山姜素对鱼源耐药性嗜水气单胞菌体外抗菌作用的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-0743

摘要/Abstract

摘要：

旨为探究山姜素对耐药性嗜水气单胞菌的体外抑菌效果及其作用机制。通过测定山姜素对嗜水气单胞菌生长、细菌形态、电导率、乳酸脱氢酶（LDH）、蛋白质代谢和DNA的影响,研究山姜素对耐药性嗜水气单胞菌的抑菌机制。山姜素对5株鱼源耐药性嗜水气单胞菌的最小抑菌浓度（MIC）和最小杀菌浓度（MBC）分别为128-256 μg/mL和512-1 024 μg/mL。与对照组相比,2MIC山姜素作用于嗜水气单胞菌CW株8 h、16 h后,菌体皱缩,细胞壁和细胞膜破损,细胞质丢失,内部空化;作用2 h后,菌悬液电导率、乳酸脱氢酶含量、DNA外渗量分别增长了3.97%、26.09%和10.50 μg/mL;作用2、4和8 h后,菌悬液核酸荧光强度和密度呈现随时间降低的趋势。山姜素对嗜水气单胞菌的体外抑菌机制为损伤菌体的细胞壁、增加细胞膜的通透性来抑制其生长繁殖。

关键词: 山姜素, 嗜水气单胞菌, 抑菌机制, 菌体形态

Abstract:

The aim of this study is to investigate the antibacterial effect and the mechanism of alpinetin against drug-resistant Aeromonas hydrophila in vitro. We investigated the mechanism of alpinetin against drug-resistant A. hydrophila by measuring the effects of alpinetin on A. hydrophila cell growth,cell morphology,electrical conductivity,lactate dehydrogenase（LDH）,protein metabolism and DNA. The minimal inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of alpinetin against 5 strains of fish-derived A. hydrophila were 128-256 μg/mL and 512-1 024 μg/mL,respectively. Compared with the control group,after treating A. hydrophila with 2 MIC alpinetin for 8 or 16 h,the bacteria showed shrinkage,disrupted cell wall and membrane,loss of cytoplasm,and interior cavitation. After treating for 2 h,bacteria suspension’s electrical conductivity,LDH activity content and DNA extravasation increased by 3.97%,26.09% and 10.50 μg/mL,respectively. After treating for 2,4 and 8 h,nucleic acid fluorescence intensity and density in the bacterial suspension showed a time-dependent decreasing tendency. The in vitro antibacterial mechanism of alpinetin against A. hydrophila is to inhibit their growth and reproduction mainly via destroying cell walls of bacteria and increasing the permeability of the cell membrane.

Key words: alpinetin, Aeromonas hydrophila, antibacterial mechanism, cell morphology

陈杰豪, 缪玉佳, 梁超, 陶雨, 欧阳萍, 汪开毓, 耿毅, 石存斌, 李宁求. 山姜素对鱼源耐药性嗜水气单胞菌体外抗菌作用的研究[J]. 生物技术通报, 2021, 37(2): 103-110.

CHEN Jie-hao, MIAO Yu-jia, LIANG Chao, TAO Yu, OUYANG Ping, WANG Kai-yu, GENG Yi, SHI Cun-bin, LI Ning-qiu. Study on the Antibacterial Mechanism of Alpinetin Against Fish-derived Drug-resistant Aeromonas hydrophila in vitro[J]. Biotechnology Bulletin, 2021, 37(2): 103-110.

图/表 8

表1 山姜素对5株不同鱼源嗜水气单胞菌的MIC值和MBC值

嗜水气单胞菌名称	来源	耐药性^*	山姜素
嗜水气单胞菌名称	来源	耐药性^*	MIC/（μg·mL^-1）	MBC/（μg·mL^-1）
CW	斑点叉尾鮰	GEN,NEO,TE,DO,AML	128	256
1G	鲫鱼	ROX,RFP,AML	256	512
Ah	长吻鮠	GEM,FFC,DO,AML	256	1024
WZ1	黄颡鱼	ROX,RFP,AML	128	512
S1D	鳜鱼	GEM,AML	256	512

图1 山姜素对菌株CW生长的影响

图2 山姜素不同作用时间对菌体细胞形态结构的影响 A：4 h对照组SEM;B：4 h 256 μg/mL山姜素SEM;C：8 h对照组TEM;D：8 h 256 μg/mL山姜素TEM;E：16 h对照组TEM;F：16 h 256 μg/mL山姜素TEM

图3 山姜素对菌悬液电导率的影响

图4 山姜素对菌株CW可溶性蛋白质含量的影响 M：标准分子量蛋白Marker;1：4 h对照组;2：4 h 256 μg/mL山姜素处理组;3：8 h对照组;4：8 h 256 μg/mL山姜素处理组;5：16 h对照组;6：16 h 256 μg/mL山姜素处理组;7：24 h 对照组;8：24 h 256 μg/mL山姜素处理组

图5 山姜素对菌株CW的LDH含量的影响

图6 山姜素对菌株CW DNA外渗量的影响

图7 山姜素作用菌株CW DNA后DAPI染色观察 A：0 h对照组;B：2 h 256 μg/mL山姜素处理组;C：4 h 256 μg/mL山姜素处理组;D：8 h 256 μg/mL山姜素处理组

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