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.
Alcoholics ; Bacillus ; Bacteria ; Bromcresol Purple ; Bromthymol Blue ; Cellobiose ; Chronic Disease ; Coloring Agents ; Diagnosis ; Gentian Violet ; Humans ; Hydrogen-Ion Concentration ; Liver Cirrhosis ; Mortality ; Peptones ; Phenolsulfonphthalein ; Sepsis* ; Survival Rate ; Thymol ; Vibrio vulnificus* ; Vibrio*

BACKGROUND: Rapid detection of carbapenemase-producing gram-negative bacilli (GNB) is required for optimal treatment of infected patients. We developed and assessed a new disk carbapenemase test (DCT). METHODS: Paper disks containing 0.3 mg of imipenem and bromothymol blue indicator were developed, and the performance of the DCT were evaluated by using 742 strains of GNB with or without carbapenemases. RESULTS: The paper disks were simple to prepare, and the dried disks were stable at -20℃ and at 4℃. The DCT detected 212 of 215 strains (98.6% sensitivity with 95% confidence interval [CI] 96.0-99.5%) of GNB with known class A (KPC and Sme) and class B (NDM, IMP, VIM, and SIM) carbapenemases within 60 min, but failed to detect GES-5 carbapenemase. The DCT also detected all two Escherichia coli isolates with OXA-48, but failed to detect GNB with OXA-232, and other OXA carbapenemases. The DCT showed 100% specificity (95% CI, 99.2-100%) in the test of 448 imipenem-nonsusceptible, but carbapenemase genes not tested, clinical isolates of GNB. CONCLUSIONS: The DCT is simple and can be easily performed, even in small laboratories, for the rapid detection of GNB with KPC, NDM and the majority of IMP, VIM, and SIM carbapenemases.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*metabolism ; Bromthymol Blue/chemistry ; Drug Resistance, Bacterial ; Gram-Negative Bacteria/drug effects/*enzymology ; Imipenem/pharmacology ; Microbial Sensitivity Tests/*methods ; Paper ; beta-Lactamases/*metabolism