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

Abstract

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).

Reverse‐Transcriptase Loop‐Mediated Isothermal Amplification as a Rapid Screening/Monitoring Tool for Salmonella Enterica Detection in Liquid Whole Eggs

“…Salmonella in carcass rinses and chicken have been successfully detected by real‐time PCR assay after pre‐enrichment for 20 h (Malorny and others 2004). A10‐h intracellular multiplication enrichment, followed by PCR could detect 10 CFU/mL of S. Enteritidis in shell eggs (Day and others 2009). Immunomagnetic separation‐PCR assay was shown to detect 47 CFU/25 g of Salmonella in inoculated ice‐cream, cheese, milk powder, egg yolk powder, and pasteurized egg yolk after at least 16‐h enrichment (Rijpens and others 1999) with 1 to 5 CFU/25 g detection in egg melange, egg melange with sugar, and dried eggs after similar enrichment (Jeníková and others 2000).…”

Enumeration of Salmonellae in Table Eggs, Pasteurized Egg Products, and Egg-Containing Dishes by Using Quantitative Real-Time PCR

“…Seo et al (18) used qPCR for quantification in pooled egg samples; however, the detection limit of 10 3 CFU/ml must be considered too high in order to be able to quantify naturally occurring levels of salmonellae in eggs (19). To lower the detection limit, other authors employed pre-qPCR processing methods such as preenrichment (27,28) or matrix lysis (29), but these methods were reported to be qualitative, with only a potential for quantitative use. In the current study, we demonstrate that quantification of salmonellae in table eggs, pasteurized egg products, and commercially available eggcontaining dishes is not only possible by using qPCR after a short nonselective 8-h preenrichment but also highly reproducible.…”

Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment

“…A novel method has recently been described for isolation and enrichment of bacterial pathogens from food matrices utilizing macrophage cell monolayers (Day and Whiting ; Day et al . ). This detection system is based on the ability of certain foodborne bacterial pathogens, namely facultative intracellular pathogens, to invade and replicate within macrophages as part of their life cycle within a host.…”