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Abstract
Vibrio vulnificus causes primary septicemia as a result of the consumption of contaminated seafood. The intestinal epithelial layer is the first host barrier encountered by V. vulnificus upon oral intake; however, epithelial translocation (invasion) of V.vulnificus has not been extensively studied. In this study, we investigated in vivo translocation of V. vulnificus using clinical (CMCP6) and environmental isolates (96-11-17M). And we analyzed physiological changes of intestinal epithelium concurrent with bacterial translocation by using polarized HCA-7 transwell culture system. The efficiency of epithelial translocation of 97-11-17M strains was significantly lower than that of pathogenic clinical isolate CMCP6 in a murine ligated ileal loop model. In an oral infection model, the survival rate was reciprocally related with efficacy of in vivo epithelial translocation. These results indicate that efficient translocation of V. vulnificus through intestinal epithelium is highly correlated with successful oral infection. We determined translocation of the bacteria from upper to lower chamber, changes of transepithelial electric resistance (TER) and cytotoxicity of the polarized HCA-7 cells to understand general features of V. vulnificus invasion. Bacterial translocation was accompanied by big decrease of TER (about 90%) and about 50% cytotoxicity of the epithelial cells. Taken together, these results indicate that V. vulnificus actively translocates the epithelium by destruction of epithelium and the efficiency of intestinal invasion by V. vulnificus is critical for successful oral infection. From this result, it is suggested that integrity of intestinal barrier is an important factor for susceptibility to oral infection of V. vulnificus.
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Figure 1.
In vivo invasion by V. vulnificus strains in a ligated ileal loop infection model. V. vulnificus (4.0 × 106 CFU/400 μl) was inoculated into ligated ileal loops of mice under anesthesia. After 3 and 6 hours, blood samples were collected from the ocular plexus from the infected mice. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates. Bacterial invasion from the ligated ileal loop into bloodstream was significantly decreased in 96-11-17M infected mice. The data represent the average values and SEM from three or four mice. *p < 0.05; **p < 0.01 compared with V. vulnificus CMCP6.
Figure 2.
Survival curve for mice infected intragastrically with V. vulnificus strains at a dose of 1.0 × 109 cfu/mouse (A) or 1.0 × 108 cfu/mouse (B). The survival rate was significantly increased in the 96-11-17M-infected mice. The survival curve was constructed according to the Kaplan-Meier method, and statistical significance was determined by the log-rank test. *p < 0.05; **p < 0.01 compared with V. vulnificus CMCP6.
Figure 3.
In vitro invasion by V. vulnificus CMCP6 and 96-11-17M in polarized HCA-7 cell monolayers. HCA-7 cells grown on transwell filters were apically infected with V. vulnificus CMCP6 or 96-11-17M. The bacterial epithelial translocation (A) was determined by measuring the number of bacterial cells that translocated from the upper chamber to the lower chamber of the transwell. The change in transepithelial electrical resistance (TER) (B) and LDH release in the upper (apical) and lower chamber (basolateral) (C) were determined. Viable bacterial cells were counted by plating on 2.5% NaCl HI agar plates.
Table 1.
Bacterial strains used in this study
Table 2.
Generation time of V. vulnificus in dialysis tubing implantation model
| Strains | Generation time (min)a | |
|---|---|---|
| Mean ± SEM | P value | |
| CMCP6 | 22.41 ± 0.62 | p > 0.05 |
| 96-11-17M | 23.29 ± 1.44 | p > 0.05 |
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