Listeria a Survivor

Product Manager Microbiology…. ivo.siegrist@sial.com

A bacteria on the rise, profiting from today’s trends in food product types

Listeriosis is a serious infection caused by Listeria monocytogenes. In recent years it has been recognized that Listeria is an important public health problem. The disease affects primarily people of advanced age, pregnant women, newborns, and adults with weakened immune systems.

Listeriosis manifests in flu-like symptoms, fever, muscle aches, and sometimes gastrointestinal symptoms such as nausea or diarrhea. If infection spreads to the nervous system, symptoms such as headache, stiff neck, confusion, loss of balance, or convulsions can occur. A severe bout of the disease may lead to blood poisoning, encephalitis and meningitis.

Infected pregnant women may experience only a mild, flu-like illness but this can lead to miscarriage or stillbirth, premature delivery or infection of the newborn.

A lot of research was done in the last years concerning mechanism which this pathogen is using to invade into the host and was found that L. monocytogenes is replicated rapidly in the cytosol of host cells like macrophages and lymphocytes (4).

Why have Listeria infections increased recently?

Today’s major problem is a change in food consumption patterns and the increased demand for longer shelf life. An increasing variety of food products and the trend for “readyto- eat” and “ready-to-cook” products are some of the reasons for such problems as well as longer storage at cool temperatures (4–8 °C). New preparation technologies, like “Cook&Chill” and “sous vide” and new processes to extend shelf life have led to increasing problems with Listeria.

Figure 1 L-form of Listeria monocytogens (source: M.Loessner and Y. Briers, ETH Zurich)

The Nature of Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, non spore forming, rod-shaped flagellate (see figure 2). It is an ubiquitous organism, it exists in plants, soil and the guts of birds, fish, shellfish and some mammals, including humans. Some studies suggest that 1-10% of humans may be intestinal carriers of L. monocytogenes. Special risk materials are raw or processed meat, raw milk products, raw or smoked fish, ready prepared salad and long stored vacuum packed food.

Figure 2 Electron micrograph of a Listeria monocytogenes bacterium in tissue.
Listeria monocytogenes is the infectious agent responsible for the food borne illness Listeriosis. In the United States, an estimated 2,500 persons become seriously ill with listeriosis each year. Of these, 500 die. (source: Dr. Balasubr Swaminathan; Peggy Hayes; CDC - Division of Bacterial and Mycotic Diseases: Listeriosis, 2002)

Listeria species are killed by heating steps, but the bacterium is relatively insensitive to high concentration of salts and acids. It also is able to multiply at fridge temperatures and inside vacuum packaging.

Biochemical Tests and Cultural Methods

The biochemical profile of Listeria includes: catalase positive, oxidase negative, fermentation of carbohydrates to acid but not to gas, hydrolysis of esculin and sodium hippurate, methyl red positive, ammonia production from arginine, negative reaction for hydrogen sulfide production, indole negative, nitrate reductase negative, no gelatin liquefaction, no hydrolysis of starch and no urea hydrolysis.

Further differentiation of Listeria ssp., specially for L. monocytogenes, by phenotypic properties is possible with additional biochemical test. All of them start with the β-hemolysis test (L. monocytogenes is positive) followed then by detection of carbohydrate fermentation ability. One possibility is to test positive for rhamnose and methyl α-D-manno pyranoside fermentation and a positive CAMP-test. In the CAMP-test some Listeria species shows the ability to enhance the haemolysis of Staphylococcus aureus. More details about this first method can be found online on the Rhamnose Broth data sheet (Fluka 80547, see also table 2d). Another possibility for phenotype identification is the testing of the fermentation ability of rhamnose, xylose and mannitol (see identification flow chart figure 7).

An interesting topic and a smart solution for confirmation of L. monocytogenes are the chromogenic media. There are diverse commercial available chromogenic media like the Agar Listeria Ottavani and Agosti (ALOA) and most of them use the following systems for differentiation:

1. Detection of β-glucosidase activity (by X-glu = 5-bromo-4-chloro-3indolyl- β-D-glucopyranoside) and also Rhamnose fermentation (by indicator phenol red) on a selective media. Listeria monocytogenes and Listeria innocua results in blue colonies with yellow background, while Listeria ivanovii shows only blue colonies.
2. Screen for the presence of β-glucosidase (by X-glu) and phosphatidylinositol specific phospholipase C on a selective media. Listeria monocytogenes and Listeria ivanovii results in greenish-blue colonies with an opaque halo, while Listeria innocua shows only greenish-blue colonies (recommended by ISO 11209-2)

Recommended Media and Tests used for the ISO Method

There is more information about the detection systems of the chromogenic and other confirmation media in table 2d. To give the media selectivity, phenyl ethanol and a high concentration of lithium chloride and sodium chloride are added to the media. As well antibiotics like moxolactam, nalidixic acid, polymyxin B sulphate, ceftazidime, amphotericin B, acriflavine, cycloheximide, colistin sulphate, cefotetan and fosfomycin are taken to inhibit growth of fungi, Gram-negative and Gram-positive bacteria.

EN ISO 11290-1 and EN ISO 11290-2: (Microbiology of Food and Animal Feeding Stuffs) describe a horizontal method for the detection and enumeration of Listeria monocytogenes. A flow chart of the process appears in figure 3. The method involves a general four-step process: enrichment, identification, isolation and confirmation.

Figure 3 ISO Protocol (EN-ISO 11290-1:1996) for detection and enumeration of Listeria monocytogenes

Sigma-Aldrich, through the innovations of chemists at its Fluka-brand, developed and commercialized reliable media and biochemical tests for many pathogens, including Listeria according to EN/ISO methodologies. The media contain the elements necessary to selectively grow and identify Listeria in food substances according to recommended and established methods. The biochemical tests are designed to get easy, quick, and reliable results.

Common media, tests and related products are more detailed and sorted in tables 2a-d and 3.

Selective Enrichment Media

Figure 4 Fraser Broth (Fluka 69198)

Figure 5 IS Listeria mono Confirmatory Agar Fluka 92302 In front Listeria moncytogenes

Identification Media

Identification Media

Confirmation Media (for Differentiation)

Figure 6 Listeria mono Differential Agar (ALOA, Fluka 77408)

Figure 7 Schematic of biochemical identification for Listeria spp. based on carbohydrate fermentation tests and hemolysis (Source: Handbook of Listeria monocytogenes, 2008)

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Materials

References

1. Food-Borne Pathogenic Microorganisms and Natural Toxins Handbook: The “Bad Bug Book” U.S. FDA/CFSAN. Center for Food Safety and Applied Nutrition, Food and Drug Administration, College park, MD (2003)
2. Cossart, P.; Bierne, H.; The use of host cell machinery in the pathogenesis of Listeria monocytogenes. Curr. Opin. Immunol. (England), 13(1), 96-103 (2001)
3. Verbrauchertipps: Schutz vor lebensmittelbedingten Infektionen mit Listerien, Bundesinstitut für Risikobewertung (2008)
4. C.L. Birmingham et al., Listeriolysin O allows Listeria monocytogenes replication in macrophage vacuoles, Nature 451: 350-354 (2008)
5. L. Dongyou, Handbook of Listeria monocytogenes, CRC Press (2008)

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