Laboratory of Microbiology and Parasitology of Aquatic Organisms, São Paulo State University (UNESP), Aquaculture Center of Unesp (Caunesp), São Paulo, Brazil.
Faculdade de Ciências Agrárias, Universidade Zambeze (UniZambeze), Tete, Mozambique.
J Appl Microbiol. 2021 Oct;131(4):1762-1774. doi: 10.1111/jam.15080. Epub 2021 Mar 29.
This study aimed to evaluate the in vitro cytotoxicity and efficacy of synthetic host defence peptides (HDPs), alone or in combination with florfenicol (FFC), oxytetracycline (OTC) or thiamphenicol (TAP), against different pathogenic bacteria isolated from diseased fish.
Solid-phase synthesis, purification and characterization of several HDPs were performed manually, using the fluorenylmethyloxycarbonyl protecting group in different resins and via high-performance liquid chromatography-mass spectrometry, respectively. The in vitro cytotoxicity and antimicrobial activity of HDPs, FFC, OTC and TAP against Nile tilapia red blood cells (RBCs) and relevant fish pathogenic bacteria (Aeromonas, Citrobacter, Edwardsiella, Streptococcus, Lactococcus and Vibrio) was determined using the haemolysis assay and broth microdilution method, respectively. The checkerboard assay was used to evaluate the synergy between the most active HDPs and other antimicrobials against the tested strains. MUC 7 12-mer, FFC, OTC and TAP were not cytotoxic to Nile tilapia RBCs, in all tested concentrations. LL-37, (p-BthTX-I) and Hylin-a1 were not cytotoxic at concentrations up to 78·13, 19·53 and 9·77 μg ml , respectively. HDPs demonstrated potent antimicrobial activity (minimum inhibitory concentration ≤31·25 µg ml ) against Aeromonas jandaei (KR-12-a5), Citrobacter freundii (Kr-12-a5; (p-BthTX-I) ; LL-37; and Hylin a1), Streptococcus agalactiae (Hylin a1; (p-BthTX-I) and LL-37), Lactococcus garviae (Hylin a1), and Vibrio fluvialis (KR-12-a5). The combinations of (p-BthTX-I) with TAP and LL-37 with FFC showed synergistic activity against C. freundii (fractional inhibitory concentration index of 0·25 and 0·50, respectively).
Synthetic HDPs have the potential as a good treatment option for bacterial diseases in aquaculture.
The in vivo effectiveness of synthetic HDPs such as KR-12-a5; LL-37; (p-BthTX-I) and Hylin a1 can be tested alone or in combination with conventional antimicrobials as a treatment option to reduce the use of antimicrobials in aquaculture.
本研究旨在评估合成宿主防御肽（HDPs）单独或与氟苯尼考（FFC）、土霉素（OTC）或噻苯咪唑（TAP）联合使用时对来自患病鱼类的不同致病性细菌的体外细胞毒性和功效。
使用不同的树脂和高效液相色谱-质谱法，手动进行了几种 HDPs 的固相合成、纯化和特性鉴定，使用芴甲氧羰基保护基。使用溶血试验和肉汤微量稀释法分别测定 HDPs、FFC、OTC 和 TAP 对尼罗罗非鱼红细胞（RBC）和相关鱼类病原菌（气单胞菌、柠檬酸杆菌、爱德华氏菌、链球菌、乳球菌和弧菌）的体外细胞毒性和抗菌活性。棋盘试验用于评估最有效的 HDPs 与其他抗菌药物联合使用对测试菌株的协同作用。在所有测试浓度下，MUC 7 12 肽、FFC、OTC 和 TAP 对尼罗罗非鱼 RBC 均无细胞毒性。在高达 78.13、19.53 和 9.77μg/ml 的浓度下，LL-37、（p-BthTX-I）和 Hylin-a1 均无细胞毒性。HDPs 对气单胞菌（KR-12-a5）、柠檬酸杆菌（Kr-12-a5；（p-BthTX-I）；LL-37；和 Hylin a1）、无乳链球菌（Hylin a1；（p-BthTX-I）和 LL-37）、乳球菌（Hylin a1）和弗氏弧菌（KR-12-a5）具有很强的抗菌活性（最小抑菌浓度≤31.25μg/ml）。（p-BthTX-I）与 TAP 的组合以及 LL-37 与 FFC 的组合对柠檬酸杆菌（分别为分数抑菌浓度指数 0.25 和 0.50）表现出协同活性。
合成 HDPs 具有作为水产养殖中细菌病的良好治疗选择的潜力。
可以单独测试 KR-12-a5、LL-37、（p-BthTX-I）和 Hylin a1 等合成 HDPs 或与常规抗菌药物联合使用作为治疗选择，以减少水产养殖中抗菌药物的使用。
