The Investigation Of Several Normal Pathogens In Viable But Nonculturable State

The viable but nonculturable (VBNC) state is a new concept in microbiology, and it draws much attention of microbiologists. The aim of this study was to investigate the viable but nonculturable state of Edwardsiella tarda CW7, Vibrio parahaemolyticus VIB799, FYZ8621.4, Vibrio alginolyticus VIB283 and Vibrio anguillarum CW1, CW4, including their morphological and virulence changes during they entered into, resuscitated from the VBNC state.We used acridine orange direct count (AODC) method, direct viable count (DVC) method and plating count (PC) method to investigate if these pathogens could enter into the VBNC state. The results showed that when these pathogens were cultured in sterilized seawater at 4℃, all of them could enter into the VBNC state. The time required to enter into the VBNC state were different. E. tarda CW7 needed 28 d, V. parahaemolyticus VIB799 needed 30 d, V. parahaemolyticus FYZ8621.4 needed 88 d, V. alginolyticus VIB283 needed 90 d, V. anguillarum CW1 needed 324 d and V. anguillarum CW4 needed 266 d to enter into this state respectively. During the experimental time, the curves of these pathogens had similar changes. Compared to the original inoculum, when the experimental strains entered into the VBNC state, all total direct counts in the studies had almost no changes. The active cells, on the other hand, declined to and remained fairly constant at the lower level. The number of the culturable bacteria was zero.The morphological changes of VBNC, normal and resuscitative pathogens were studied with epifluorescence microscope, scanning electron microscope and flow cytometry. The results showed that when the cells of these pathogens entered into the VBNC state, they all gradually changed in shape from rods, short rods or curved rods to coccoid and decreased in size. With the method of flow cytometry, it was found that the resuscitative V. parahaemolyticus VIB799 cells and V. alginolyticus VIB283 cells did not show any obvious differences with their normal cells respectively; the resuscitative E. tarda CW7 were much smaller than its normal cells.We also resuscitated E. tarda CW7, V. parahaemolyticus VIB799 and V. alginolyticus VIB283 by the method of temperature upshift with or without the presence of nutrition and chick embryos experiments. The results showed that E. tarda CW7 could be resuscitated by the method of temperature upshift with the presence of nutrition and chick embryos experiments, the resuscitative time were 8 d and 6 d respectively; V. parahaemolyticus VIB799 could be resuscitated by the method of temperature upshift with the presence of nutrition, the resuscitative time were 2 d; V. alginolyticus VIB283 could be resuscitated by the method of temperature upshift with or without the presence of nutrition, the resuscitative time were 8 d and 16 h respectively.Turbots were used to investigate the retention of virulence in the VBNC and the resuscitative E. tarda CW7 cells. The turbots inoculated with the VBNC E. tarda CW7 cells kept alive during the experimental time, and there was no microorganisms isolated from these experimental turbots. The turbots inoculated with the resuscitative E. tarda CW7 died on the 7th day. The results showed that the VBNC E. tarda CW7 cells might keep their pathogenic potential, when resuscitated from the VBNC state, these E. tarda CW7 cells retained their pathogenicity and caused disease.The biochemical characteristics of the normal and the resuscitated V. alginolyticus VIB283 cells by the method of temperature upshift with or without the presence of nutrition were identified using the API 20E test strip. The results showed that the resuscitated cells by temperature upshift with the presence of nutrition had the capacity of citrate utilization compared to the normal cells and the cells resuscitated by temperature upshift.