The Investigation Of Vibrio Harveyi In Viable But Nonculturable State

The viable but nonculturable state (VBNC state) is a new concept in microbiology, bacteria in the VBNC state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies. Cells in the VBNC state typically demonstrate very low levels of metabolic activity, but on resuscitation are again culturable. The aim of this study was to investigate the VBNC state of Vibrio harveyi SF1, VIB295 and CW2, including their morphological and virulence during they entered into, resuscitated from the VBNC state.Vibrio harveyi SF1, VIB295 and CW2 were induced into the VBNC state by incubated in sterilized aged seawater (ASW) at 4℃. The direct viable counts declined from 106 to 104CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC). The VBNC cells could be resuscitated by two methods: addition of Tween 20 and compound vitamin B supplemented with yeast extract respectively. Temperature upshift with and without the presence of nutrition did not result in recovery from nonculturability. The morphological changes of VBNC, normal and resuscitative bacterium cells were studied with scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative and the normal cells showed no morphological differences. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately and the LD50 of the resuscitative cells was close to the normal cells, whereas no fish died after injected with the VBNC cells. Then we investigated the maintenance of potential virulence of Vibrio harveyi in VBNC state by reverse transcription-polymerase chain reaction (RT-PCR), no expression of the hemolysin gene was observed. These first data indicated that the pathogenic strains would not maintain the expression of the hemolysin gene in VBNC state.We also compared the normal bacterial cells with the resuscitative cells in biochemical characterization and antibiotic resistance, there were no significant differences between them.