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Applied and Environmental Microbiology, April 2002, p. 1988-1993, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1988-1993.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Inducible Gene Expression by Nonculturable Bacteria in Milk after Pasteurization

Thusitha S. Gunasekera,^1* Anders Sørensen,^2, Paul V. Attfield,¹ Søren J. Sørensen,² and Duncan A. Veal¹

Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia,¹ Department of General Microbiology, Institute of Molecular Biology, University of Copenhagen, DK 1307K Copenhagen, Denmark²

Received 3 August 2001/ Accepted 21 January 2002

The viability of bacteria in milk after heat treatments was assessed by using three different viability indicators: (i) CFU on plate count agar, (ii) de novo expression of a gfp reporter gene, and (iii) membrane integrity based on propidium iodide exclusion. In commercially available pasteurized milk, direct viable counts, based on dye exclusion, were significantly (P < 0.05) higher than viable cell counts determined from CFU, suggesting that a significant subpopulation of cells in pasteurized milk are viable but nonculturable. Heating milk at 63.5°C for 30 min resulted in a >4-log-unit reduction in the number of CFU of Escherichia coli and Pseudomonas putida that were marked with lac-inducible gfp. However, the reduction in the number of gfp-expressing cells of both organisms under the same conditions was <2.5 log units. These results demonstrate that a substantial portion of cells rendered incapable of forming colonies by heat treatment are metabolically active and are able to transcribe and translate genes de novo.

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* Corresponding author. Mailing address: Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. Phone: 61-2-98508157. Fax: 61-2-98508253. E-mail: tgunasek{at}rna.bio.mq.edu.au.

Present address: Statens Veterinære Serumlaboratorium, DK-1790, Copenhagen V, Denmark.

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Applied and Environmental Microbiology, April 2002, p. 1988-1993, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1988-1993.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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