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Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion
1Department of Laboratory Medicine, Nagasaki University School of Medicine, Japan.
Abstract:
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 antibacterial 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 methicillin-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.
Insights
Human defensins (HNP-1 to HNP-3) show antibacterial activity against various bacteria, with HNP-1 and HNP-2 being more potent than HNP-3. The radial diffusion assay effectively measures bacterial susceptibility to these defensins.
Area of Science:
- Immunology
- Microbiology
- Biochemistry
Background:
- Defensins are cationic antimicrobial peptides found in polymorphonuclear neutrophils (PMNs).
- Human PMNs contain four types of defensins: HNP-1, HNP-2, HNP-3, and HNP-4.
- These peptides play a crucial role in the innate immune system against bacterial infections.
Purpose of the Study:
- To evaluate the antibacterial activity of purified human neutrophil defensins (HNP-1, -2, -3) against Gram-positive and Gram-negative bacteria.
- To compare the efficacy of individual defensins (HNP-1, -2, -3) with a crude mixture (crude HNPs).
- To assess the suitability of the radial diffusion assay for determining bacterial susceptibility to defensins.
Main Methods:
- Purification of crude human neutrophil defensins (HNPs) from PMN extracts using reversed-phase high-pressure liquid chromatography (RP-HPLC).
- Separation of individual defensins (HNP-1, -2, -3) using RP-HPLC and gel permeation chromatography.
- Antibacterial activity testing using a radial diffusion assay against various strains of Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus.
Main Results:
- HNP-1 and HNP-2 exhibited antibacterial activity comparable to crude HNPs against all tested bacterial strains.
- HNP-3 showed less potent activity compared to HNP-1 and HNP-2.
- Minimal Inhibitory Doses (MIDs) were determined for different bacterial species; P. aeruginosa generally required higher MIDs, indicating lower susceptibility.
- The radial diffusion assay demonstrated sensitivity, simplicity, and good reproducibility for screening bacterial susceptibility to defensins.
Conclusions:
- Human neutrophil defensins HNP-1 and HNP-2 are potent antibacterial agents against a range of clinically relevant bacteria.
- HNP-3 possesses weaker antimicrobial activity compared to HNP-1 and HNP-2.
- The radial diffusion assay is a valuable and practical tool for assessing bacterial susceptibility to defensins, aiding in the understanding of innate immunity and potential therapeutic strategies.