Immunomagnetic Capture of Bacillus anthracis Spores from Food

Abstract

<jats:p>Food is a vulnerable target for potential bioterrorist attacks; therefore, a critical mitigation strategy is needed for the rapid concentration and detection of biothreat agents from food matrices. Magnetic beads offer a unique advantage in that they have a large surface area for efficient capture of bacteria. We have demonstrated the efficient capture and concentration of Bacillus anthracis (Sterne) spores using immunomagnetic beads for a potential food application. Magnetic beads from three different sources, with varying sizes and surface chemistries, were functionalized with monoclonal antibodies and polyclonal antibodies from commercial sources and used to capture and concentrate anthrax spores from spiked food matrices, including milk, apple juice, bagged salad, processed meat, and bottled water. The results indicated that the Pathatrix beads were more effective in the binding and capture of anthrax spores than the other two bead types investigated. Furthermore, it was observed that the use of polyclonal antibodies resulted in a more efficient recovery of anthrax spores than the use of monoclonal antibodies. Three different magnetic capture methods, inversion, the Pathatrix Auto system, and the new iCropTheBug system, were investigated. The iCropTheBug system yielded a much higher recovery of spores than the Pathatrix Auto system. Spore recoveries ranged from 80 to 100% for the iCropTheBug system when using pure spore preparations, whereas the Pathatrix Auto system had recoveries from 20 to 30%. Spore capture from food samples inoculated at a level of 1 CFU/ml resulted in 80 to 100% capture for milk, bottled water, and juice samples and 60 to 80% for processed meat and bagged salad when using the iCropTheBug system. This efficient capture of anthrax spores at very low concentrations without enrichment has the potential to enhance the sensitivity of downstream detection technologies and will be a useful method in a foodborne bioterrorism response.</jats:p>

Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

“…The Pathatrix and NRC magnetic beads were functionalized with anti- Y. pestis antibody polyclonal rabbit anti- Y. pestis (Tetracore, Rockville, MD, USA) or Y. pestis monoclonal Clone# M996145 (Fitzgerald Industries International, Acton, MA, USA) at a concentration of 1 mg/mL using the Pathatrix custom-coating kit with slight modifications [20]. The functionalized beads were adjusted to a final concentration of 20 mg/mL and stored at 4°C until use.…”

“…The two methods for capturing Y. pestis , Pathatrix Auto system (Life Technologies, Carlsbad, CA, USA) and i CropTheBug systems (FiltaFlex Ltd., Almonte, ON, Canada), were used as previously described [20]. To compare each machine for capture efficiency of Y. pestis, 1 mg of Pathatrix immunomagnetic beads (IMBs) functionalized with rabbit anti- Y. pestis and 50 mL of BPW (pH 7.2) containing ~5 CFU/mL of Y. pestis were mixed for 1 h, after which the beads were magnetically captured from the solution.…”

“…Whole milk (3.25% milk fat), processed meat (black forest ham), and prewashed bagged salad (romaine lettuce) were purchased from a local grocery store and used for the food-spiking experiments as previously described [20]. Briefly, Y. pestis cultures were grown to a concentration of 10 7  CFU/mL and serially diluted to 10 2 –10 4  CFU/mL.…”

“…The beads were mixed and captured according to the i CropTheBug method as previously described [20]. Experiments involving each food matrix and bacterial concentration were done in duplicate.…”

“…We have recently developed an immunomagnetic separation (IMS) assay for the efficient concentration of Bacillus anthracis spores from different food matrices [20] and a novel sequence-based assay using pyrosequencing for the specific detection and antimicrobial resistance gene profiling of Y. pestis [21]. Here, we present the application of IMS and pyrosequencing based assays for the rapid, specific, and sensitive detection and identification of Y. pestis from food matrices such as milk, bagged salad, and processed meat.…”