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Applied and Environmental Microbiology, January 2002, p. 11-19, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.11-19.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori

Hans-Olof Nilsson,1 Jens Blom,2 Waleed Abu Al-Soud,1 Åsa Ljungh,1 Leif P. Andersen,3 and Torkel Wadström1*

Department of Medical Microbiology, Dermatology and Infection, Lund University, SE-223 62 Lund, Sweden,1 Laboratory for Electron Microscopy, State Serum Institute,2 Department of Microbiology, Rigshospitalet, Copenhagen, Denmark3

Received 11 June 2001/ Accepted 27 September 2001

Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.

* Corresponding author. Mailing address: Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-223 62 Lund, Sweden. Phone: 46 46 173240. Fax: 46 46 152564. E-mail: Torkel.Wadstrom{at}mmb.lu.se.

Applied and Environmental Microbiology, January 2002, p. 11-19, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.11-19.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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