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Updated: Jun 16, 2026

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method
Published on: April 18, 2019
Synergistic antibacterial effect of CATH-2 and D-amino acids against mastitis causing gram-positive bacteria
Edwin J A Veldhuizen1, Sasja T van der Velden1, David Alons1
1Department Biomolecular Health Sciences, Utrecht University, Utrecht, Netherlands.
Introduction:
To effectively treat bacterial diseases caused by antibiotic-resistant strains, new antimicrobial agents are needed to overcome this resistance. This study explored whether the combination of the antimicrobial peptide CATH-2 with positively charged D-amino acids can be used as a potential alternative to antibiotics for treatment of mastitis in cows.
Methods:
Antibacterial assays and bacterial growth kinetics were assessed in the presence of CATH-2 combined with either D-arginine or D-lysine. Synergistic interactions between the two components were evaluated using checkerboard assays. Cytotoxicity of the tested compounds was determined through WST, LDH release, and trypan blue exclusion assays on multiple mammalian cell lines, including bovine udder epithelial cells.
Results:
The combination of CATH-2 and D-arginine showed strong synergistic antibacterial activity against Gram-positive bacteria, including all tested multidrug-resistant clinical Staphylococcus aureus isolates, in both LB medium (FIC = 0.37) and fresh raw milk (FIC < 0.12). No detectable cytotoxic effects on bovine udder epithelial cells or other epithelial cell lines were observed at concentrations that were effective against bacterial growth.
Conclusions:
This novel combination of an antimicrobial peptide with a positively charged D-amino acid offers promising potential for treating bacterial infections, particularly those caused by antibiotic-resistant strains.
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