Results, only the antibacterial activity of MRSA (ATCCBAA-41) were described.
In Methods, Biofilm activity assay was mentioned. But I didn’t see any results.

Discussion:

The author mentioned “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 death”. There has no experimental evidence in the manuscript to support this conclusion.
The FIC≤ 0.5 was interpreted as synergistic activity. In table 2, the FIC of the peptide and vancomycin was the lowest (0.5), why the author claimed “the results showed a significant increase in effectiveness with Levofloxacin, Chloramphenicol, Rifampicin, Clarithromycin and Doxycycline”?

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

To find and design bioactive peptides with antimicrobial and anti-inflammatory activities.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

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[2] 2. Mainous AG, Rooks BJ, Carek PJ: Methicillin-Resistant Staphylococcus Aureus Colonization and Mortality Risk Among Community Adults Aged 40-85. J. Am. Board. Fam. Med. 2021; 34(2): 439–441. Publisher Full Text

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Bactericidal activity of newly synthesized antimicrobial peptides against methicillin-resistant staphylococcus aureus and biofilm-forming methicillin-resistant staphylococcus aureus

Abstract

Keywords

Introduction

Methods

Bacterial cultures

Synthesis of ultra-short antimicrobial peptides (USAMPs)

Biofilm activity assay

Determination of the minimum inhibitory & bactericidal concentrations (MIC/MBCs)

Erythrocyte hemolytic assay

Stastical analysis

Results

Figure 1. The structure of the peptide.

Figure 2. Percentage of viable cell after the treatment with KWKWKW-NH2 peptide.

Table 1. The minimum inhibitory concentration (MIC) (in μM) and MBC of the the antibiotics test panel.

Table 2. Minimum inhibitory concentration (MIC) in μM and the FIC index of combinations of peptide & the antibiotics against all the tested bacterial strains.

Table 3. The hemolysis activity of the peptide in human erythrocytes in vitro.

Discussion

Conclusions

Data availability

Underlying data

Acknowledgements

References

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Figure 2. Percentage of viable cell after the treatment with KWKWKW-NH₂ peptide.