浙江农业学报 ›› 2022, Vol. 34 ›› Issue (7): 1412-1422.DOI: 10.3969/j.issn.1004-1524.2022.07.08
吴玉如a(), 梁天雨a, 梁超a, 谭媛元a, 柳源a, 潘星羽a, 黄小丽b, 陈德芳b, 耿毅a, 欧阳萍a,*(
)
收稿日期:
2021-06-25
出版日期:
2022-07-25
发布日期:
2022-07-26
通讯作者:
欧阳萍
作者简介:
* 欧阳萍,E-mail: ouyang.ping@live.cn基金资助:
WU Yurua(), LIANG Tianyua, LIANG Chaoa, TAN Yuanyuana, LIU Yuana, PAN Xingyua, HUANG Xiaolib, CHEN Defangb, GENG Yia, OUYANG Pinga,*(
)
Received:
2021-06-25
Online:
2022-07-25
Published:
2022-07-26
Contact:
OUYANG Ping
摘要:
为探究百里香酚对鱼源耐药性维氏气单胞菌(Aeromonas veronii)的抑菌效果和作用机制,从患病黄颡鱼(Pelteobagrus fulvidraco)体内分离得到一株维氏气单胞菌,药敏试验结果显示,其对氟苯尼考、环丙沙星和恩诺沙星耐药。通过测定最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和生长曲线来评价百里香酚对耐药性维氏气单胞菌的抑菌活性;通过测定百里香酚对其细胞膜通透性、可溶性蛋白、乳酸脱氢酶活性和DNA的影响,结合电镜观察细菌超微结构的变化研究其作用机制。结果显示,百里香酚对耐药性维氏气单胞菌的抑菌效果明显,MIC为256 μg·mL-1,MBC为512 μg·mL-1。经512 μg·mL-1的百里香酚作用1 h后,维氏气单胞菌液电导率极显著(P<0.01)上升,DNA外渗量迅速上升至(115.6±0.5)mg·L-1。经512 μg·mL-1的百里香酚作用后,维氏气单胞菌的可溶性蛋白明显变少,细菌乳酸脱氢酶活性在2、4、6、8 h分别极显著(P<0.01)降低了(32.8±0.7)%、(46.2±0.3)%、(46.1±1.6)%、(60.0±1.0)%,DAPI(4',6-二脒基-2-苯基吲哚)染色的荧光强度和密度降低,电镜下可见菌体表面溶解塌陷,皱缩变形,细胞壁和细胞膜分离,细胞质丢失,内部空化。上述结果表明,百里香酚对维氏气单胞菌具有较强的抗菌活性,主要通过增加细胞膜通透性导致胞内物质流失造成细菌死亡。百里香酚可以作为治疗耐药性维氏气单胞菌感染的备选药物进行研究。
中图分类号:
吴玉如, 梁天雨, 梁超, 谭媛元, 柳源, 潘星羽, 黄小丽, 陈德芳, 耿毅, 欧阳萍. 百里香酚对鱼源耐药性维氏气单胞菌的体外抑菌效果及其机制研究[J]. 浙江农业学报, 2022, 34(7): 1412-1422.
WU Yuru, LIANG Tianyu, LIANG Chao, TAN Yuanyuan, LIU Yuan, PAN Xingyu, HUANG Xiaoli, CHEN Defang, GENG Yi, OUYANG Ping. Antibacterial effect of thymol against fish-derived drug-resistant Aeromonas veronii in vitro and its mechanism[J]. Acta Agriculturae Zhejiangensis, 2022, 34(7): 1412-1422.
抗生素 Antibiotic | 基于抑菌圈直径(mm)的耐药性判定标准 Criterion for drug resistance based on diameter (mm) of inhibition zone | 抑菌圈直径 Diameter of inhibition zone/mm | 判定结果 Result | ||
---|---|---|---|---|---|
R | I | S | |||
环丙沙星Ciprofloxacin | ≤15 | 16~20 | ≥21 | 12 | R |
氟苯尼考Florfenicol* | ≤12 | 13~17 | ≥18 | 12 | R |
恩诺沙星Enrofloxacin* | ≤13 | 14~27 | ≥28 | 13 | R |
庆大霉素Gentamicin* | ≤12 | 13~14 | ≥15 | 14 | I |
美罗培南Meropenem | ≤13 | 14~15 | ≥16 | 15 | I |
四环素Tetracycline | ≤14 | 15~18 | ≥19 | 16 | I |
阿奇霉素Azithromycin | ≤13 | 14~17 | ≥18 | 17 | I |
头孢克洛Cefaclor | ≤14 | 15~17 | ≥18 | 18 | S |
头孢噻肟Cefotaxime | ≤14 | 15~17 | ≥18 | 19 | S |
复方新诺明 | ≤15 | 16~23 | ≥24 | 20 | I |
Compound sulfamethoxazole | |||||
多黏菌素B Polymyxin B | ≤8 | 8~11 | ≥12 | 21 | S |
磺胺异恶唑Sulfaisoxazole | ≤18 | 19~31 | ≥32 | 22 | I |
表1 维氏气单胞菌SC株的耐药性
Table 1 Drug resistance of A. veronii SC strain
抗生素 Antibiotic | 基于抑菌圈直径(mm)的耐药性判定标准 Criterion for drug resistance based on diameter (mm) of inhibition zone | 抑菌圈直径 Diameter of inhibition zone/mm | 判定结果 Result | ||
---|---|---|---|---|---|
R | I | S | |||
环丙沙星Ciprofloxacin | ≤15 | 16~20 | ≥21 | 12 | R |
氟苯尼考Florfenicol* | ≤12 | 13~17 | ≥18 | 12 | R |
恩诺沙星Enrofloxacin* | ≤13 | 14~27 | ≥28 | 13 | R |
庆大霉素Gentamicin* | ≤12 | 13~14 | ≥15 | 14 | I |
美罗培南Meropenem | ≤13 | 14~15 | ≥16 | 15 | I |
四环素Tetracycline | ≤14 | 15~18 | ≥19 | 16 | I |
阿奇霉素Azithromycin | ≤13 | 14~17 | ≥18 | 17 | I |
头孢克洛Cefaclor | ≤14 | 15~17 | ≥18 | 18 | S |
头孢噻肟Cefotaxime | ≤14 | 15~17 | ≥18 | 19 | S |
复方新诺明 | ≤15 | 16~23 | ≥24 | 20 | I |
Compound sulfamethoxazole | |||||
多黏菌素B Polymyxin B | ≤8 | 8~11 | ≥12 | 21 | S |
磺胺异恶唑Sulfaisoxazole | ≤18 | 19~31 | ≥32 | 22 | I |
图2 百里香酚(512 μg·mL-1)对维氏气单胞菌SC株菌液电导率的影响 “**”表示差异极显著(P<0.01)。下同。
Fig.2 Effect of thymol (512 μg·mL-1) on conductivity of A. veronii SC strain solution “**” means significant difference at P<0.01. The same as below.
图3 百里香酚(512 μg·mL-1)对维氏气单胞菌SC株可溶性蛋白的影响 M代表Marker(蛋白质量标记),泳道1、3、5、7分别对应于对照组4、8、16、24 h的菌体蛋白,泳道2、4、6、8分别对应于添加百里香酚(512 μg·mL-1)处理组4、8、16、24 h的菌体蛋白。
Fig.3 Effect of thymol (512 μg·mL-1) on soluble protein of A. veronii SC strain M, Marker (protein weight marker); Lanes 1, 3, 5, 7, Proteins of SC strain in the control group at 4, 8, 16, 24 h, respectively; Lanes 2, 4, 6, 8, Proteins of SC strain in the experiment group (addition of 512 μg·mL-1 thymol) at 4, 8, 16, 24 h, respectively.
图6 百里香酚(512 μg·mL-1)对维氏气单胞菌SC株DNA染色荧光强度的影响 A,对照组(0 h);B~D,分别对应于百里香酚(512 μg·mL-1)处理2、4、8 h。
Fig.6 Effect of thymol (512 μg·mL-1) on fluorescence intensity of A. veronii SC strain A, Control (0 h); B-D, Treatment with thymol (512 μg·mL-1) for 2, 4, 8 h, respectively.
图7 百里香酚(512 μg·mL-1)对维氏气单胞菌SC株菌体形态结构的影响 A~B,扫描电镜(SEM)观察结果;C~F,透射电镜(TEM)观察结果。A,对照组,4 h;B,百里香酚(512 μg·mL-1)处理4 h,箭头指示菌体表面溶解塌陷;C,对照组,8 h;D,百里香酚(512 μg·mL-1)处理8 h,箭头指示细胞染色不均,并开始出现空泡化;E,对照组,16 h;F,百里香酚(512 μg·mL-1)处理16 h,箭头指示细胞膜与细胞壁分离,细胞皱缩坏死。
Fig.7 Effect of thymol (512 μg·mL-1) on morphology of A. veronii SC strain A-B, Scanning electron microscope (SEM) result; C-F, Transmission electron microscope (TEM) result. A, Control group, 4 h; B, Treatment with thymol (512 μg·mL-1) for 4 h, arrow indicates the dissolution and collapse of the bacterial cell surface; C, Control group, 8 h; D, Treatment with thymol (512 μg·mL-1) for 8 h, arrow indicates that the cells are stained unevenly, and vacuoles have begun to appear; E, Control group, 16 h; F, Treatment with thymol (512 μg·mL-1) for 16 h, arrow indicates that the cell membrane is separated from the cell wall, with cell shrinking and necrosis.
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