A Basic Study for the Development of Effective Preventive Measure against Vibrio vulnificus Septicemia: Bactericidal Mechanism of Osmotic Shock

Rhee, Joon Haeng; Lee, Shee Eun; Chung, Sun Sik; Shin, Sung Heui; Shin, Boo Ahn

Abstract

The establishment of effective preventive measure against V. vulnificus septicemia is urgently required. It was reported that V. vulnificus osmotically shocked by distilled water lost viability rapidly but regain viability after appropriate resuscitation (RS) procedure. But V. vulnificus was reported to be completely killed when osmotically shocked in the presence of ethylenediaminetetraacetic acid (EDTA). This study was carried out to uncover the bactericidal mechanism of osmotic shock and the mechanism of potentiation of osmotic shock by EDTA. When about 2.0 x 10(7) CFU/ml of V. vulnificus were inoculated in distilled water, the number of viable cells abruptly decreased to 2.5 x 10(3) CFU/ml in 1 min. and slowly thereafter to 1.0 x 10(1) CFU/ml in 5 min. After RS, there was a increase in the number of surviving bacteria by 10(3) to 10(4) fold. When the bacteria were inoculated in 1 mM EDTA solution, osmotic concentration of which is about 30 mEq./1, no colony could be observed even in 1 minute. The turbidity decreased abruptly as soon as the bacteria were inoculated in distilled water or in the 1 mM EDTA solution, but rather slowly thereafter. When V. vulnificus whose cellular constituents were labeled with 3H-L-amino acid mixture was inoculated in distilled water or in the 1 mM EDTA solution, about 35% of the whole cell radioactivity was released in the 1 mM EDTA solution in 30 sec while about 6% of the whole cell radioactivity was released to the supernatant in distilled water in 5 minutes. The cell surface hydrophilicity decreased significantly by osmotic shock. The decrease was more significant when the bacteria were inoculated in 1 mM EDTA solution than in distilled water. Bacterial cell volume analysis with a flow cytometer revealed that the osmotic shock balloons V. vulnificus. The increase in the cell volume was more prominent in 1 mM EDTA solution. When the cytoplasmic RNA content in the osmotically shocked bacteria was measured by a flow cytometer, the frequency of the cells with decreased RNA content increased after osmotic shock, and the degree of increase was more prominent in 1 mM EDTA solution. Number of non-staining cells also increased after osmotic shock, and the degree of increase was more prominent in the 1 mM EDTA solution. To see whether the susceptibility to osmotic shock is unique to V. vulnificus, bactericidal kinetic curves of other Vibrio species were observed after inoculating in distilled water. V. cholerae and V. mimicus were more resistant to the osmotic shock than V. vulnificus. V. parahaemolyticus, V. furnissii, V. fluvialis, V. damsela, and V. harveyi showed similar susceptibility to osmotic shock as V. vulnificus. V. alginolyticus and V. hollisae were more susceptible than V. vulnificus. The concentration of NaCl in culture media influenced the susceptibility of V. vulnificus to osmotic shock. V. vulnificus grown in 0.5% NaCl was more resistant to the osmotic shock than that grown in 2.5% NaCl. Taken together, it was concluded that osmotic shock causes leakage of the cytoplasmic contents(ribosomes etc.). And EDTA was supposed to quantitatively potentiate the bactericidal effect of the osmotic shock. Susceptibility to osmotic shock was influenced by the osmolarity of culture media and appeared to be a phenotypic property of V. vulnificus.