The food microbiological analyst: pairing tradition with the future

Keywords: food safety, food spoilage, foodborne disease, genomics, method validation, PCR, rapid methods, WGS.

Food safety is important for all. The WHO has estimated that globally 600 million people get sick from foodborne illnesses each year including an estimated 420 000 deaths and loss of 33 million heathy years of life.¹ Apart from food safety, food spoilage and waste are also global issues. It has been estimated that one-third of all food produced globally is lost or wasted.² Not only is a large-scale spoilage issue detrimental to a brand but discarded spoiled food products cause losses in waste of energy input, land resources, water, shipping costs and more.³ Therefore, the role of the food microbiology analyst is critical in ensuring foodborne outbreaks are solved, food is constantly monitored for microbial safety and spoilage of food is minimised. This has a major effect on people’s health, safety, and the economy. However, identification of microbes in food presents many challenges that need to be understood by food microbiology analysts. Food often contains many microorganisms in a complex food matrix. Finding the elusive pathogen that caused a foodborne outbreak or caused spoilage in a batch of food can be like finding a ‘needle in a haystack’. Critical decisions need to be made by the trained food microbiologist to ensure the laboratory has the ability to choose the appropriate method to produce accurate, sensitive and specific results that truly reflect the microorganisms present in the food sample submitted for analysis.

The decision about which method to use will be based on the food microbiologist asking the right questions such as: what is an appropriate sample size; is the sample homogenous; what is an appropriate subsample for analysis; how consistent are the subsamples; which portion or area of the sample to target or include in the process; and is there matrix interference such as with garlic or spices, that requires inhibition mitigation or extra dilution, due to their antimicrobial effects on the target organisms. Decisions also need to be made if qualitative or quantitative analysis is required based on infective dose and pathogen virulence. An important consideration for qualitative analysis is the type of enrichment performed to ensure enough of the pathogen is present to be detectable in subsequent steps, especially for severe pathogens with a low infective dose. Uniquely in microbiology, the test target can exist in a wide spectrum of viable states, that is, target cells may be damaged to varying degrees, and this is often related to the effects of food processing. This, in turn, means it is important to consider method choices to ensure maximum recovery of stressed microorganisms, such as using broths versus plates, or adding selective steps later rather than sooner in the method. In addition, the design and use of selective and differential agars can have limitations related to visually separating the irrelevant microbes likely to be present in a food sample from the possible culprit. Measurement Uncertainty (MU) is another issue to be addressed.⁴ All routine test methods have MU, and as such, any numerical result should be seen as existing within a range of possible values within which the true value of the measurement lies. This must also be considered when obtaining a result, including the MU overlap of a specified or applied guideline. Numerous methods may be available for the same target organism. Does one method have lesser uncertainty (e.g. measurement of E. coli by acid production versus presence of a specific enzyme in chromogenic agars)? It is strongly advisable to consider MU when applying specific guidelines to produced results.

Food microbiology analysts need to be able to identify these issues when developing or implementing new technologies by being well trained in strict validation protocols. Although these questions have been addressed in designing international standards for the detection and identification of a handful of well-documented foodborne bacterial and viral pathogens, the CDC state that researchers have identified at least 250 foodborne diseases caused by a mix of bacteria, viruses and parasites,⁵ many of which would not have current validated standard food microbiological methods. This requires future method development addressing many of the questions above and deciding which of the developed and emerging methods might be appropriate.

Over the last few decades, analytic food microbiology laboratories have embraced automated technologies using culture-independent diagnostic platforms based on immunological principles or principles of nucleic acid amplification. The advantages of pathogen detection kits that rely on nucleic acid amplification techniques (such as but not limited to PCR) include significantly increased throughput, the potential to combine testing for multiple pathogens at one time, and the ability to detect viable but non-culturable (VBNC) microorganisms and others that are difficult to isolate by traditional culture techniques.⁶ Procedures for use of the ‘black box’ equipment for rapid automated technologies can be quite straight forward with good instructions, but there is still a very important need for training of analysts to understand the underlying science and method limitations. As an example, the sensitivity of a PCR test in foods can be greatly reduced in a complex food matrix, such that a false negative could be reported if the food contains ingredients that are PCR inhibitors.⁷ Another implication of automated technologies for the food microbiologist is to be able to change from observing a colony growing on a plate to reading a response, spike, or curve on a screen.

Many medical laboratories now use fully automated culture-independent diagnostic techniques, but most food microbiology analytic laboratories still do many tests using the traditional standard methods supplemented by modern automated methods. This is because microbial analysis of food can be significantly more challenging due to the mix of microorganisms typically present and possible low numbers of target pathogens compared to, say, identification of an infectious agent in normally sterile urine, CSF, or blood. In most cases a negative food pathogen result using the above technologies, such as a PCR test of sample taken directly from an enrichment broth, is enough to report the food sample result as negative and therefore the food as safe or compliant (notwithstanding the overriding importance of eliminating the likelihood of a false-negative); but if the test is positive, it is necessary to go back to the food sample (or at least the enrichment broth) and retest or confirm the result via an approved standard method that relies on isolating viable colonies.^8,9 This retesting or additional analysis is necessary as it proves the food sample contained a living pathogen and not just left-over strands of genetic material from a pathogen captured using genetic analysis. This would lead to a false positive result, when in fact the food processing steps may have been correctly designed to kill any microorganisms in the raw or pre-processed food. Therefore, again, understanding the importance of validation and intended use and consequences of using these methods cannot be understated.

In further advancement, genomic technologies are now rapidly replacing culture methods⁶ as this technology advances and the cost of sequencing is continually decreased. Sequencing the genes that are diagnostic for a presumptive positive foodborne isolate growing on an agar plate is now routine in public health laboratories using commercially available sequencing equipment, but the interpretation of the sequencing reads requires people trained in bioinformatics. The advantage of genomics is that it can rapidly detect multiple genes or transcription products, which is invaluable in subtyping bacteria and for collecting epidemiological data. This is now critical for tracking foodborne outbreaks locally, nationally, and internationally in sufficient time to act. As the genomic analysis of SARS-CoV-2 has highlighted, gene sequencing is very important in assessing the evolutionary pathway of strains. In the same way, the sequencing of isolates to link clinical, food and environmental samples is invaluable in providing information about the origin of outbreaks, the path(s) of the pathogen from farm to fork,⁶ and ultimately in implementing change to improve food safety in a timely way.

The GenomeTrakr Network is an international collaboration of government, public health and academic laboratories that collect and openly share genomic and geographic data from foodborne pathogens for the benefit of public health. It is the most extensive and best-known application of Whole Genome Sequencing (WGS) to food safety.¹⁰ It includes the U.S. Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDC), U.S. Department of Agriculture, U.S. National Center for Biotechnology Information (NCBI), state health departments, and international partners.^11,12 It is vital to support public health and for the diagnosis and epidemiology of emerging pathogens, microbial genome variation and evolution, and new gene discovery.¹³

One recent technology that sits between traditional methods and genomics is matrix-assisted laser desorption-ionisation time of flight mass spectrometry (MALDI-TOF MS). Again, food microbiology analysts who rely on the results of this technology should understand the underlying science and limitations. Its advantages are being useful for screening presumptive pathogenic or spoilage colony isolates as it generates rapid results, is cost effective and easy-to-use. However, identification of the isolated target relies heavily on the database of peptide mass fingerprints containing the spectra of known organisms¹⁴ and while databases are improving, they are not perfect, due to limitations of lack of sufficient spectra in the database and an inability to discriminate between some related species.¹⁵

There is a need for more rapid and precise methods for microbial food analysis and the use of genomics will eventually become more mainstream. However, this requires ongoing education around sequencing platforms and bioinformatics analysis to be able to correctly interpret the results. These methods, as with any new method including novel molecular methods, will require robust validation to determine the sensitivity, selectivity and in particular, reproducibility, to be applicable in a global framework. The end goal is for regulators, manufacturers and public health authorities to make clear and confident decisions based on these results. However traditional culturing also remains important to determine pathogen viability, for enumeration, and as a proof of experimental concept for data obtained from genomic methods.⁶

Being a food microbiologist is a truly fascinating career path with many strings to its bow, but to ensure the upcoming student who wishes to become a specialist in food microbiological analysis has the widest possible career options, tertiary education must now encompass not only traditional food microbiology analysis but also cutting edge molecular, genomic and associated bioinformatic technologies.

Data sharing is not applicable as no new data were generated or analysed during this study.

The authors declare no conflicts of interest.

This research did not receive any specific funding.