Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR

Abstract

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low-and highspeed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10 1 to 10 3 CFU/g using the RTi-qPCR assay, and amounts as small as 10 0 to 10 1 CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10 1 to 10 2 CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.

Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration

“…Some recent works are summarized in Table 1. For example, Wolffs et al used a two-step filtration protocol followed by a real-time PCR assay, allowing quantitation of 75 CFU/g of Salmonella and identification of as low as 0.22 CFU/g in chicken rinse within 3 h (7); Fukushima et al combined filtration, centrifugation, and buoyant density gradient centrifugation, concentrating cells by 250-fold, and enabling detection of 10 1 to 10 3 CFU/g of Salmonella assay in naturally contaminated chicken using real-time qPCR (RTi-qPCR) (8). Here, when processing higher-volume samples (250 ml) using an automated instrument, significant numbers of microorganisms are recovered from chicken natural microbiota (10 3 to 10 6 CFU/ml) with a mean recovery of 76.2% of viable cells.…”

“…Microfiltration represents a conceptually simple way to reduce large samples to a small volume and effectively increase cell concentration without lengthy culturing and enrichment steps. There have been various recent reports on this approach as summarized in Table 1 (7)(8)(9)(10)(11). However, there were still several challenges to be overcome in order to develop a rapid filtration approach for the microorganism concentration and recovery from foods.…”

Enzymatic Digestion for Improved Bacteria Separation from Leafy Green Vegetables

“…The major challenges include the impact of components in the complex food matrix and the potential interference with the detection system. Bacterial separation and concentration is often the bottleneck of an effective detection because it depends on the food matrix and analytes (23).…”

Quantification of Periodontopathic Bacteria in Saliva Using the Invader Assay

“…However, the use of real-time PCR in clinical practice is impractical for the quantification of periodontopathic bacteria in spite of its high sensitivity and specificity (11), because real-time PCR involves gene amplification and requires expensive equipments such as thermal cyclers. Furthermore, because the oral cavity's complex aerobic and anaerobic flora contain more than 300 different bacterial species (12), bacterial counts may be underestimated due to false positive results (13,14). When bacteria that have sequences similar to those of the periodontopathic bacteria are contaminated, they could be amplified and detected even at low concentrations (10 2 -10 4 /tube).…”