Department of Biochemistry, Faculty of Science, Protein Proteomic Research Group, Khon Kaen University, Khon Kaen, Thailand.
Ann Clin Microbiol Antimicrob. 2012 Jul 30;11:22. doi: 10.1186/1476-0711-11-22.
The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains.
Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity.
Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates.
The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.
暹罗鳄（Crocodylus siamensis）是一种极度濒危的淡水鳄种。鳄类动物生活中会受到机会性细菌感染，但通常不会受到不良影响。它们并非完全免疫微生物感染，但对其的抵抗力非常有效。在这项研究中，从暹罗鳄的全血中提取的粗提和纯化血浆，针对临床分离的人类病原菌株和相关参考株进行了抗菌活性检测。
通过差速沉淀从暹罗鳄的全血中制备粗提血浆。通过液体生长抑制测定法检测粗提血浆的抗菌活性。通过扫描电子显微镜观察粗鳄血浆对伤寒沙门氏菌 ATCC 11778 细胞的影响。通过 MTT 测定法检测粗鳄血浆对细胞活力的影响。此外，通过阴离子交换柱层析用 DEAE-Toyopearl 650 M 对血浆进行纯化，并检测纯化血浆的抗菌活性。
从暹罗鳄的全血中制备了粗提血浆，对金黄色葡萄球菌、伤寒沙门氏菌、大肠杆菌、霍乱弧菌、铜绿假单胞菌和表皮葡萄球菌等 6 种参考菌株以及表皮葡萄球菌、铜绿假单胞菌、伤寒沙门氏菌和霍乱弧菌等 4 种临床分离株的生长抑制率超过 40%。特别是，伤寒沙门氏菌、霍乱弧菌和表皮葡萄球菌的参考菌株以及伤寒沙门氏菌和霍乱弧菌的临床分离株的生长抑制率超过 80%。粗血浆对伤寒沙门氏菌的细菌细胞有影响，可能有某种抗菌物质逐渐渗透到细胞质空间，扰乱和破坏细菌膜。用巨噬细胞样细胞 RAW 264.7 进行的黄色四唑溴盐（MTT）测定法表明，粗血浆没有毒性。通过柱层析获得了四个合并的馏分，命名为馏分 D1-D4，只有馏分 D1 对参考株和临床致病性分离株显示出生长抑制作用。
暹罗鳄的粗提和纯化血浆对病原菌株和参考株表现出显著的抗菌活性，通过损伤靶细菌细胞的细胞膜。通过 MTT 测定法，暹罗鳄血浆对细胞没有细胞毒性。
