Effect Of Sodium Bicarbonate On The Growth And Virulence Of Aeromonas Hydrophila

Aeromonas hydrophila is an important pathogen of fresh aquatic fish, which frequently causes the outbreaks of fish diseases such as bacterial septicemia during the relatively high temperature season or in the highly-intensive aquaculture water, leading to the great economic losses for farmer. At the present, antibiotics are the most common way used to prevent and control the diseases caused by A. hydrophila. However, the pathogenic bacteria have acquired the resistance to these antibiotic drugs. Moreover, the application of the abusive antibiotics also brings substantial damages to environment and human beings. It has been indicated by the previous studies that the organic carbon source could affect the growth and virulence of A. hydrophila. However, the effect of inorganic carbon sources on A. hydrophila has not been reported yet. Sodium bicarbonate has been reported to possess the antimicrobial activity on bacteria such as Escherichia coli, Pseudomonas aeruginosa etc. However, whether sodium bicarbonate has the same inhibitory effect on A. hydrophila is still unknown. Therefore, this study focused on investigation of the inhibitory effects and inhibitory mechanisms of sodium bicarbonate on A. hydrophila, as well as the effect of sodium bicarbonate on pathogenicity and the expression of main virulence genes in A. hydrophila, aiming to provide some theoretical basis and data support for controlling the diseases caused by A. hydrophila by using sodium bicarbonate reasonably. The main results obtained from this study as the follows:1. Effect of sodium bicarbonate on the growth of A. hydrophilaIn order to investigate the inhibitory effects of sodium bicarbonate on the growth of A. hydrophila and the underlying mechanism, the effects of different concentrations（0.2, 0.4, and 0.8M） of NaHCO₃ as well as sodium chloride（NaCl） and potassium bicarbonate（KHCO₃） at equimolar concentrations on the growth of A. hydrophila were examined. Results showed that the growth of A. hydrophila was more and more slowly with the increasing of the concentration of the NaHCO₃. NaHCO₃ possessed bactericidal activity when its concentration was increased to certain levels. With increasing NaHCO₃ concentration, the A. hydrophila mortality was getting more higher, and the faster rate of the organism was killed. No A. hydrophila survived in the medium containing NaHCO₃ at 0.4 and 0.8M when being exposed for twelve hours. Only a few bacteria exposed in NaHCO₃ at 0.2M survived（lethality was lower than 0.01%） as well. The NaHCO₃ at 0.2 and 0.4M could effectively inhibit the growth of A. hydrophila, but the growth of bacteria was not influenced by NaCl at equimolar concentrations. Bacterial growth was also inhibited distinctly by KHCO₃ at in equimolar concentrations. NaHCO₃ and KHCO₃ showed similar inhibitory activity, suggesting that the antimicrobial activity of NaHCO₃ was due to bicarbonate ion instead of the osmotic stress. These results provide the theoretical basis for the effective control of the diseases caused by A. hydrophila.2. Effect of sodium bicarbonate at different concentrations on pathogenicity of A. hydrophilaIn order to examine the effect of sodium bicarbonate on pathogenicity of A. hydrophila, the bacterial cells grown in the medium containing sodium bicarbonate at different concentrations were used to infect zebra fish. At the same time, we also compared the effects of infections of zebra fish with A.hydrophila cultured in medium containing sodium bicarbonate, glucose and sucrose respectively, and measured the virulence of A.hydrophila by median lethal dose for zebra fish. The results indicated that the higher of the median lethal dose means the lower effectiveness of the bacterial virulence. The result showed that compared with the A. hydrophila without sodium bicarbonate, median lethal dose of A. hydrophila with sodium bicarbonate was much higher, indicating that sodium bicarbonate can reduce the virulence of A.hydrophila when the concentrations of sodium bicarbonate were in the range of 0.024 to 0.096 M. With increasing sodium bicarbonate concentration, the virulence of A.hydrophila was weakened gradually. When the concentrations were at the range of 0.024 to 0.096 M, the virulence of A. hydrophila grown with inorganic carbon source sodium bicarbonate was weaker than that of A.hydrophila grown with organic carbon source such as glucose and sucrose. In addition, median lethal dose of A. hydrophila grown with glucose was higher than that grown with sucrose, indicating that that glucose is more effective in reducing the virulence of A. hydrophila than sucrose is.3. Effect of sodium bicarbonate on the expression of main virulence genes in A. hydrophila.To explore why sodium bicarbonate could reduce the virulence of A. hydrophila, the relative expression levels of three major virulence genes, including hemolysin gene（AHH-1）, aerolysin gene（AerA）, and elastinase gene（ahyB） in A. hydrophila grown with sodium bicarbonate at 0.024, 0.048, 0.072 M, 0.096 M and 0.12 M were analyze by real-time quantitative PCR technique and compared with those of A. hydrophila without sodium bicarbonate as control. The result revealed that the expression levels of AHH-1,AerA and ahyB in A. hydrophila grown with sodium bicarbonate were significantly lower than those of A. hydrophila without sodium bicarbonate. With increasing sodium bicarbonate level, the expression levels of these genes showed the decreasing trends. What’s more, hemolysin and aerolysin both have hemolytic activity, elastinase can decompose elastic protein, therefore, the results lead us to speculate that sodium bicarbonate can reduce the toxicity of A. hydrophila by reducing its hemolytic activity and capacity of decomposing elastic protein.In conclusion, this study has revealed that sodium bicarbonate has excellent inhibitory effect on the growth and virulence. Further study found that A. hydrophila maybe reduce bacterial virulence by lowering the mRNA expression level of genes which related to hemolytic substance and elastinase. The results could be of significance in providing the guidance for preventing and treating disease caused by A. hydrophila.