| Vibrio alginolyticus is one of the primary etiological agents for the death of aquatic animals during nursery and grown-up stages. It can infect fish, causing bleeding, ulcer, and blood poisoning; it can infect shrimps, causing shrimp postlarva bacterial vitrified syndrome, acute hepatopancreatic necrosis disease, rotted gill disease, and white fecal syndrome; it can infect crabs, causing milk disease and toothpaste disease; it can infect sea cucumbers, causing skin ulcer syndrome; and also it can infect shellfish, thereby bringing great economic loss to the marine aquaculture industry. Antibiotics, disinfectants, and microecologics are usually used in the aquaculture production to prevent or cure these infectious diseases, but the effect is unsatisfactory. In addition, irrational drug use easily gives rise to the increase of risks in environmental pollution, bacterial drug resistance, and drug residue. Compared with antibiotics, traditional Chinese medicines have the advantages of antibacterial property, immunoregulation, slight toxic and side effect, little drug resistance and drug residue, etc. Therefore, they have gradually aroused people's attention in recent years.
Taking Vibrio alginolyticus and Vibrio parahemolyticus isolated from penaeid shrimp larvae with bacterial vitrified syndrome as research objects, this study performed the screening of bacteriostatic activity of 50 traditional Chinese medicines, and the results showed that the four traditional Chinese medicines, namely terminalia chebula, galla chinensis, pomegranate peel, and sanguisorba officinalis, had a good bacteriostatic effect. Gallic acid (GA) is a main active component of the four traditional Chinese medicines. It has been reported that GA has good antibacterial, antiviral, anti-inflammatory, and antioxidative properties and can protect liver and improve the body's immunological function, so it is of positive significance for the prevention and cure of multiple animal diseases.
Although it has been reported that GA has a good antibacterial effect on various bacteria, the bacteriostatic activity and possible mechanism of GA against Vibrio alginolyticus are still not reported. By measuring the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and growth curve of GA against Vibrio alginolyticus, this paper made a research on the bacteriostatic activity of GA against Vibrio alginolyticus. Moreover, the changes in the AKP activity and electrical conductivity of the supernatant fluid of bacteria solution and in the biofilms, moveability, and aggregation capacity of Vibrio alginolyticus were determined before and after the interaction between different concentrations of GA solution and Vibrio alginolyticus, so as to investigate the bacteriostatic mechanism of GA against Vibrio alginolyticus.
The results revealed that the MIC and MBC of GA against Vibrio alginolyticus were 4 mg/mL and 8 mg/mL, respectively, and these two concentrations of GA could completely inhibit the growth of Vibrio alginolyticus, while 2 mg/mL GA could significantly suppress the growth of Vibrio alginolyticus, suggesting that the inhibitory effect of GA on Vibrio alginolyticus is dose-dependent. Thus, 4 mg/mL and above GA should be selected and used to achieve an effective bacteriostatic effect on the growth of Vibrio alginolyticus.
The cell wall of bacteria is an important structure to maintain their morphology and protect their body. Biofilms are composed of various extracellular materials secreted by bacteria, such as protein, exopolysaccharides, lipid, and extracellular DNA, and they can not only enhance bacteria's resistance to adverse external environment, but also increase their resistance to antibacterial agents. However, traditional Chinese medicines can restrain the formation and development of bacteria's biofilms, destroy their cell wall and cell membrane, affect the synthesis of their protein and nucleic acid, a genetic material, promote oxidative stress, and inhibit the expression of virulent factor to suppress or kill bacteria. The changes in AKP and electrical conductivity of bacterial culture serve as an index to verify whether bacteria's cell wall is destroyed or whether its permeability increases. The results of this research indicated that 1, 2, 4, and 8 mg/mL GA could all destroy the cell wall within 2 h and cause AKP leakage, and the destroying degree of the cell wall of Vibrio alginolyticus was positively correlated with GA concentrations; 2, 4, and 8 mg/mL GA could remarkably increase electrical conductivity of the supernatant fluid of Vibrio alginolyticus; compared with the positive control group, 4 and 8 mg/mL GA had a significant inhibitory effect on the formation of the biofilms of Vibrio alginolyticus, with the inhibition ratio of 83.26% (P<0.05) and 77.80% (P<0.05), respectively, and meanwhile 4 and 8 mg/mL GA could obviously eliminate mature biofilms, with the elimination ratio of 68.01% (P<0.05) and 67.54% (P<0.05), respectively; 4 and 8 mg/mL GA could completely suppress the growth of Vibrio alginolyticus on the LB swimming motility agar plate; and 1, 2, 4, and 8 mg/mL GA could significantly inhibit the aggregation capacity of Vibrio alginolyticus, with the aggregation rate reducing to 18.68% (P<0.05), 19.19% (P<0.05), 25.70% (P<0.05), and 37.41% (P<0.05), respectively.
In conclusion, GA has a strong inhibitory effect on Vibrio alginolyticus by restraining its growth, destroying its cell wall, increasing its cell membrane permeability, suppressing the formation of its biofilms, eliminate its mature biofilms, and inhibiting its moveability and aggregation capacity. This study lays a good foundation for exploration on the action mechanism of GA in suppressing Vibrio alginolyticus and provides a theoretical basis for GA in the prevention and cure of infectious diseases caused by Vibrio alginolyticus in aquatic animals. |
