Salmonella control is a top priority for regulatory agencies, public health organizations, and food production companies based on the steady number of associated foodborne illnesses. While much effort has focused on better understanding salmonellosis and managing Salmonella during food production, the estimated 1.2 million cases that occur in the U.S. each year are well above current public health goals of about 36,000 cases nationwide.
The basic ecology of agricultural and animal-derived food products results in a normal association with Salmonella. Accordingly, Salmonella management is a major challenge for the food production industry. Understanding salmonellosis is difficult because the number of Salmonella cells required is unclear. Scientists also do not understand whether every strain of Salmonella can cause illness. There are thousands of strains found in nature. Previous risk assessments have indicated that there is a dose-response relationship between the number of Salmonella present and the severity and number of individuals infected after consumption of contaminated poultry products. The ability to identify points in processing with higher levels of contamination would greatly assist processors in better managing Salmonella levels present in finished food products.
Public health data illustrate an important association between raw poultry and salmonellosis. Accordingly, the USDA Food Safety and Inspection Service (FSIS) and the poultry industry are working on programs to improve Salmonella control. This August, USDA-FSIS published the Final Rule for Modernization of Poultry Inspection. The new regulation is expected to have a direct impact on the number of Salmonella-associated illnesses each year with advancing inspection practices and more science-based detection methods. With a more preventive focus, this new inspection process will allow USDA-FSIS to verify safety programs and provide a more comprehensive assessment of process control by examining sanitation procedures, reviewing records, and collecting test samples for microbiological analysis.
Furthermore, poultry processors must consider Salmonella a food safety hazard. Failure to implement and manage control procedures could result in regulatory action. Therefore, processors will be expected to engage in food safety management, including sampling and testing programs for Salmonella.
The Need for New Techniques
Rapid detection assays have been used by the food processing industry for many years to detect extremely low levels of Salmonella. Based on currently available commercial technology, a sample enrichment period is required to reliably and qualitatively detect low levels of Salmonella in food products. Qualitative detection of foodborne pathogens indicates presence or absence in the test portion analyzed and is not intended to provide quantitative information on the starting levels of the pathogen. Thus, if a series of independent samples were determined to be positive, it would be unknown how many cells were originally present at the time of sampling and testing. Quantitative data, which would allow for a better understanding of contamination levels, would be useful to food processors of raw agricultural products that consistently have pathogens present. It can also be used to better understand the extent of contamination for a given sample type and point in the process or environment.
Quantitative pathogen analysis has typically been conducted using conventional microbiological methods. A most probable number (MPN) analysis is typically used to enumerate low concentrations of pathogens, while direct plating would be used for higher levels. These methods are time-consuming, labor intensive, and expensive. Their accuracy can also be confounded by the dynamics associated with enrichment and bacterial isolation. Specifically, MPN analysis relies on enrichment of a series of cultures that have been serially diluted and subjected to detection to yield a ratio of positive and negative samples. This ratio is then used to estimate the number of starting cells per gram or milliliter in the original, un-enriched sample. This approach is labor intensive and expensive due to the number of dilutions, enrichments, detections, and confirmations required per sample.
Direct plating typically occurs by preparing a homogenate of the test sample and using a portion to inoculate agar plates selective for a given pathogen. Background organisms that can grow on the same agar can confound the process, making it difficult to isolate the target pathogen. Also, injured or stressed cells may not grow at all. Thus, the reliability of this method varies based on the type of sample, state of the target pathogen, and presence of other microbial flora.
Both of these enumeration methods are time-consuming because of culture growth requirements, confounding factors, and requirement for hands-on technical interpretation. Food processing needs rapid, cost-effective methods that provide quantitative information on the contamination level to better support process control and pathogen management.
Evaluating a Quantitative Solution
Based on the association of Salmonella with raw poultry products and the limitations of conventional quantitative methods for Salmonella evaluation, processors face a big challenge in understanding overall process control and potential for high contamination levels. Despite limitations in the science for direct enumeration of low pathogen levels, a threshold determination based on little or no enrichment is possible with existing detection technology.
Vanguard Sciences (formerly AEGIS Food Testing Laboratories, Inc.) provides technical consultation and microbiological testing services. Vanguard Sciences in collaboration with Bio-Rad Laboratories and a poultry processor evaluated the feasibility of Bio-Rad’s iQ-Check Salmonella II standard real-time polymerase chain reaction (PCR) method for direct detection of Salmonella at a threshold of 100 colony-forming units (CFU)/milliliter (2.00 logCFU/ml). Feasibility and method (matrix) validation trials were performed using poultry carcass rinsate samples prepared as part of a routine Salmonella sampling and testing program in a commercial poultry processing facility.
For the feasibility trials, 10 carcass rinsate samples that tested Salmonella negative were shipped via overnight courier to Vanguard Sciences under refrigeration for inoculation within 24 hours of receipt. Samples were analyzed for background flora based on aerobic plate count (APC) by direct plating on 3M PetriFilm following AOAC method 990.12. In addition, Salmonella-negative status was verified with the iQ-Check direct PCR standard method prior to inoculation. A poultry isolate of Salmonella was used to inoculate 10 prepared poultry carcass rinsates and samples were serially diluted to above and below 100 CFU/ml (6 concentrations per rinsate; n=60). They were also directly analyzed without enrichment for the presence of Salmonella using the iQ-Check Salmonella II standard real-time PCR method.
After trials one and two, optimizations to the assay were made to improve accuracy and sensitivity, including adjusting the sample size and volume of lysate buffer. All inoculated rinsates were simultaneously direct plated on Bio-Rad’s RAPID’Salmonella chromogenic agar at the time of testing to verify the presence of Salmonella at the target threshold level. Of the 60 rinsates analyzed during trial three using the optimized protocol, a total of 20 had Salmonella present on the plates, indicating levels of 20 to 1,000 CFU/ml (1.30 to 3.00 logCFU/ml). Of those samples, 18 were positive for Salmonella using the iQ-Check Salmonella II standard real-time PCR assay, ranging between 30 to 1,000 CFU/ml (1.48 to 3.00 logCFU/ml). Two samples at 80 and 20 CFU/ml (1.9 and 1.3 logCFU/ml) were PCR negative. A total of 10 rinsates were not inoculated and served as negative controls, and all were negative by direct plating and PCR. Background flora (APC) ranged from 10 to 8,600 CFU/ml. These data indicate the feasibility of this approach as a direct detection method for Salmonella at a specified threshold level.
Upon feasibility determination, a matrix validation was performed whereby representative bone-in, skin-on thighs (n=20) were inoculated with Salmonella and subjected to standard rinsate procedures to allow for recovery of approximately 100 CFU/ml (2.00 logCFU/ml) in the rinsate. These rinsates were then subjected to analysis using the optimized protocol for the iQ-Check Salmonella II real-time PCR assay in conjunction with a reference method following USDA-FSIS procedures published in the Microbiology Laboratory Guidebook. In addition, samples were direct plated on RAPID’Salmonella chromogenic media. A total of five samples were processed as negative controls. All samples were also processed for indigenous Salmonella and background APC.
Test samples were negative for indigenous Salmonella and had an average APC value of 2.7 logCFU/gram. For the 20 samples inoculated, Salmonella was recovered at 80 to 490 CFU/ml by direct plating. All direct unenriched samples were positive by PCR following the iQ-Check Salmonella II standard real-time PCR method and by the USDA-FSIS reference methods after standard enrichment procedure.
Collectively, these data illustrate that the iQ-Check Salmonella II standard real-time PCR method with modification can be used directly and reliably to detect Salmonella at a threshold of 100 CFU/ml (2.00 log CFU/ml) in poultry rinsates without enrichment.
Warren has provided technical support and guidance to the food and infection control industries for over 15 years and is currently vice president of government and regulatory affairs at Vanguard Sciences (formerly AEGIS Food Testing Laboratories). Reach her at firstname.lastname@example.org.
References Furnished Upon Request