| Vibrio alginolyticus is a primary etiological agent for aquatic animal death. V. alginolyticus can infect fish, causing bleeding, ulcer, and blood poisoning; shrimps, causing shrimp post-larvae bacterial vitrified syndrome, acute hepatopancreatic necrosis disease, rotted gill disease, and white fecal syndrome; crabs, causing milk and toothpaste diseases; sea cucumbers, causing skin ulcer syndrome; and shellfish, thereby leading to substantial economic loss in the marine aquaculture industry. Antibiotics, disinfectants, and microecologics are usually used in the aquaculture to prevent or cure such infectious diseases however, their outcomes remain unsatisfactory. In addition, irrational drug use increases risks, such as environmental pollution, bacterial drug resistance, and drug residue. Compared with antibiotics, traditional Chinese medicines (TCMs) present certain advantages, such as antibacterial properties, immunoregulation, slight toxic and side effect, and low drug resistance or residue. Therefore, TCMs have garnered increasing attention in recent years.
Using V. alginolyticus and V. parahemolyticus isolated from penaeid shrimp larvae with bacterial vitrified syndrome as research objects, we screened the bacteriostatic activity of 50 TCMs. Four kinds of TCM, namely Terminalia chebula, Galla chinensis, pomegranate peel, and Sanguisorba officinalis, demonstrated good bacteriostatic effects. Gallic acid (GA) is a main active component of these four TCM compounds. GA has good antibacterial, antiviral, anti-inflammatory, and antioxidative properties and can protect the liver and improve the immunological function of the body, thus it may be used to treat and prevent multiple animal diseases.
Although GA has antibacterial effects on various bacteria, the bacteriostatic activity and possible mechanism against V. alginolyticus remains unelucidated. By measuring the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and growth curve of GA against V. alginolyticus, we evaluated the bacteriostatic activity of GA against V. alginolyticus. Moreover, the changes in the AKP activity and electrical conductivity of the supernatant fluid of bacteria solution and biofilms, moveability, and aggregation capacity of V. alginolyticus were determined before and after GA treatment to investigate the bacteriostatic mechanism of GA against V. alginolyticus.
The MIC and MBC of GA against V. alginolyticus were 4 mg/mL and 8 mg/mL, respectively. These two GA concentrations could completely inhibit V. alginolyticus growth, while 2 mg/mL GA significantly suppressed V. alginolyticus growth, suggesting that the inhibitory effect of GA on V. alginolyticus was dose-dependent. Thus, GA concentrations greater than 4 mg/mL should be selected and used to achieve an effective bacteriostatic effect on V. alginolyticus.
The bacterial cell wall is an important structure that maintains cell morphology and facilitates cell protection. Biofilms comprise various extracellular materials, such as proteins, exopolysaccharides, lipids, and extracellular DNA. Biofilms not only enhance the resistance of bacteria to adverse external environment, but also increase their resistance to antibacterial agents. However, TCM can restrain bacterial biofilm formation and development, destroy their cell wall and membrane, affect protein and nucleic acid synthesis, promote oxidative stress, and inhibit virulent factor expression to ultimately suppress or kill bacteria. The changes in AKP and electrical conductivity of bacterial culture serve as an index to verify whether the bacterial cell wall was destroyed or whether its permeability increased. GA (1, 2, 4, and 8 mg/mL) destroyed the cell wall within 2 h and caused AKP leakage. Furthermore, the degree of V. alginolyticus cell wall destruction was positively correlated with GA concentrations; 2, 4, and 8 mg/mL GA could remarkably increase electrical conductivity of the supernatant fluid of V. 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 V. alginolyticus, with an inhibition ratio of 83.26% (P<0.05) and 77.80% (P<0.05), respectively. Meanwhile, 4 and 8 mg/mL GA notably eliminated mature biofilms, with an elimination ratio of 68.01% (P<0.05) and 67.54% (P<0.05), respectively; 4 and 8 mg/mL GA completely suppressed V. alginolyticus growth on LB swimming motility agar plates; and 1, 2, 4, and 8 mg/mL GA significantly inhibited V. alginolyticus aggregation capacity, with aggregation rates reduced 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 V. alginolyticus by growth restraint, cell wall destruction, cell membrane permeability increase, biofilm formation suppression, mature biofilm elimination, and moveability and aggregation inhibition. This study presents a foundation for exploring the action mechanism of GA in suppressing V. alginolyticus growth and provides a theoretical basis for GA in preventing and curing infectious diseases caused by V. alginolyticus in aquatic animals. |
