Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR.
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 high-speed 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.
Fukushima
Hiroshi
H
Shimane Prefectural Institute of Public Health and Environmental Science, 582 Nishihamasada, Matsue, Shimane 690-0122, Japan. fukushima-hiroshi@pref.shimane.lg.jp
Katsube
Kazunori
K
Hata
Yukiko
Y
Kishi
Ryoko
R
Fujiwara
Satomi
S
D000818:Animals; D016123:Campylobacter jejuni / Q000302:isolation & purification; D002417:Cattle; D002499:Centrifugation, Density Gradient / Q000379:methods; D002645:Chickens / Q000382:microbiology; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D003611:Dairy Products / Q000382:microbiology; D005374:Filtration; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000382:microbiology*; D006090:Gram-Negative Bacteria / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000302:isolation & purification; D006801:Humans; D008460:Meat / Q000382:microbiology; D010473:Perciformes / Q000382:microbiology; D016133:Polymerase Chain Reaction; D019779:Salmonella enterica / Q000145:classification / Q000302:isolation & purification; D013997:Time Factors
D004269:DNA, Bacterial