How to Use Bacterial Enrichment to Accelerate Food Pathogen Detection

According to the World Health Organization, 600 million cases of foodborne illness occur every year worldwide, causing 420,000 deaths. About 60% of these cases are caused by pathogenic bacteria. As such, the detection of bacterial contamination in foods represents an important health safety concern and a great challenge to global food security.

To prevent foodborne illness, food safety regulations require that manufacturers ensure their food meets highly specific microbiological standards, but pathogenic bacteria, such as Salmonella spp., Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), and Cronobacter spp., are usually present only in a very small amount within a food sample. Therefore, manufacturers need to enrich their samples prior to testing in order to detect pathogens.

The Need for Bacterial Enrichment Prior to Food Analysis

A reliable method for detecting pathogenic bacteria must support their growth and enable their identification based on physiological, metabolic, or molecular characteristics. It also must be sensitive enough to detect one pathogenic bacterium in a 25-gram food portion for the food to be considered safe. However, no method, whether microbiological, immunological, or molecular, is sensitive enough to directly and reliably detect such a low concentration of bacteria, especially in the complex environment of a food sample. For this reason, regardless of the detection method used, all samples must undergo preliminary enrichment to grow the target bacteria to detectable levels.

Enrichment poses different challenges than pathogen detection. For example, during enrichment, pathogen growth might be interfered with by important and various competitive background microflora. The growth of these competitive species must be limited using selective conditions; however, the target bacteria might also be sensitive to these conditions, especially if they’ve been exposed to environmental stressors, such as heat, drying, freezing, or exposure to acids or to sanitary compounds during food processing treatments. These stressors can cause damage to bacterial cell membranes, delaying exponential growth. Consequently, the detection method must enable a rapid resuscitation of the stressed bacteria and promote their growth by inhibiting other competitive microorganisms.

How Harmonized Enrichment Works

Pathogen detection methods traditionally employ an enrichment step that targets one pathogenic genus or species; however, pathogen detection would be more efficient if one could enrich multiple bacterial species at once. About 20 years ago, the concept of simultaneous pathogen detection emerged with the development of the universal pre-enrichment broth (UPB). This medium was initially designed for the co-detection of Salmonella spp. and Listeria spp., two pathogens commonly found in dairy products, meat, and poultry. Then, the protocol was extended to STEC, another organism commonly found in these food products.

This co-enrichment strategy offers tremendous cost savings, as it reduces the number of sample preparations, the need for supplies and reagents, the space needed in incubators, and hands-on time. UPB is highly buffered and low in carbohydrates to prevent a rapid drop in pH and to support strong recovery of the stressed target pathogens but may lead to the overgrowth of background microflora, especially in challenging foods.

Table 1. Comparison of time to results between UPB use and simultaneous selective enrichment of two bacterial species.

Intrinsic differences between Gram-negative and Gram-positive bacteria make their simultaneous enrichment impossible, but by using a single selective medium or a second specific enrichment broth, scientists can still enrich multiple organisms with similar characteristics. For example, harmonized enrichment involves detecting all of the genera that share the same properties, such as Gram-negative bacteria that exhibit similar growth rates, in one enrichment step. Harmonized enrichment encapsulates a wider number of species, thereby increasing efficiency. Reducing the number of analysis steps using harmonized enrichment decreases costs, both by reducing the need for media and by streamlining laboratory workflows.