Bacterial cultures
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).
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. Purity was determined using reverse phase high performance liquid chromatography (RPHPLC), and validated using mass spectrometry and electrospray ionization mass spectrometry (ESIMS)13
Biofilm activity assay
For biofilm formation we used the Calgary biofilm device, as previously reported14 with Staphylococcus aureus (ATCC 33591).
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 triplicate15.
Antibiotic Used In This Study
Levofloxacin, Chloramphenicol, Rifampicin Amoxicillin, Clarithromycin,Doxycycline,Vancomycin and and Cefixime was obtained from sigma Aldrich .
Mic And Mbc Determination Of Antibiotics Alone
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.
Mic Determination Of Peptides-antibiotics Combinations
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.
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 technique18.
Erythrocyte Hemolytic Assay
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).