EVALUATION OF SUSCEPTIBILITY OF GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA TO HUMAN DEFENSINS BY USING RADIAL DIFFUSION ASSAY

Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs co ntain four defensins termed HNP-1 to HNP-4. We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects o f human defensins against grampositive and -negative bacteria. A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HP LC). The different components were later separated by RP-HPLC and gel permeation chromatography. We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeru ginosa, methicillin-susceptible Staphylococcus aureus, and methicillin -resistant S. aureus strains using the radial diffusion assay, The ant ibacterial activities of HNP-1 and HNP-2 against all strains tested we re similar to those of the crude HNPs, but the activity of HNP-3 was l ess than those of the other defensins. To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minim al inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptib le S. aureus, and 12 strains of methicillin-resistant S. aureus isolat es, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species, However, the MIDs for P. a eruginosa were higher than those for the other organisms tested. The r adial diffusion assay is suitable as a screening test for measuring th e susceptibilities of isolates to defensins, because it is sensitive a nd simple and has good reproducibility.