June 14, 2005 Salmonella is a leading cause of food borne illness in the United States. The Food and Drug Administration estimates two to four million Salmonella infections occur annually. Additionally, Salmonella species have been used for domestic terrorism in the past, making hundreds of people ill. Current preferred methods for isolation and detection of Salmonella take a minimum of three days, and in some cases up to five. However, more rapid procedures are essential for faster diagnoses, administration of therapy and response to an outbreak, whether it is an act of terrorism or naturally occurring. It is hypothesized that using Immunomagnetic Separation (IMS), to selectively enrich for Salmonella from contaminated food, will considerably reduce the time required to obtain the pure culture required for further analysis. The different food matrices yielded variable results, suggesting that IMS cannot be used to isolate Salmonella from all food types. A larger sampling of food matrices needs to be done to determine the types of foods that work best for this selective enrichment procedure, thus allowing quicker diagnosis, and more thorough case tracking.
Teresa Cutting's experiments were designed and lead by PhD. candidate, Aparna Tatavarthy, with input from her other colleagues at Center for Biological Defense (CBD), Dr. Jacqueline Cattani (Principal Investigator), Dr. Andrew Cannons (Co-Principal & Scientific Director), and Dr. Philip Amuso (Co-Principal Investigator and Florida Department of Health Laboratory Director). The CBD at the University of South Florida conducts research in the areas of detection, molecular typing and source tracking of pathogenic bacteria. When an outbreak occurs, source tracking for containment, as well as specific identification of bacteria to determine the appropriate treatment of affected individuals is crucial. For these reasons, researchers at the CBD have been collaborating with the Florida Department of Health Bureau of Laboratories to develop more efficient, cost effective methods for rapid detection and isolation of Salmonella species from several food types.
The goal of this study was to decrease the time required to obtain a pure culture of Salmonella from the current three days to one day. Conventional isolation methods are designed to detect one colony-forming unit (CFU) of Salmonella in 25 grams of food. To achieve high sensitivity, in this study we spiked 25 grams of food samples with 1 to 10 CFU of Salmonella. However, the incubation period was lowered to only six hours. Considering the possibility of terrorism affecting the food supply, food samples that were pre-prepared or pre-cooked, and available from local grocery stores, were chosen. This included ready-cooked chicken cuts, hamburger patties, egg salad, and blueberries.
IMS employs tiny magnetic beads coated with antibodies specific to the cells of interest, in this case Salmonella. The beads are then separated from the cell suspension with a magnet and plated on agar that is selective for Salmonella. The number of Salmonella CFUs were then counted and the recovery was determined. Homogenized food samples were also tested by real time PCR methods to check for the sensitivity and specificity. To ensure the method was highly specific for Salmonella, this study also conducted mixed spiking experiments. E. coli, Citrobacter and Proteus species were used in the mixed spiking experiments, as these organisms can sometimes be mistaken for as Salmonella in the initial screening tests on selective/differential media.
The average recovery of Salmonella CFU from different foods spiked with either low numbers of Salmonella alone, or Salmonella mixed with other bacteria, was determined. Salmonella could be isolated from spiked foods such as chicken and egg salad without IMS. However, IMS increased the sensitivity by approximately twofold. Cheese required an overnight incubation for valid results and data suggests that IMS did not increase selectivity. This may be due to the affects of background flora. The most successful recoveries of Salmonella were obtained from hamburger samples using the IMS protocol. The poorest recoveries were from orange juice samples irrespective of whether IMS was used for isolation. Similar results were obtained using the real time PCR for detection. These results suggest that no single method can be used for optimal recovery of Salmonella from foods. Rather, the food type dictates which protocol should be used. Salmonella could still be isolated from all food types tested and the isolates could be used for further analyses including, molecular typing and antimicrobial resistance/sensitivity profiles.
The CBD is part of the USF College of Public Health in Tampa, Florida and works in close collaboration with the Florida Department of Health Tampa Laboratory. Funded by the Department of Defense (Contract Number DAAD13-02-C-0043) since October of 2000, the USF CBD conducts bio-defense research in the laboratory and provides education and training in the field, all of which protect the United States against the threat of bio-terrorism from bacterial agents. This work will be presented at the American Society of Microbiology (ASM) 2005 General Meeting in Atlanta, GA on June 8.
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