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Applied and Environmental Microbiology, August 2009, p. 5321-5327, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.02422-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR{triangledown}

J. B. Day,* U. Basavanna, and S. K. Sharma

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland 20740

Received 22 October 2008/ Accepted 24 May 2009

Salmonella enterica serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify Salmonella serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify Salmonella serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of Salmonella serotype Enteritidis. Exposure of macrophages to Salmonella serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of Salmonella serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the Salmonella serotype Enteritidis genes sefA and orgC. Salmonella serotype Enteritidis could also be distinguished from other non-serogroup D Salmonella serotypes by using the sefA- and orgC-specific primer and probe sets. Confirmatory identification of Salmonella serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies Salmonella serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002).

* Corresponding author. Mailing address: FDA, Center for Food Safety and Applied Nutrition, HFS-712, 5100 Paint Branch Parkway, College Park, MD 20740. Phone: (301) 436-2013. Fax: (301) 436-2632. E-mail: james.day{at}fda.hhs.gov

{triangledown} Published ahead of print on 26 June 2009.

Applied and Environmental Microbiology, August 2009, p. 5321-5327, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.02422-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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