Abstract

Antimicrobial peptides have been isolated from mucosal and myeloid tissues of many vertebrates and invertebrates.^1,2,3,4,5 These peptides are one of the host's first defense mechanisms to invading organisms such as bacteria. While these peptides have been well documented in mammals, their isolation in fish is a relatively new focus of research. A novel family of antimicrobial peptides (Piscidins), has been recently isolated from mast cells of hybrid striped bass (Morone saxatilis male x M. chrysops female).⁵ The purpose of this study was to determine the susceptibility of human and fish bacterial pathogens to another novel antimicrobial peptide, also isolated from the gills of hybrid striped bass.

Bacterial susceptibility was tested using a standard microtiter dilution assay.⁵ Briefly, the bacteria were grown on Mueller Hinton agar and then a single colony was transferred to Mueller Hinton broth (MHB). Following incubation, this culture was diluted in MHB to obtain a final bacterial concentration of 5 X 10⁵ CFU/ml. Serial dilutions of the novel peptide were made in 2% bovine serum albumin and 0.01% acetic acid. The minimum inhibitory concentration (MIC) was determined as the lowest dilution with no visible growth. The MBC was the lowest concentration that killed at least 99.9% of the CFUs contained in the original inoculum, as determined by plating 20 μl of the three lowest dilutions with no visible growth.

The novel peptide is inhibitory and bactericidal for the majority of bacteria tested. This includes gram (+) and (-) human and fish pathogens. The novel peptide is therefore a relatively broad-spectrum antibiotic. Two bacteria, Enterococcus facecalis and Proteus mirabilis, are not susceptible at a peptide concentration of 200 μg/ml and are therefore likely resistant. This resistance may be due to an innate property of the bacteria, the mechanism of action of the peptide, or an interaction between the two. Additionally, the MBC of most bacteria is equal to the MIC, indicating that the thresholds for inhibition and death are quite similar. This may be related to and may aid in elucidating the mechanism of action of the novel peptide.

The pathogens were less susceptible to the novel peptide than to the Piscidins. One possible explanation for this difference may be the target organism. Perhaps the novel peptide is targeted more at other pathogens such as fungi or parasites and less at bacteria. Further research will help in determining if this is indeed the case.

While there is still much research to be done, our results suggest that modifications of this novel peptide may be useful for treating microbial infections in humans. This peptide might also be an important defensive molecule in hybrid striped bass. With the increasing concern of antibiotic resistance, the discovery of novel broad-spectrum antimicrobial peptides may prove essential in veterinary and human medicine in the future.

Table 1. The susceptibility of fish and human bacterial pathogens to the novel peptide. MIC=Minimum inhibitory concentration, MBC=Minimum bactericidal concentration

This project was supported by Sea Grant, USDA-NRI, and the United States-Israel Bi-national Agricultural Research and Development Fund.

References

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2.  Khoo L, DW Robinette, EJ Noga. 1999. Callinectin, an antibacterial peptide from blue crab, Callinectes sapidus, hemocytes. Marine Biotechnology 1:44-51.

3.  Noga EJ, Z Fan, U Silphaduang. 2001. Histone-like proteins from fish are lethal to the parasitic dinoflagellate Amyloodinium ocellatum. Parasitology 123: 57-65.

4.  Park IY, CB Park, MS Kim, SC Kim. 1998. Parasin I, an antimicrobial peptide derived from Histone H2A in the catfish, Parasilurus asotus. FEBS Letters 437:258-262.

5.  Silphaduang U, EJ Noga. 2001. Peptide antibiotics in mast cells of fish. Nature 414:268-269.