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Applied and Environmental Microbiology, April 2005, p. 1890-1898, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1890-1898.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cytotoxicity Potential and Genotypic Characterization of Escherichia coli Isolates from Environmental and Food Sources

Yadilka Maldonado,1 Jennifer C. Fiser,2 Cindy H. Nakatsu,2 and Arun K. Bhunia1*

Molecular Food Microbiology Laboratory, Department of Food Science,1 Department of Agronomy, Purdue University, West Lafayette, Indiana2

Received 10 August 2004/ Accepted 11 November 2004

The presence of Escherichia coli isolates in the environment is a potential source of contamination of food and water supplies. Moreover, these isolates may harbor virulence genes that can be a source of new forms of pathogenic strains. Here, using multiplex PCR, we examined the presence of virulence gene markers (stx1, stx2, eaeA, hlyA) in 1,698 environmental isolates of E. coli and 81 isolates from food and clinical sources. The PCR analysis showed that ~5% (79 of 1,698) of the total environmental isolates and 96% (79 of 81) of the food and clinical isolates were positive for at least one of the genes. Of the food and clinical isolates, 84% (68 of 81 isolates) were positive for all four genes. Of the subset of environmental isolates chosen for further analysis, 16% (13 of 79 isolates) were positive for stx2 and 84% (66 of 79 isolates) were positive for eaeA; 16 of the latter strains were also positive for hlyA. The pathogenic potentials of 174 isolates (81 isolates from food and clinical sources and 93 isolates from environmental sources) were tested by using a cytotoxicity assay based on lactate dehydrogenase release from Vero cells. In general, 97% (79 of 81) of the food and clinical isolates and 41% (39 of 93) of the environmental isolates exhibited positive cytotoxicity. High cytotoxicity values correlated to the presence of stx genes. The majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. Isolates were also tested for sorbitol utilization and were genotyped by ribotyping and by repetitive extragenic palindromic PCR (REP-PCR) as potential means of quickly identifying virulent strains from the environment, but none of these methods could be used to distinguish cytotoxic environmental isolates. Only 31% of the isolates were negative for sorbitol fermentation, and none of the isolates had common ribotypes or REP-PCR fingerprints. This study suggests that overall higher cytotoxicity values correlated with the production of stx genes, and the majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. This study demonstrated that there is widespread distribution of potentially virulent E. coli strains in the environment that may be a cause of concern for human health.

* Corresponding author. Mailing address: Department of Food Science, Purdue University, 745 Agricultural Mall Dr., West Lafayette, IN 47907-2009. Phone: (765) 494-5443. Fax: (765) 494-7953. E-mail: bhunia{at}purdue.edu.

Applied and Environmental Microbiology, April 2005, p. 1890-1898, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.1890-1898.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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