Mechanism of Action of Bacitracin Bacitracin is an antibiotic produced by the strain of Bacillus subtilis. It acts by inhibiting bacterial cell wall synthesis. It is active against a variety of gram positive and a few gram negative organisms. Mechanism of Action of Neomycin Aminoglycosides like neomycin "irreversibly" bind to specific 30S-subunit proteins and 16S rRNA. Specifically neomycin binds to four nucleotides of 16S rRNA and a single amino acid of protein S12. This interferes with decoding site in the vicinity of nucleotide 1400 in 16S rRNA of 30S subunit. Mechanism of Action of Polymyxin B Polymyxin B sulfate has a bactericidal action against almost all gram-negative bacilli except the Proteus group. Polymyxin B sulfate interacts with the lipopolysaccharide of the cytoplasmic outer membrane of Gram-negative bacteria, altering membrane permeability and causing cell death. It does not need to enter the cell.