New Selective Medium for Rapid Identification of Vibrio vulnificus from Patients with V. vulnificus Sepsis

You, Dong O; Jeong, Sang Won; Park, Seok Don

Abstract

BACKGROUND
Vibrio(V.) vulnificus is a halophilic, gram-negative bacillus that causes a fatal sepsis in patients with underlying chronic disease such as liver cirrhosis and alcoholic abuse. Because V. vulnificus infection has a fulminant course and high mortality rate, early recognition and rapid diagnosis with prompt therapy are necessary to improve survival rate. OBJECTIVE: The purpose of this study was to develop a new selective medium for rapid identification of V. vulnificus through color change of medium according to pH from patients suspected of having V. vulnificus sepsis. METHODS: Rapid isolation and identification of V. vulnificus can be possible by modifying the component of PNC(5% peptone, 1% NaCl, and 0.08% cellobiose [pH 8.0]) broth medium. From this PNC broth, a basal broth(5% peptone+1% NaCl+cellobiose) was prepared and used to evaluate additional medium supplements(cellobiose concentration [0.08, 0.2, 0.1%], pH [6.8, 7.5, 8.0] and pH indicator dye [bromthymol blue, thymol blue, phenol red, bromcresol purple, crystal violet, cresol red, and neutral red]). To examine the rapid identification and selectivity of this basal medium according to various conditions, V. vulnificus was tested by using saline and normal human blood containing these bacteria(1, 000 bacteria/ml), respectively at 37degrees C. A positive reaction(V. vulnificus growth) appeared as color change. The selectivity and identification capacity of this new broth was tested by using other 6 Vibrio species and 14 strains of other bacteria. RESULTS: Color change appeared only in the medium including bromthymol blue and thymol blue as a pH indicator dye. It was called the basal medium containing blue dyes as PNCB(peptone, NaCl, cellobiose and blue dye) medium. It took an average time of 4.8hr for becoming aware of yellow color change in PNCB broth after cultivating with saline mixed with V. vulnificus and 6hr in PNCB broth after cultivating with blood mixed with V. vulnificus. One Vibrio species and another 3 bacteria produced color change. So we confirmed that the final composition and pH of PNCB broth medium was 5% peptone, 1% NaCl, 0.2% cellobiose, 0.0004% bromthymol blue and 0.0004% thymol blue [pH 7.5] CONCLUSIONS: PNCB broth could be used as a selective and differential medium for rapid isolation and identification of V. vulnificus in patients with V. vulnificus sepsis.