Immobilization with Metal Hydroxides as a Means To Concentrate Food-Borne Bacteria for Detection by Cultural and Molecular Methods

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Abstract
The application of nucleic acid amplification methods to the detection of food-borne pathogens could be facilitated by concentrating the organisms from the food matrix before detection. This study evaluated the utility of metal hydroxide immobilization for the concentration of bacterial cells from dairy foods prior to detection by cultural and molecular methods. Using reconstituted nonfat dry milk (NFDM) as a model, two food-borne pathogens ( Listeria monocytogenes and Salmonella enterica serovar Enteritidis) were concentrated from 25-ml samples by the sequential steps of clarification and high-speed centrifugation (designated primary concentration) and immobilization with zirconium hydroxide and low-speed centrifugation (designated secondary concentration). Sample volume reduction after immobilization with zirconium hydroxide was 50-fold, with total bacterial recoveries ranging from 78 to 96% of input for serovar Enteritidis and 65 to 96% of input for L. monocytogenes . Immobilized bacteria remained viable and could be enumerated by standard cultural procedures. When followed by RNA extraction and subsequent detection by reverse transcription (RT)-PCR, detection limits of 10 1 to 10 2 CFU/25 ml of reconstituted NFDM were achieved for both organisms. The bacterial-immobilization step was relatively nonspecific, resulting in recovery of >50% of the input cells when evaluated on a panel of representative bacterial strains of significance to foods. The method could be adapted to more complex dairy products, such as whole milk and ice cream, for which bacterial recoveries after immobilization ranged from 64 to >100%, with subsequent RT-PCR detection limits of ≥10 2 CFU/ml for whole milk and ≥10 1 CFU for ice cream for both serovar Enteritidis and L. monocytogenes . The bacterial-immobilization method is easy, rapid, and inexpensive and may have applications for the concentration of a wide variety of food-borne bacteria prior to detection by both conventional and alternative methods.