MALDI-TOF – Developments in epidemiology and laboratory testing for listeria monocytogenes

Listeriosis remains a prominent foodborne disease, not because of the number of cases but because of the high case fatality rate. In recent years the foods involved in outbreaks of listeriosis seem to have diversified and the demographics of cases show a distinct trend. Testing for the organism in food as part of due diligence, or in the food production environment, remains important, and technological innovations continue to reduce testing times significantly.

Listeriosis is usually caused by the species Listeria monocytogenes and opinion is that around 99% of cases are the result of foodborne transmission. Most cases are in one of the ‘at risk’ groups; the Young, Old, Pregnant or Immunocompromised (YOPI). The clinical consequences can include miscarriage, still-birth, bacteraemia, meningitis and death. Another manifestation of listeriosis is ‘febrile gastroenteritis’ which generally does not cause fatalities and follows the consumption of foods containing concentrations greater than 105 CFU/g of the organism.

According to data produced by Public Health England there was an average of 180 reported listeriosis cases in England and Wales per annum over the decade up to 2014, so the number of cases is not high compared to most other foodborne diseases. However, the case fatality rate at 20-40% is high, and so the number of deaths resulting from the small number of infections is of concern. In 2014 there was a 5.3% increase in the number of cases compared to the preceding year, and the sub-population experiencing increasing incidence is that of people over 60 years of age where the primary clinical presentation is bacteraemia.

Trends in listeriosis

Up until quite recently the listeriosis story seemed to have become somewhat stereotyped: disease mainly occurred in the at risk groups following the consumption of long shelf life refrigerated ready-to-eat (RTE) meat, dairy and seafood products in which the pathogen had grown to concentrations around 100 CFU/g (this being accepted as the level of concern). Long shelf life foods were a problem as L. monocytogenes can grow under refrigeration and so, if present at the start of a product’s shelf life, can multiply steadily until the food is eaten.

However, there have been several recent instances where outbreaks of disease have been linked to a broadening variety of foods; for example melons, caramel apples, stone fruit, ice cream and bean sprouts. In fact the outbreak involving whole cantaloupe melons caused the second largest number of fatalities of any listeriosis outbreak in the US (147 cases, 33 deaths and one miscarriage were directly attributable). The question is whether the association of these foods with listeriosis is new or whether it has just recently been recognised (bearing in mind that the first food to be linked with listeriosis was coleslaw).

The rise in the proportion of cases in the over 60 year olds suffering from bacteraemia is also something of a mystery. It could be due to a change in medical treatments, the emergence of a virulent strain, a behavioural risk factor or a change in exposure patterns (i.e. a food is more frequently contaminated, contaminated at a higher concentration than it was before, or is being more frequently consumed). The ACMSF considered these possibilities but noted that there were insufficient data to draw firm conclusions, and recommended further studies to clarify the relative contributions¹.

There are other changes which might be influencing the epidemiology of listeriosis. For example there have been changes to the formulation of foods to reduce salt², and this could allow L. monocytogenes to grow faster or result in hurdle technologies becoming less effective. Another potential factor is the use of antacids to treat indigestion which may allow L. monocytogenes cells to survive passage through the stomach³.

A peculiarity of the UK is the link between listeriosis outbreaks (N.B. most cases are not associated with outbreaks but are termed ‘sporadic’) and the consumption of sandwiches available in hospitals (Table 1). Sandwiches in general do seem to be one of the most frequently contaminated foods with respect to the available data (Table 2), but do not seem to harbour abnormally large concentrations of the organism in a significant proportion of samples. If, however, contaminated sandwiches were temperature abused then there is potential for low concentrations to grow to a level where they would be considered hazardous. Even the butter outbreak (Table 2), involved the preparation of sandwiches.

Testing for L. monocytogenes

The control of L. monocytogenes in RTE foods is difficult to achieve and a full discussion beyond the scope of this article⁴. End-product testing needs to be done to verify that the HACCP programme being used is controlling the hazard. In addition, environmental monitoring is necessary as it should detect problems in the food processing plant before food becomes contaminated. Environmental testing results alongside genetic analysis of isolates provide a powerful tool for industry to identify and negate potential sources of contamination⁵.

In a modern ISO 17025 compliant contract laboratory routinely testing food and environmental samples, all screening methods currently in use to isolate and identify L. monocytogenes are based on an initial selective enrichment step in liquid culture which provides favourable growth conditions for the target organism but suppresses the growth of most of the other bacteria present. This is generally followed by a detection stage in the form of either growth on Petri dishes containing a very selective agar which favours the growth of Listeria (generally termed ‘culture methods’) or the processing through an automatic detector that ‘senses’ growth in the liquid medium itself (known as ‘rapid methods’). At the end of this second stage the laboratory is able to alert the customer of a potential (‘presumptive’) Listeria positive sample. The problem is that out of around 12 different species of Listeria currently identified, L. monocytogenes is the only species regulated by law. The final ‘confirmation step’ of screening is therefore critical as here the laboratory must determine whether L. monocytogenes is present or whether the sample contains a different and non-harmful species. Confirmation conventionally takes two days, one to purify the culture and another to run the biochemical tests needed to identify the species.

This period of time is critical because the customer knows there is a potential problem with their sample but doesn’t have all the information needed for further action. This is highly inconvenient and can lead to stressful contingencies where, for instance, a recall of a product would be necessary when L. monocytogenes is confirmed, but confirmation is still two days away.

An exciting new opportunity has recently arisen with the introduction of robust and reliable Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry for Listeria screening in the commercial food testing laboratory⁶. MALDI-TOF is a well-established rapid bacterial identification technique that has been in routine use in the clinical environment for over six years, and has recently made its debut in the food testing field. At its simplest, it exploits the unique profile (or ‘fingerprint’) generated by specific proteins in a bacterial cell when bombarded by a laser at 337nm. It does so by comparing this fingerprint to a database of known ‘suspects’, not unlike some police fingerprint identification software. (See Figure 1).

This new approach is simple, robust and takes around 30 minutes to give a definitive species identification, thus eliminating the uncertainty associated with a presumptive Listeria positive result without its accompanying species identification. The fact that the profiles used by this technique are generated from the most prevalent organism in the sample mean that it can be performed directly from the selective agar plates, bypassing the need to purify colonies and thus saving time. This means that the conventional culture method 24-48 hour presumptive stage is not needed, and an identification made quickly.

Rapid methods such as lateral flow devices take slightly longer at this stage because they test the broth. To obtain isolates for confirmation there is a need for subsequent culture on agar plates which requires more time. However, this just means an overnight incubation, still reducing the waiting times for a confirmation.

MALDI-TOF is revolutionary in the sense that, by not relying on sugar fermentation and other biochemical identification methods it is not easily confused by factors such as growth conditions, bacterial stress levels and growth media used. This makes it extremely unlikely for an unreliable identification to be obtained, so eliminating the need to repeat testing.

The advantages of MALDI-TOF mean that it is rapidly becoming adopted and many new identification techniques are being validated for different bacteria such as Salmonella, Legionella and Campylobacter. Table 3 shows the results for L. monocytogenes obtained from readings performed at 24 and 48 hours on ALOA and Oxford agars. An average score of ≥ 2.0 is regarded as a reliable identification.

The challenge is comprehensively validating this novel technique and obtaining acceptance of its use for bacterial confirmation by a formal accreditation body such as UKAS. This is because there is not much data existing to support the validation of a new technology and therefore the group wishing to prove the effectiveness of a new technique needs to plan and carry out a comprehensive validation plan that covers all aspects. This can easily lead to confusion due to the large number of variables involved. An effective method of validating such technique is a step by step approach by challenging each variable in turn before moving on to the next. In one such approach⁶ the equivalence to current methods was addressed first, thus proving equivalence to what is considered the gold standard. This allowed the validation to progress to address the more advanced stages, such as the use of more than one ‘fingerprint’ library, (e.g. that provided by the instrument manufacturer and the user’s own library obtained from samples received and authenticated); the impact of environmental conditions and the stage at which the technique can be routinely employed once its performance against the current methods had been documented. The final stage is the method’s documented use on a series of routine samples, and in proficiency tests, and routine use in the laboratory to provide a final clear demonstration of its effectiveness.

The main factors determining the success of MALDI-TOF as a commercial technique is its speed; relative inexpensive running costs and its flexibility. During internal factory contamination events or outbreaks of a particular organism it is of vital importance for a company or public institution to be able to track and characterise the organism responsible for the outbreak and compare it with other known bacteria. This can mean comparison with known and previously characterised (or ‘typed’) internal ‘offenders’ (e.g. in a factory) to establish whether is the same strain causing the outbreak or if it is a completely new one, or comparison between different clinical samples to track the movements and evolution of a certain organism in case of outbreak. During a recent Shiga-Toxigenic Escherichia coli O104:H4 (STEC) outbreak in Germany a team of scientists was able to demonstrate successfully how MALDI-TOF was a useful tool to identify and track a particular STEC strain during an ongoing outbreak⁷. However the use of MALD-TOF for isolate typing is not yet well established.

The adaptability, robustness and ease-of-use of MALDI-TOF mean that this technique is on its way to become the benchmark for rapid bacterial identification in food analysis in the very near future.

References

1. Advisory Committee on the Microbiological Safety of Foods. Report on the increased incidence of listeriosis in the UK. Food Standards Agency, 2009 Contract No.: FSA/1439/0709.
2. Taormina PJ. Implications of salt and sodium reduction in microbial food safety. Critical Reviews in Food Science and Nutrition. 2010;50:209-27.
3. Friesema IH, Kuiling S, van der Ende A, Heck ME, Spanjaard L, van Pelt W. Risk factors for sporadic listeriosis in the Netherlands, 2008 to 2013. Euro Surveill. 2015;20(31):pii=21199.
4. Luber P, Crerar S, Dufour C, Farber J, Datta A, Todd ECD. Controlling Listeria monocytogenes in ready-to-eat foods: Working towards global scientific consensus and harmonization – Recommendations for improved prevention and control. Food Control. 2011;22(9):1535-49.
5. Rückerl I, Muhterem-Uyar M, Muri-Klinger S, Wagner KH, Wagner M, Stessl B. monocytogenes in a cheese processing facility: Learning from contamination scenarios over three years of sampling. International Journal of Food Microbiology. 2014;189:98-105.
6. Capocefalo M, Ridley EV, Tranfield EY, Thompson KC. MALDI-TOF: A rapid microbiological confirmation technique for food and water analysis. In: Cook N, D’Agostino M, Thompson KC, editors. Molecular Microbial Diagnostic Methods Pathways to implementation in the food and water industries. 1st ed: Academic Press; 2015.
7. Christner M, Trusch M, Rohde H, Kwiatkowski M, Schlüter H, Wolters M, et al. Rapid MALDI-TOF mass spectrometry strain typing during a large outbreak of Shiga-Toxigenic Escherichia coli. PLoS ONE. 2014;9(7): e101924.

About the authors

Andrew has a BSc (Hons) from Bristol University and doctorate from the University of Waikato in New Zealand. His initial foray into food microbiology was at the Meat Industry Research Institute of New Zealand producing predictive growth models for pathogens. Later, he joined New Zealand’s Institute of Environmental Science and Research Ltd where he was a science leader in the Food Safety Programme. The main area of interest was the control of foodborne pathogens using bacteriophages. He was also involved in research and consultancy projects in foodborne pathogens with particular focus on Campylobacter, pathogenic Escherichia coli, Listeria and Yersinia. In 2015 he moved back to the UK to take up his present role at Fera as Head of Microbiology in Food Quality and Safety. He is a Fellow of both the New Zealand Institute of Food Science and Technology, and the Institute of Food Science and Technology. Andrew has published more than 90 peer-reviewed papers, reviews and book chapters on various topics in food microbiology.

Prof K. Clive Thompson is a Chartered Chemist; Fellow of the Royal Society of Chemistry; Fellow of the Institute of Food Science & Technology; Fellow of the Royal Society of Public Health; Member of the Water Management Society; Member of the American Society for Microbiology; Fellow of the Chartered Institute of Water and Environmental Management; Member of Board of Trustees of the Society of Chemical Industry; Society of Chemical Industry (SCI) Environmental Medal (2003); Member of AOAC; Member of the Society of Environmental Toxicology and Chemistry; Distinguished Service Certificate, British Standards in appreciation of long and valued contributions to the development of British, European and International Standards; and Visiting Professor at Brunel University. He is currently Chief Scientist at ALcontrol Laboratories UK, which analyses a very wide range of samples including food, drinking water, process waters, and effluents for both chemical and microbiological parameters. He has previously worked for Yorkshire Water and Severn Trent Water. He has co-edited a wide range of chemical and microbiological books.

Matteo Capocefalo is an associate member of the Royal Society of Biology. Matteo graduated in Microbiology with Immunology at The University of Leeds in 2010. Before that he attended a highly specialised college in his home town Turin, Italy, focusing on Microbiology and Biochemistry. He has lived in Yorkshire since 2006 and has been working for ALcontrol for the past two years, beginning as Team Leader and then moving on to become Laboratory Manager. Aside from the running of a busy pathogens laboratory his main focus is the development and validation of new rapid confirmation techniques through MALDI-TOF. He was heavily involved in the successful validation of rapid MALDI-TOF Listeria confirmation (recently granted UKAS accreditation) and he is now continuing to explore the many possibilities offered by this technique.