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Applied and Environmental Microbiology, March 1999, p. 1110-1116, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Physiological Characterization of Viable-but-Nonculturable Campylobacter jejuni Cells

J. L. Tholozan,1,* J. M. Cappelier,2 J. P. Tissier,1 G. Delattre,1 and M. Federighi2

Laboratoire de Génie des Procédés et de Technologie Alimentaire, Institut National de la Recherche Agronomique, F-59651 Villeneuve d'Ascq Cedex,1 and Unité Associée INRA d'Hygiène Alimentaire, Ecole Nationale Vétérinaire de Nantes, F-44307 Nantes Cedex 03,2 France

Received 6 April 1998/Accepted 5 November 1998

Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (107 cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 µl mg of protein-1 for the culturable form and 10.96 µl mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.

* Corresponding author. Mailing address: Laboratoire de Génie des Procédés et de Technologie Alimentaire, Institut National de la Recherche Agronomique, 369 rue J. Guesde, BP 39, F-59651 Villeneuve d'Ascq Cedex, France. Phone: 33-(0)3-20-43-54-24. Fax: 33-(0)3-20-43-54-65. E-mail: tholozan{at}lille.inra.fr.

Applied and Environmental Microbiology, March 1999, p. 1110-1116, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.