Antibacterial effect of thymol against fish-derived drug-resistant Aeromonas veronii in vitro and its mechanism

doi:10.3969/j.issn.1004-1524.2022.07.08

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (7): 1412-1422.DOI: 10.3969/j.issn.1004-1524.2022.07.08

• Animal Science • Previous Articles Next Articles

Antibacterial effect of thymol against fish-derived drug-resistant Aeromonas veronii in vitro and its mechanism

WU Yuru^a(), LIANG Tianyu^a, LIANG Chao^a, TAN Yuanyuan^a, LIU Yuan^a, PAN Xingyu^a, HUANG Xiaoli^b, CHEN Defang^b, GENG Yi^a, OUYANG Ping^a^,^*()

a. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
b. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China

Received:2021-06-25 Online:2022-07-25 Published:2022-07-26
Contact: OUYANG Ping

Abstract

Abstract:

In order to investigate the antibacterial effect and mechanism of thymol against fish-derived drug-resistant Aeromonas veroniae, a strain of A. veronii was isolated from diseased Pelteobagrus fulvidraco. Drug sensitivity test showed that this strain was resistant to florfenicol, ciprofloxacin and enrofloxacin. The antibacterial activity of thymol against drug-resistant A. veronii was studied by determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and growth curve, and the mechanism was explored by measuring its influence on membrane permeability, soluble protein, lactate dehydrogenase activity, DNA and cell ultrastructural changes. The results showed that thymol had inhibitory effect on A. veronii with MIC of 256 μg·mL^-1 and MBC of 512 μg·mL^-1, respectively. After treatment with 512 μg·mL^-1 thymol for 1 h, the electrical conductivity in strain solution was significantly (P<0.01) increased, and the DNA content in strain solution was increased to (115.6±0.5) mg·L^-1. After treatment with 512 μg·mL^-1 thymol, the soluble protein of this strain decreased, the activity of lactate dehydrogenase significantly (P<0.01) decreased by (32.8±0.7)%, (46.2±0.3)%, (46.1±1.6)%, (60.0±1.0)% at 2, 4, 6, 8 h, respectively, the fluorescence intensity and density of DAPI (4',6-diamidino-2-phenylindole) staining were also decreased, and the surface of this strain seemed to dissolve and collapse, shrink and deform, cell wall and membrane separated, with cytoplasm loss and internal cavitation under electron microscope. All these results suggested that thymol had strong antibacterial activity against A. veronii. The increase of membrane permeability with thymol treatment would cause the loss of intracellular substances, and result in bacterial death. Therefore, thymol could be used as a candidate for the treatment of antibiotics-resistant A. veronii infection.

Key words: drug-resistant Aeromonas veronii, thymol, antibacterial mechanism, cell membrane permeability

CLC Number:

S859.3

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.

Figures/Tables 8

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
抗生素 Antibiotic	R	I	S	抑菌圈直径 Diameter of inhibition zone/mm	判定结果 Result
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

Fig.1 Effect of different concentrations of thymol on growth of A. veronii SC strain

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.

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.

Fig.4 Effect of thymol (512 μg·mL-1) on lactate dehydrogenase (LDH) activity of A. veronii SC strain

Fig.5 Effect of thymol (512 μg·mL-1) on DNA extravasation of A. veronii SC strain

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.

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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Antibacterial effect of thymol against fish-derived drug-resistant Aeromonas veronii in vitro and its mechanism

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