An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing.
OBJECTIVE
To induce pathogenic Vibrio bacteria into a changed physiological state, in response to cold temperatures in sea water, and assess their sensitivity to heating and freezing, as compared with normal cells.


RESULTS
Cells of exponential phase Vibrio vulnificus, V. cholerae and V. parahaemolyticus were washed and inoculated into flasks of sea water, which were stored at 20 and 4 degrees C. Cells stored at 20 degrees C could be recovered after 60 d on non-selective agar (heart infusion agar; HIA) and on the selective agar (thiosulphate citrate bile salts agar) which is used in most Vibrio detection methodology. At 4 degrees C cells became non-culturable on both agars over time. The non-culturable cells appeared to be metabolically active and maintained their membrane integrity, whilst undergoing a change in morphology from rod-shaped to coccoid cells. Resuscitation was possible, in some cases, by an upshift in temperature before plating and the addition of catalase to HIA plates was found to increase recovery. Studies were carried out to assess the sensitivity of the non-culturable cells to heating and freezing compared with the normal cells. Vibrio organisms, whether culturable or in the non-culturable form, were not inactivated by freezing to -20 degrees C. Heating studies showed that V. parahaemolyticus was very heat resistant at low temperatures. However, a pasteurization regime of 2 min at 70 degrees C was found to be effective against all three strains. Experiments showed that the non-culturable cells of all three strains were similar in their heat resistance or, in some cases, were more heat sensitive than cells in the normal form.


CONCLUSIONS
Cells in the changed physiological form would not be detected in fish or seafood products by the current Vibrio detection methods. Freezing had no effect in reducing cell numbers. Vibrio parahaemolyticus was very heat resistant in the low temperature pasteurization studies. The higher pasteurization regime of 70 degrees C for 2 min was effective against all three pathogens. Non-culturable cells had similar heat sensitivity or were more heat sensitive than cells in the normal state. SIGNIFICANCE OF IMPACT OF THE STUDY: The study has highlighted a need for the development of better Vibrio detection methods. The low temperature pasteurization of oysters, which has been recommended in the USA, would not be adequate against the strain of V. parahaemolyticus used in this study. Heating regimes which were found to control cells in the normal form will also be effective for the control of the cells with changed physiology.
Johnston
M D
MD
Unilever Research, Colworth Laboratory, Sharnbrook, Bedfordshire, UK. Moira.Johnston@Unilever.com
Brown
M H
MH
D000362:Agar; D001431:Bacteriological Techniques; D002374:Catalase / Q000494:pharmacology; D015169:Colony Count, Microbial; D005615:Freezing; D006358:Hot Temperature; D008854:Microscopy, Electron; D014734:Vibrio cholerae / Q000254:growth & development* / Q000502:physiology* / Q000648:ultrastructure; D014736:Vibrio parahaemolyticus / Q000254:growth & development* / Q000502:physiology* / Q000648:ultrastructure
D000362:Agar; D002374:Catalase