Conclusion

Since the pioneering studies from Colwell's laboratory,24,25 it has become evident that many gram-negative bacteria enter into a state of dormancy in response to one or more of a variety of environmental stresses, including both low and high temperatures. VBNC cells remain metabolically active, although they can not be cultured by standard methods. Cells undergo a variety of morphological, physiological, and biochemical changes as they enter this state, all of which may allow the cells to survive what could otherwise be lethal conditions. On removal of the inducing stress, these cells can resuscitate from the VBNC state, again becoming metabolically active.

Fig. 1.10. Fluorescence due to the green fluorescent protein in P. fluorescens, as detected by flow cytometry. Shown is the percent of control cell fluorescence of cells incubated at 5°C, 30°C, and 37.5°C for up to 19d. Cells incubated at 37.5°C were completely nonculturable by day 15 in this study. M. Lowder and J.D. Oliver (unpublished).

Fig. 1.10. Fluorescence due to the green fluorescent protein in P. fluorescens, as detected by flow cytometry. Shown is the percent of control cell fluorescence of cells incubated at 5°C, 30°C, and 37.5°C for up to 19d. Cells incubated at 37.5°C were completely nonculturable by day 15 in this study. M. Lowder and J.D. Oliver (unpublished).

Unfortunately, it appears that cells in the VBNC state may undergo changes in their chromosomal DNA which prevents their detection through PCR amplification. Further, it is possible that VBNC cells may participate in genetic exchange with bacteria making up the normal microflora of a release environment. Such an event could involve gain or loss of plasmids, an event having potentially dramatic consequences on the conditions inducing the VBNC state. Whether these dormant cells can be genetically transformed or transduced is not know at this time, but if so, such events could lead to significant genetic modifications in these nonculturable cells.

Thus, the VBNC state presents a number of potential concerns to the researcher and regulator alike, making detection of the introduced cells difficult, and presenting a situation wherein gene exchange could potentially occur, yet be undetectable. Whether or not new technologies such as the use of the gfp marker gene will at least provide a means for monitoring such cells is currently under investigation.

Acknowledgments

J.D. Oliver is a member of the MAREP Concerted Action sponsored by the European Commission Biotechnology Programme, DGXII.

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