Development of a macrophage cell culture method to isolate and enrich Francisella tularensis from food matrices for subsequent detection by real-time PCR

Abstract

Francisella tularensis is a gram-negative bacterium that can cause gastrointestinal or oropharyngeal tularemia in humans from ingestion of contaminated food or water. Despite the potential for accidental or intentional contamination of foods with F. tularensis, there are no techniques currently available to detect this organism in specific food matrices. In this study, a macrophage cell culture system is combined with real-time PCR to identify F. tularensis in food matrices. The method utilizes a mouse macrophage cell line (RAW 264.7) as host for the isolation and intracellular replication of F. tularensis. Exposure of macrophages to F. tularensis-contaminated food matrices results in uptake and intracellular replication of the bacteria, which can be subsequently detected by real-time PCR analysis of the DNA released from infected macrophage cell lysates. Macrophage monolayers were exposed to infant formula, liquid egg whites, and lettuce contaminated with varying quantities of F. tularensis. As few as 10 CFU/ml (or CFU per gram) F. tularensis was detected in infant formula and lettuce after 5 h postinfection. As few as 10 CFU/ml F. tularensis was detected in liquid egg whites after 18 h postinfection. Intracellular F. tularensis could also be isolated on Mueller-Hinton medium from lysates of macrophages infected with the bacteria in infant formula, liquid egg whites, and lettuce for subsequent confirmatory identification. This method is the first to successfully identify F. tularensis from select food matrices.