Multi-Pathogen Enrichment Media for Food Testing

Detecting foodborne bacteria has never been easy. Pathogenic bacteria are often sparsely distributed throughout food batches and can be present in very low numbers in randomly collected food samples. Further, the diversity of normal flora found in various food matrices creates a complex and dynamic microbial ecosystem that could interfere with the detection of target pathogens. Microbial dynamics are also influenced by food’s microstructure and chemical composition, which adds additional complexity to the detection process. Consequently, the detection of foodborne pathogens requires, first and foremost, accurate and reliable techniques to effectively maintain food safety.

Conventional culture-based methods, which were developed and implemented several decades ago, continue to deliver a reliable but conservative solution, capable of detecting as few as one target cell per 25g to 325g food sample. These methods are still widely regarded as gold standards for detecting foodborne pathogens, due to their precision and accuracy; however, these traditional methodologies are time-consuming, taking more than a week to provide a final result. Moreover, each identified pathogen requires independent protocols, which is neither convenient nor compatible with today’s intense production needs.

To address the shortcomings of traditional detection methods, numerous molecular techniques have been developed and used effectively in the past decade to detect foodborne pathogens. The development of real-time quantitative polymerase chain reaction (qPCR) has revolutionized microbiological analysis by enabling the detection of pathogenic microorganisms in food without the need for the labor-intensive and time-consuming procedures of isolation and identification. This method has dramatically reduced the time-to-result, which is a critical performance standard used to evaluate the effectiveness of a detection tool, alongside assay sensitivity.

The qPCR method not only provides faster, more sensitive, and specific results than traditional PCR methods, but also offers the potential for multiplexing, which means it can simultaneously detect multiple pathogens in the same reaction, enhancing operational efficiency and reducing overall costs. Numerous food commodities, including shellfish, fresh fruits and vegetables, dairy, and meat products, have been found to be contaminated with multiple pathogens of concern, such as Salmonella enterica, Listeria monocytogenes, and Escherichia coli, along with diverse species of Shigella, Campylobacter, and Vibrio. Consequently, simultaneous detection of multiple pathogens on a single-assay platform aligns with contemporary food industry trends and could also mitigate industry and regulatory needs in the mandatory testing of food products for a range of pathogens prior to distribution.

The Enrichment Step

While the potential advantages of qPCR multiplexing may seem apparent, it’s important to consider the sensitivity of these detection platforms. To guarantee the achievement of legal limits (absence in 25g for most bacterial pathogens), an enrichment step using microbiological culture media is still required prior to qPCR detection. Integrating both traditional microbiological enrichment and molecular pathogen detection serves as a useful bridge that links traditional and molecular microbiology. This approach offers combined benefits while also reducing some of the limitations associated with each method.

If performed appropriately, a short enrichment step is typically sufficient to “produce enough DNA” for subsequent qPCR detection. Moreover, the enrichment process not only increases the concentration of target pathogens in the sample but also revitalizes physiologically stressed or injured microbial cells. Selective enrichment is also crucial for suppressing the naturally occurring background microorganisms, enhancing detection efficiency, and preventing false positive outcomes; however, some of the drawbacks of selective enrichment media include the inhibitory nature of selective agents, which may slow down the growth or even suppress recovery of healthy or stressed target pathogens, ultimately impacting the detection process.

Numerous microbiological culture media with optimized selectivity have been validated and commercialized for short, single-step enrichments for the detection of foodborne pathogens such as Salmonella, Listeria, E. coli, and Campylobacter, across a variety of simplex qPCR assay platforms.