LABTips: Overcoming Food Microbial Testing Challenges

Food microbial testing is essential for public health and safety, preventing harmful bacteria and other microorganisms from reaching consumers, where they can cause illness or even death. Microbiology testing of food is also important to prevent product recalls, which are costly and can damage brand reputation. Pathogen testing, whether through traditional cell culture methods, immunoassays or PCR, comes with many challenges, including a number of different interference mechanisms associated with complex and diverse food matrices. These matrix effects create a difficulty for the food industry due to the costs and risks associated with both false positives and false negatives.

The following tips provide an overview of some of the matrix challenges to look for in food microbial testing, as well as best practices to overcome them and prevent inaccurate results.

1. Optimize Pre-enrichment to Prevent False Negatives from Antimicrobial Ingredients

Many food products contain ingredients that can have an inhibitory effect on the growth of target bacteria like E. coli and Salmonella. Common antimicrobial inhibitors found in food products include salt, acids, onions, garlic, cocoa and herbs and spices like cinnamon, oregano, cloves and allspice.¹ Ingredients with antimicrobial properties may seem like a great thing for food safety, but from a pathogen testing perspective, they can also be a recipe for false negative results. This is due to the fact that while some pathogens may be able to survive in small amounts in foods containing these inhibitors, such as low-moisture spice blends, they can be prevented from growing to detectable amounts when antimicrobials are present in enrichment broth. So even though unsafe target microbes are present in the food in reality, the antimicrobial agents would make it appear as though they are not present during testing.

Fortunately, there are methods for mitigating this problem through specific pre-enrichment techniques. Strategies include dilution, partitioning, or neutralization of the inhibiting agent.¹ These strategies should be selected and optimized based on the types of inhibitors you are dealing with. Regulatory agencies and organizations like the FDA and ISO provide guidance on dealing with certain inhibitors and enabling target pathogens to grow unencumbered in enrichment media. For foods with high sodium chloride content (>10% w/w), a dilution bringing the total concentration of NaCl to ≥1% is recommended. Dilution beyond antimicrobial toxicity is also the preferred method for many herbs and spices as well, according to the FDA Bacteriological Analytical Manual (BAM). For example, the agency recommends a 1:100 dilution in the pre-enrichment broth for cinnamon, oregano and allspice, and a 1:1000 dilution for cloves when testing for Salmonella.

For acid ingredients, the pH of the pre-enrichment mixture can be adjusted using NaOH or double-strength Buffered Peptone Water. An addition of 0.5% w/v K2SO3 has been found to be effective for neutralizing the toxic disulfide compounds found in onions and garlic.² Lastly, partitioning methods can be used to separate the antimicrobials from the target pathogen. Adding 2% corn oil to the pre-enrichment medium followed by shaking incubation is one method that has been described in the research literature. This procedure was found to increase Salmonella recovery from oregano samples by greater than 50%.³

2. Select Appropriate Sample Prep Methods to Remove PCR Inhibitors

Even if colonies of target bacteria are able to grow and thrive following enrichment, false negatives can still arise due to interferences from food matrix components during downstream PCR. PCR inhibitors are found in many food matrices and include calcium ions in dairy products, lipids, polysaccharides and polyphenols. Such components may interfere with PCR through a variety of mechanisms, such as co-precipitation with the nucleic acids, interactions with nucleic acids that change their chemical properties, or inhibition of PCR enzymes (DNA polymerase and reverse transcriptase).⁴ To prevent these problems and reduce the risk of false negative PCR results, it is essential to utilize sample preparation methods that are effective at removing these inhibitors.

Knowing your sample and the nature of the matrix is important to understand the steps necessary to purify the target pathogen from interfering components. For example, berries are often rich in polyphenols, which can have a significant effect on PCR reactions due to cross-linking with nucleic acids and inhibition of nucleic acid resuspension.⁵ Understanding which inhibitors are at work in the food matrix can help you select a robust and effective preparation protocol. In the case of berries, and other plant-based foods with high polyphenol content, using an extraction buffer with polyvinylpyrrolidone (PVP) can prevent the interaction between phenols and nucleic acids.

In general, many extraction methods for food safety testing, such as those available in kit format, already have built-in steps designed to minimize the impact of common inhibitors like pectin, polysaccharides, proteases or calcium ions. But ensuring that a method is fit-for-purpose to deal with your specific matrix is key for ensuring that certain inhibitors don’t fall through the cracks. One example is the case of certain seafoods, specifically mollusks and crustaceans, which contain hemocyanins - proteins that can inhibit PCR even at relatively low concentrations.⁶ With this in mind, it’s important to ensure your sample preparation method is truly robust enough to effectively clean up all potential inhibition sources associated with your specific matrix.

3. Suppress, Separate or Eliminate Background Flora to Prevent Both False Positives and False Negatives

There are many types of bacteria found in food matrices that are harmless to consumers and often present at much higher concentrations than target pathogens. These non-target background species can cause problems for food safety testing, either as cross-reacting species that result in false positives, or as competing organisms that prevent target pathogens that are present from growing in enrichment media, causing false negatives. It is important to take measures to suppress, separate or eliminate these interfering microbes and recognize when background flora might be causing a problem in your analysis.

One important factor in preventing both false positives and false negatives is the selectivity of enrichment media. It is especially important to ensure that media are sufficient to support the growth of the target pathogen while preventing the proliferation of similar microorganisms that can produce false positive results, like Proteus and Citrobacter bacteria in the case of Salmonella testing. Strategies for formulating selective enrichment broth have included the inclusion of antibiotics and bacteriophages. Bacteriophages in particular offer the advantage of strict host specificity, which overcomes problems with antibiotic resistance in non-target background flora.⁷ Identifying problematic non-target bacteria in samples coming through your lab could aid in the identification of phages that would improve enrichment selectivity.⁸

Immunomagnetic separation (IMS) is another powerful technique commonly used to isolate target bacteria prior to detection. This method uses magnetic beads coated with antibodies that bind to the target bacteria, separating them from other background flora and as well as concentrating them onto the beads. While the selectivity of the antibodies used for IMS must still be assessed to prevent false positives, it can serve as a valuable approach for purifying target bacteria and improve the selectivity of immunochemical detection methods like ELISA.⁹

4. Only Use Methods That Are Fit-for-Purpose for Your Specific Matrix

It may seem obvious that any method used for enrichment, extraction and detection of foodborne pathogens should be validated for the sample type you are testing at your lab. However, what might not be as obvious is whether a method validated for one matrix can be applied to another matrix that is similar but not identical. One example would be the difference in testing milk chocolate versus dark chocolate.¹⁰ Both of these products fall under the category of chocolate, but their differences in formulation could result in variable results in the testing process. One formulation may include greater antimicrobial or PCR inhibiting qualities than the other. Thus, it would not be wise to assume the same exact method can be adopted for the new product without further verification that the results would be accurate.

Fortunately, this does not necessarily mean you need to undergo a complete validation process for every possible variation within a matrix category. When a new product (ex. dark chocolate) is a sub-category of a validated category (ex. chocolate), a less intensive verification process is often sufficient to address the potential effects of different product formulations.¹⁰ Performing a verification study for your method to accommodate matrix variations ensures that the method remains fit-for-purpose to accurately assess food safety.

References

1. "Salmonella Detection Methods and Laboratory Best Practices for Seasonings, Herbs, and Spice Matrices," White Paper, Microbiology Methods Task Force, American Spice Trade Association (2020). https://www.astaspice.org/wordpress/wp-content/uploads/dlm_uploads/2021/01/SalmonellaWhitePaper_FINAL_V2_Reduced.pdf
2. Heredia, N.; Wesley, I.; García S. Microbiologically Safe Foods; John Wiley & Sons: Hoboken, N.J., 2009.
3. Jean-Gilles Beaubrun, J.; Flamer, M.-L.; Addy, N.; Ewing, L.; Gopinath, G.; Jarvis, K.; Grim, C.; Hanes, D. E. Evaluation of Corn Oil as an Additive in the Pre-Enrichment Step to Increase Recovery of Salmonella Enterica from Oregano. Food Microbiology 2016, 57, 195–203. https://doi.org/10.1016/j.fm.2016.03.005.
4. Schrader, C., Schielke, A., Ellerbroek, L. and Johne, R. (2012), PCR inhibitors – occurrence, properties and removal. J Appl Microbiol, 113: 1014-1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x
5. Sun, B.; Bosch, A.; Myrmel, M. Extended Direct Lysis Method for Virus Detection on Berries Including Droplet Digital RT-PCR or Real Time RT-PCR with Reduced Influence from Inhibitors. Journal of Virological Methods 2019, 271, 113638. https://doi.org/10.1016/j.jviromet.2019.04.004.
6. Suther, C.; Moore, M. D. Quantification and Discovery of PCR Inhibitors Found in Food Matrices Commonly Associated with Foodborne Viruses. Food Science and Human Wellness 2019, 8 (4), 351–355. https://doi.org/10.1016/j.fshw.2019.09.002.
7. Kim, J.; Hur, J. I.; Ryu, S.; Jeon, B. Bacteriophage-Mediated Modulation of Bacterial Competition during Selective Enrichment of Campylobacter. Microbiology Spectrum 2021, 9 (3). https://doi.org/10.1128/spectrum.01703-21.
8. "Preventing False-Positive Results in Pathogen Testing," Article by Stefan Widmann, Romer Labs (2016). https://www.romerlabs.com/en/knowledge-center/knowledge-library/articles/news/preventing-false-positive-results-in-pathogen-testing/
9. K. Málková , P. Rauch , G. M. Wyatt & M. R. A. Morgan (1998) Combined Immunomagnetic Separation and Detection of Salmonella enteritidis in Food Samples, Food and Agricultural Immunology, 10:3, 271-280, DOI: 10.1080/09540109809354990
10. "Overcoming the challenges of pathogen testing in 'difficult' foods," Webinar Presented by Chanelle Adams, PhD, Roka Bioscience (2015). https://www.youtube.com/watch?v=WfJMscAM-J0