Listeria in the lab

With listeriosis hitting the headlines again this year, Lisa Green takes a look at the causative organism and discusses the contribution of more effective microbiological culture media to its isolation and identification

Colonies on Harlequin Listeria Chromogenic Agar

Twenty years ago, the Public Health Laboratory Service published a report that outlined a sharp rise, followed by a sharp fall, in reported cases of listeriosis across England and Wales. At the time, the exact reasons behind the outbreak were unclear but the subsequent decrease in cases coincided directly with a public health campaign about foodborne Listeria monocytogenes and the risks to certain sectors of the population. Following the success of the campaign, it was noted within the Communicable Disease Report that: “There is no cause for complacency, however, because the reasons for these changes are poorly understood. A new risk factor may emerge or warnings about high risk foods may be increasingly disregarded as the media publicity wanes; the trends could, therefore, reverse again.” 1 

Today Listeria infection still remains a cause for concern. In 2007 The Food Standards Agency reported a large increase in the number of cases of listeriosis compared with the previous year, and also indicated a change to the pattern of disease. In the 1990s it was mostly associated with pregnant women, but in 2007 approximately 70% of cases were in people over sixty years of age2. This year’s National Food Safety Week (held in June) focused specifically on the need for correct storage and handling of food in the home, targeting in particular people over sixty. Overall, the incidence of Listeria infection in this age group has doubled since 2001 and is the cause of more deaths than Salmonella and E. coli O157 combined. A recent call by government ministers for there to be clearer labelling and a better understanding of ‘best-before’ and ‘use-by’ dates on food has also opened up debate. The results of a recent survey suggest that a dismissive approach to use-by dates coupled with a reluctance to throw away food may be contributing to the rising incidence of listeriosis in the over sixties3. Added to this are current national concerns about the large amounts of food wasted each year, further fuelling discussion about guidance and labelling.

There are six species of Listeria, but only the pathogenic Listeria monocytogenes is known to cause disease in man. This Gram-positive bacillus occurs widely in nature, livestock and the environment, and, until the 1980s when there were several large outbreaks, listeriosis was regarded as a rare human disease. Although killed by cooking food and by pasteurisation, the organism is unusual in its ability to grow at low temperatures, thriving in normal refrigerator conditions. The primary transmission route to humans is regarded as being through foods contaminated during production. Most likely to be affected are unpasteurised cheeses, cold cuts of meat, pâtés and smoked fish, or pre-cooked ready meals that have been chilled for some time.

For most people, Listeria infection results at most in a mild and relatively harmless flu-like illness or gastroenteritis, and it exists in a harmless carrier state in a small proportion of the population. However, in vulnerable groups it can have serious consequences. Pregnant women, older people and those with compromised immune systems are much more susceptible. In pregnancy it is especially dangerous because although the illness is unlikely to be harmful for the mother, it can cause miscarriage, premature delivery or severe illness in a newborn child. In non-pregnant adults it is an important cause of septicaemia and bacterial meningitis. Drawn from figures published by the HPA4, figure 1 indicates overall figures for human cases of Listeria monocytogenes in England and Wales.

The challenge in the food industry is to achieve rapid and routine monitoring for L. monocytogenes. Strict regulation designed to minimise the incidence of Listeria infection is now in place. The European Regulation EC 2073/2005 "Microbiological Criteria for Foodstuffs"5 states that all "Ready-to-eat" foods (RTE being defined as food that is sold ready for consumption without the need for cooking or reheating) must be tested for Listeria. Ready-to-eat food for infants and special medical purposes must demonstrate absence of Listeria monocytogenes in 25 grams during product shelf-life and other RTE foods must demonstrate the absence of Listeria monocytogenes in 25 grams before product leaves the factory and must remain below 100 colony forming units (cfu) per gram throughout its shelf-life.

Traditional isolation of Listeria monocytogenes from food and environmental samples begins with pre-enrichment broth followed by enrichment and then sub-culture onto selective solid medium. Figure 2 shows the FDA (United States Food and Drug Administration)6, ISO 11290 (International Standards Organisation)7 and USDA (United States Department of Agriculture)8 approved protocols.

In the UK, a large number of laboratories work to the ISO protocol, which includes enrichment in Fraser Broths, followed by plating to a primary isolation medium with further confirmatory testing as required. Issued in 2004, this was the first ISO standard to specify the use of a chromogenic medium, reducing the time to presumptive positive results.

Since the detection and isolation of L. monocytogenes is such a critical issue for food producers there is considerable interest not only in developing direct tests, but also for further improving existing ISO compliant culture media. The latest Fraser Broth formulations and chromogenic media are good examples.

Figure 1: Cases of Listeria monocytogenes in humans reported to the HPA Centre for Infections between 1983 and 2008. NB: 1) a single pregnancy-associated case includes both mother and neonate. 2) 2008 data is provisional only. Assembled using data from the HPA [4]

Fraser Broth contains the selective agents acriflavine and nalidixic acid, together with lithium chloride to suppress the growth of Enterococci. Aesculin in the medium is hydrolysed by Listeria species to form aesculetin. This reacts with ferric ammonium citrate to give a black precipitate and a visible positive reaction. Modifications (Fraser BrothPLUS and Half Fraser BrothPLUS from Lab M) to the traditional formulation, whereby the ferric ammonium citrate is added to the tempered broth after autoclaving, have improved the selectivity of the medium with both pure cultures and food samples. Furthermore, there are indications that it can lead to more stable results and fewer false-positives in subsequent ELISA testing.

Chromogenic media now also play a major role in Listeria testing, offering faster detection compared with classical culture media and reducing the requirement for subculture and confirmatory tests. The principle behind the technology of chromogenic media is straightforward: the activity of bacterial enzymes specific to the microorganism to be identified are used to cleave synthetic chromogenic substrates incorporated into the culture media. Importantly, the chromogenic substrates themselves are colourless; it is only when they are cleaved by specific enzymes that they release a characteristic coloured product. This turns growing colonies containing the relevant enzymes a distinct colour.
The benefits of ‘intelligent’ chromogenic media are now widely accepted; by providing the potential for target microorganism identification and reducing the need for subculture and/or confirmatory tests, chromogenic media offer improvements in efficiency, not only saving time, but also reducing the cost of consumables. However, despite the simplicity of the technology on which they are based, their development and manufacture is a sophisticated process. Success is highly dependent on a combination of targeted selectivity, specific chromogenic substrate and a complex blend of nutritional components.

Figure 2: Listeria isolation protocols

The contribution of chromogenic media to L. monocytogenes isolation and presumptive identification is illustrated using Lab M’s Harlequin Listeria Chromogenic Agar (Agosti & Ottaviani formulation) as an example. Here, lithium chloride in the base medium and supplementary antimicrobial compounds ceftazidime, polymyxin, nalidixic acid and cycloheximide provide selectivity. Chromogenic activity results from a chromogenic substrate for the detection of the β-glucosidase enzyme common to all Listeria species. A lecithin substrate detects the phospholipase enzyme present only in L. monocytogenes colonies, resulting in a halo of precipitation around them (Figure 3). With a combination of chromogenic and phospholipase reactions it is possible to differentiate L. monocytogenes from other Listeria species. Supplied in dehydrated form, this medium can be prepared as and when required, to fit any lab testing schedules.

After a fall during the 1990s, we are again faced in the UK with a rising incidence of Listeria food poisoning, especially in the older age groups. Various UK bodies are concerned to educate consumers on how to minimise the risk of infection by handling, preparing and using food correctly. Food industry vigilance is supported by the availability of enhanced microbiological methods that simplify and accelerate routine testing for L. monocytogenes.

References 1. L Newton, S M Hall, M Pelerin, J McLauchlin, Communicable Disease Report, Listeriosis surveillance: 1990, Public Health Laboratory Service (PHLS),  Vol 1 Review No 10, 13 Sept 1991 2. Andrew Wadge, Chief Scientist, Food Standards Agency http://www.fsascience.net/2007/06/06/listeriosis 3. Food Standards Agency Management Report, J1834 JF/KME/MD, COI ref: 29103, Food Hygiene Creative Development Research - Working with Older People, February 2009, http://www.food.gov.uk/multimedia/pdfs/olderresearch.pdf 4. Listeria monocytogenes Human cases in England and Wales reported to the HPA Centre for Infections, 1983 – 2008, http://www.hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1195733749778?p=1191942172760 5.  Commission Regulation (EC) No 2073/2005. 15 November 2005.  Microbiological criteria for foodstuffs. 6.  Bacteriological Analytical Manual (January 2003).  Chapter 10  Detection and Enumeration of Listeria monocytogenes in Foods.  7. ISO 11290-1:1997 (Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of Listeria monocytogenes - part 1, Incorporating Amendment 1.) 8  USDA/FSIS. 2002. Microbiology laboratory guidebook, 3rd ed., revision 3,  chapter 8.