Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells

Campylobacter jejuni is a pathogenic, microaerophailic, gram-negative, meso philic bacterium. Three strains isolated from humans with enteric campyloba cteriosis were able to survive at high population levels (10(7) cells ml(-1 )) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC fo rms of the three C,jejuni strains were enumerated and characterized by usin g 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole stai ning. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in s tationary-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 notabl e increase in cell volume was observed with the VBNC state; the average cel l volumes were 1.73 yl mg of protein(-1) for the culturable form and 10.96 mu l mg of protein-l after 30 days of incubation in microcosm water. Both t he internal potassium content. and the membrane potential were significantl y lower in the VBNC state than in the culturable state. Culturable cells we re 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 progress ively with time of incubation in microcosm water. Measurements of the cellu lar adenylate nucleotide concentrations revealed that the, cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microco sm water, and AMP was the only nucleotide detected in the three strains aft er 30 days of incubation in microcosm,vater.