1. Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.
- Author
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Satei P, Ghaznavi-Rad E, Fahimirad S, and Abtahi H
- Subjects
- Animals, Anti-Bacterial Agents biosynthesis, Antimicrobial Cationic Peptides biosynthesis, Antimicrobial Cationic Peptides genetics, Cloning, Molecular, Drug Synergism, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Male, Methicillin-Resistant Staphylococcus aureus growth & development, Methicillin-Resistant Staphylococcus aureus pathogenicity, Microbial Sensitivity Tests, Rats, Rats, Wistar, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins pharmacology, Skin drug effects, Skin injuries, Skin microbiology, Staphylococcal Infections microbiology, Staphylococcal Infections pathology, Staphylococcal Skin Infections microbiology, Staphylococcal Skin Infections pathology, Wound Healing drug effects, Wounds, Nonpenetrating microbiology, Wounds, Nonpenetrating pathology, Anti-Bacterial Agents pharmacology, Antimicrobial Cationic Peptides pharmacology, Methicillin-Resistant Staphylococcus aureus drug effects, Staphylococcal Infections drug therapy, Staphylococcal Skin Infections drug therapy, Wounds, Nonpenetrating drug therapy
- Abstract
Purpose: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA)., Method: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested., Results and Conclusion: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log
10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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