Vaccine cadidate strains were selected through tests of minimal media, antibiotic susceptibility patterns, plasmid profiles and mouse virulence from 120 Salmonella typhi strains, which were collected from university hospital in Korea.
For the production of deleted gene mutants, first of all, we have selected 37 strains growth on minimal media and have selected KNIH 352 among the 37 strains by antibiotic susceptibility patterns and virulence tests. In the antibiotic
susceptibility
tests, isolated strains have appeared lower antibiotics resistance frequency than other enteric bacteria. In plasmid pattern, all of isolated strains except KNIH 352 strain have showed one band, approximately 24 Kb plasmid. But, only KNIH 352
strain has
showed various plasmid patterns and the high toxicity in virulence test.
@EN The establishement 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 te mechanism of potentiation of osmotic shock by EDTA.
When about 2.0×10E7 CFU/ml of V. vulnificus were inoculated in distilled water, the number of viable cells abruptly decreased to 2.5×10E3 CFU/ml in 1 min. and slowly thereafter to 1.0×10E1 CFU/ml in 5 min. After RS, there was a increase in the
number
of surviving bacteria by 10E3 to 10E4 fold. When the bacteria ware 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 thw 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
ballons 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 was more suceptible than V. vulnificus. The
concentration
of NaCI in culture media influenced the suceptibility of V. vulnificus to osmotic shock. V. vulnificus growth 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 ofthe 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.
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