Immobilization with metal hydroxides as a means to concentrate food-borne bacteria for detection by cultural and molecular methods

The application of nucleic acid amplification methods to the detection of f ood borne pathogens could be facilitated by concentrating the organisms fro m the food matrix before detection. This study evaluated the utility of met al hydroxide immobilization for the concentration of bacterial cells from d airy foods prior to detection by cultural and molecular methods. Using reco nstituted nonfat dry milk (NFDM) as a model, two food-borne pathogens (List eria monocytogenes and Salmonella enterica serovar Enteritidis) were concen trated from 25-ml samples by the sequential steps of clarification and high -speed centrifugation (designated primary concentration) and immobilization ,vith zirconium hydroxide and low-speed centrifugation (designated secondar y concentration). Sample volume reduction after immobilization with zirconi um hydroxide was 50-fold, with total bacterial recoveries ranging from 78 t o 96% of input for serovar Enteritidis and 65 to 96% of input for L. monocy togenes. Immobilized bacteria remained viable and could be enumerated by st andard cultural procedures. When followed by RNA extraction and subsequent detection by reverse transcription (RT)-PCR, detection limits of 10(1) to 1 0(2) CFU/25 mi of reconstituted NFDM were achieved for both organisms. The bacterial-immobilization step was relatively nonspecific, resulting in reco very 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 b acterial recoveries after immobilization ranged from 64 to > 100%, with sub sequent RT-PCR detection limits of greater than or equal to 10(2) CFU/ml fo r whole milk and greater than or equal to 10(1) CFU for ice cream for both serovar Enteritidis and L. monocytogenes. The bacterial-immobilization meth od is easy, rapid, and inexpensive and may have applications for the concen tration of a wide variety of food borne bacteria prior to detection by both conventional and alternative methods.