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Applied and Environmental Microbiology, September 2005, p. 5022-5028, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5022-5028.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

R. Virto, P. Mañas, I. Álvarez, S. Condon, and J. Raso*

Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, 50.013 Zaragoza, Spain

Received 4 November 2004/ Accepted 29 March 2005

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.

* Corresponding author. Mailing address: Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50.013 Zaragoza, Spain. Phone: 34 976 76 15 81. Fax: 34 976 76 15 90. E-mail: jraso{at}unizar.es.

Applied and Environmental Microbiology, September 2005, p. 5022-5028, Vol. 71, No. 9
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.9.5022-5028.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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