Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay

Hiromu Takemura, Mitsuo Kaku, Shigeru Kohno, Yoichi Hirakata, Hironori Tanaka, Ryoji Yoshida, Kazunori Tomono, Hironobu Koga, Akihiro Wada, Toshiya Hirayama, Shimeru Kamihira

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46 Citations (Scopus)


Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs contain 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 of human defensins against gram-positive 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-HPLC). 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 aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus strains using the radial diffusion assay. The anti-bacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less 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 minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptible S. aureus, and 12 strains of methiciliin-resistant S. aureus isolates, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species. However, the MIDs for P. aeruginosa were higher than those for the other organisms tested. The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.

Original languageEnglish
Pages (from-to)2280-2284
Number of pages5
JournalAntimicrobial agents and chemotherapy
Issue number10
Publication statusPublished - 1996 Oct
Externally publishedYes

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases


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