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

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).Nontyphoidal salmonellosis is an invasive intestinal disease contracted predominately by ingestion of food contaminated with serotypes of the gram-negative bacterial species Salmonella enterica. Gastroenteritis caused by Salmonella spp. represents a large portion of the natural food-borne illnesses that occur worldwide each year. Bacterial virulence is established in part by the bacterium''s ability to invade and survive within host cells (20). S. enterica is capable of survival within a wide array of host cells, including epithelial cells, dendritic cells, and macrophages in both animal and cell culture models (16, 17, 18, 19). However, survival in macrophages is required for initiation of systemic infection (24). Two chromosomal pathogenicity islands, SPI-1 and SPI-2, which are present in all Salmonella enterica serotypes, are essential for the invasion of epithelial cells and intracellular replication in macrophages, respectively (13, 14).There are currently over 2,500 distinct serotypes of S. enterica (http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf). Of these, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are most commonly associated with food-borne illness in humans (4). Raw and undercooked shell eggs have been implicated as vehicles for the transmission of both of these serotypes of Salmonella enterica (9, 38). However, Salmonella serotype Enteritidis infection has been more frequently linked to shell egg consumption, whereas Salmonella serotype Typhimurium infection is more often associated with the consumption of contaminated chicken meat (8). Of the 309 documented outbreaks of Salmonella serotype Enteritidis in the United States from 1990 to 2001, 241 were attributed to the consumption of raw or undercooked eggs (6). Salmonella serotype Enteritidis phage types 4, 8, and 13 have been implicated in the majority of salmonellosis cases from the consumption of egg products (5). In addition, Salmonella serotype Enteritidis is able to colonize laying hen reproductive organs and developing eggs and has been shown to persist in eggs after they have been laid (23).A variety of methods have been developed in order to expedite the detection of salmonellae in eggs, including GeneQuence DNA hybridization, PCR analysis, and enzyme-linked immunosorbent assay (3, 27, 37). However, these methods require lengthy enrichment steps prior to the application of the respective methods. Real-time PCR (RT-PCR) is a promising new method currently used for detection of a wide variety of bacterial pathogens in food matrices (12, 15, 22, 34, 40). However, this technique can be ineffective for the detection of Salmonella serotype Enteritidis in foods such as eggs due to the presence of PCR-inhibitory components (41).In this study, we developed a novel detection system to allow for the specific identification of viable Salmonella serotype Enteritidis in raw shell eggs. The method developed is based on the ability of Salmonella to invade and replicate within macrophages as part of its life cycle within a host. In theory, cultured eukaryotic cell lines exposed to Salmonella-contaminated foods will allow the penetration and replication of Salmonella while confining food particles and noninvasive bacteria to the extracellular environment, allowing the isolation and enrichment of intracellular Salmonella for subsequent detection by commercially available techniques, such as RT-PCR. In practice, a suitable mammalian cell monolayer is exposed to a particular food matrix suspected of harboring salmonellae. The exposure is promoted for sufficient time to allow cell contact and engulfment of salmonellae. The mammalian cell monolayer is then washed sufficiently to remove the food matrix and extracellular microorganisms. The infected cell monolayer is reconstituted with fresh medium and further incubated to allow for intracellular multiplication of Salmonella (postinfection). After the infection is terminated, the culture medium is discarded, the infected cells are disrupted, and the DNA present in the resultant lysates is analyzed by RT-PCR using primers and probes specific for unique Salmonella DNA sequences. We utilized this method for the presumptive and confirmatory identification of Salmonella serotype Enteritidis in raw shell eggs.