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Applied and Environmental Microbiology, June 2009, p. 3593-3597, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02750-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Recovery and Detection of Escherichia coli O157:H7 in Surface Water, Using Ultrafiltration and Real-Time PCR{triangledown}

Bonnie Mull1,2* and Vincent R. Hill1

Centers for Disease Control and Prevention, Atlanta, Georgia,1 Association of Public Health Laboratories/Centers for Disease Control and Prevention, Emerging Infectious Diseases Laboratory Fellowship Program, Atlanta, Georgia2

Received 2 December 2008/ Accepted 31 March 2009

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the stx1, stx2, and rfbE gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect rfbE, stx1, and stx2 genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.

* Corresponding author. Mailing address: Centers for Disease Control and Prevention, National Center for Zoonotic, Vector-Borne, and Enteric Diseases, Division of Parasitic Diseases, 4770 Buford Highway, Mail Stop F-36, Atlanta, GA 30341-3724. Phone: (770) 488-4194. Fax: (770) 488-4253. E-mail: BMull{at}cdc.gov

{triangledown} Published ahead of print on 10 April 2009.

Applied and Environmental Microbiology, June 2009, p. 3593-3597, Vol. 75, No. 11
0099-2240/09/$08.00+0     doi:10.1128/AEM.02750-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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