The ability of TP3 and TP4 to protect tilapia from a lethal <i>V</i>. <i>vulnificus</i> challenge in the co-treatment experiments.

<p>(a) There were six groups in this experiment: a control, either injected with only PBS or only <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish), or injection with a mixture of <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and TP3 peptide (0.1, 1, 10, or 20 μg/fish). The survival rates were recorded after 3, 6, 12, 24, and 48 hours for up to seven days. (b) Survival rates from experiments in which the fish that survived the above trial were re-injected and a control group was injected with a mixture of <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and TP3 peptides (20 μg/fish); the results after seven days are shown. After being re-challenged, the surviving fish from the previous experiment were continuously monitored to determine their survival rates for seven days. (c) There were six groups in this experiment: a control group, groups injected with only PBS or only <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish), and groups treated with a mixture of <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and TP4 peptides (0.1, 1, 10, or 20 μg/fish). The survival rates were recorded as a percentage after 3, 6, 12, 24, and 48 hours for up to seven days. (b) The survival rates of fish from the first trial that were re-challenged with a mixture of <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and TP4 peptides (20 μg/fish), which were injected simultaneously after seven days. After being re-challenged, the surviving fish were continuously monitored to determine their survival rates for seven days. Results marked with *** (P<0.001) or * (P<0.05) differed significantly among the treatments.</p

Study of the Antimicrobial Activity of Tilapia Piscidin 3 (TP3) and TP4 and Their Effects on Immune Functions in Hybrid Tilapia (<i>Oreochromis</i> spp.)

<div><p>To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from <i>Oreochromis niloticus</i>, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (<i>Oreochromis</i> spp.) with <i>Vibrio vulnificus</i> and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. <i>In vivo</i> experiments showed that co-treatment with <i>V</i>. <i>vulnificus</i> and TP3 (20 μg/fish) or TP4 (20 μg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and <i>V</i>. <i>vulnificus</i> into tilapia and then re-challenged the fish with <i>V</i>. <i>vulnificus</i> after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 μg/fish) or TP4 (20 μg/fish) for 30 minutes prior to <i>V</i>. <i>vulnificus</i> infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 μg/fish or 30 μg/fish) or TP4 (20 μg/fish) 30 minutes after <i>V</i>. <i>vulnificus</i> infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of <i>V</i>. <i>vulnificus</i> bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with <i>V</i>. <i>vulnificus</i> were 7.8 and 62.5 μg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 μg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed that <i>V</i>. <i>vulnificus</i> disrupted the outer membranes of cells, resulting in the loss of cell shape and integrity. We examined whether TP3 and TP4 increased the membrane permeability of <i>V</i>. <i>vulnificus</i> by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). Treating fish with TP3 and TP4 under different pH and temperature conditions did not significantly increase MIC values, suggesting that temperature and the acid-base environment do not affect AMP function. In addition, the qPCR results showed that TP3 and TP4 influence the expression of immune-responsive genes, including interleukin (IL)-1β, IL-6, and IL-8. In this study, we demonstrate that TP3 and TP4 show potential for development as drugs to combat fish bacterial infections in aquaculture.</p></div

TP3 and TP4 induce membrane permeation and cause membrane disruption in V. vulnificus.

<p>(a) Treatment with TP3 or TP4 resulted in the appearance of cell injuries in <i>V</i>. <i>vulnificus</i> and the formation of electron-dense structures on the surfaces of the cells. The blue arrow indicates damage to the plasma membranes of <i>V</i>. <i>vulnificus</i> cells. The results of scanning electron microscopy (SEM) are shown on the left, and transmission electron microscopy (TEM) results are shown on the right. (b) We determined <i>V</i>. <i>vulnificus</i> membrane permeability by measuring the fluorescence resulting from the uptake of 1-N-phenyl-naphthylamine (NPN). We used cultures of <i>V</i>. <i>vulnificus</i> cells alone or <i>V</i>. <i>vulnificus</i> incubated with NPN as the control groups. Cells were treated with TP3, TP4, or kanamycin at different concentrations, and the resulting NPN fluorescence intensities were recorded to determine their correlations with membrane permeability. The data are shown as the means±SE of three independent experiments.</p

Time-dependent effects of pre-treatment with <i>V</i>. <i>vulnificus</i> followed by injection with TP3 or TP4.

<p>(a) Tilapia were infected with <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and then injected with TP3 (20 μg/fish) or PBS (control) after 0.5, 1, or 2 hours. Another control group was injected with only <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish). (b) Tilapia were first infected with <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish) and then injected with TP4 (20 μg/fish) or PBS (control) after 0.5, 1, or 2 hours, and another control group was injected with only <i>V</i>. <i>vulnificus</i> (2×10⁵ CFU/fish). (c) The same experimental method described in (a) was used, but the fish were treated with TP3 (30 μg/fish). (d) The same experimental method described in (b) was used, but the fish were treated with TP4 (30 μg/fish). (e) The same experimental method described in (a) was used, but the fish were treated with TP3 (40 μg/fish). (f) The same experimental method described in (b) was used, but the fish were treated with TP4 (40 μg/fish). ns, not significantly different. Results marked with ** (P<0.01) or * (P<0.05) differed significantly among the treatment groups.</p

Analysis of protease effects on TP3 and TP4 antimicrobial activity.

<p>Analysis of protease effects on TP3 and TP4 antimicrobial activity.</p

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TP3, TP4, kanamycin, and ampicillin on <i>V</i>. <i>vulnificus</i>.

<p>Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TP3, TP4, kanamycin, and ampicillin on <i>V</i>. <i>vulnificus</i>.</p

Analysis of the effect of pH on TP3 and TP4 antimicrobial activity.

<p>Analysis of the effect of pH on TP3 and TP4 antimicrobial activity.</p

Primer used in this study.

<p>Primer used in this study.</p

Synergistic activity of ampicillin and kanamycin in combination with TP3 or TP4.

<p>(a) The synergistic effects of TP3 and ampicillin against <i>V</i>. <i>vulnificus</i>. (b) The synergistic effects of TP3 and kanamycin against <i>V</i>. <i>vulnificus</i>. (c) The synergistic effects of TP4 and ampicillin against <i>V</i>. <i>vulnificus</i>. (d) The synergistic effects of TP4 and kanamycin against <i>V</i>. <i>vulnificus</i>. Each bar represents the mean value obtained from three experiments. Treatment results (mean±SE) marked with different numbers were significantly different (P<0.05) from each other.</p

Time-kill analysis.

<p>Time-kill analysis showing the effects of (a) TP3 and (b) TP4 against <i>V</i>. <i>vulnificus</i>. Peptides at 0.5xMIC, 1xMIC, or 2xMIC were added to the bacterial cultures, which were monitored for 24 hours. Aliquots were collected at 1, 2, 3, 6, 9, 12, and 24 hours to count the bacteria. The data are the means±SE.</p