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Listeria a Survivor

By: Jvo Siegrist, Microbiology Focus Edition 2.2

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 “ready-to-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 ß-hemolysis test (L. monocytogenes is positive) followed then by detection of carbohydrate fermentation ability. One possibility is to test positive for rhamnose and methyl a-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 (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 ß-glucosidase activity (by X-glu = 5-bromo-4-chloro-3indolyl- ß-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 ß-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

Description Brand Cat. No.
Products for Enrichment Steps    
Fraser Broth, Base Sigma-Aldrich 69198
Fraser Selective Supplement Sigma-Aldrich 18038
Fraser Supplement Sigma-Aldrich 90836
 
Products for Plating    
Oxford Agar Sigma-Aldrich 75805
Oxford-Listeria Selective Supplement Sigma-Aldrich 75806
PALCAM Listeria Selective Agar Plate Sigma-Aldrich 75977
PALCAM Listeria Selective Supplement Sigma-Aldrich 03396
 
Purification Medium    
Tryptone Soya Yeast Extract Agar Sigma-Aldrich 93395
 
Products for Confirmation    
Gram Staining Kit Sigma-Aldrich 77730
Listeria Motility Medium Sigma-Aldrich 55265
Carbohydrate Consumption Broth Sigma-Aldrich 07410
Blood Agar base No. 2 Sigma B1676
 
Table 1: Products used for 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 its chemists, 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

Cat. No. Brand Description Package Size
69198 Sigma-Aldrich Fraser Broth, Base (see Figure 4) 500g
18038 Sigma-Aldrich Fraser Selective Supplement 5 vials
90836 Sigma-Aldrich Fraser Supplement 10 vials
F6672 Sigma-Aldrich Fraser secondary enrichment broth base 500g
F2674 Sigma Fraser enrichment supplement 5 vials
62353 Sigma-Aldrich Listeria Enrichment Broth according to FDA/IDF-FIL 500g
62351 Sigma-Aldrich Listeria Selective Supplements according to IDF-FIL 16 vials
62348 Sigma-Aldrich Listeria Selective Supplement according to FDA 16 vials
59859 Sigma-Aldrich PALCAM Listeria Selective Enrichment Broth, Vegitone (see Figure 5) 500g
91986 Sigma-Aldrich PALCAM Listeria Selective Supplement according to Van Netten et al. 10 vials
94485 Sigma-Aldrich UVM Listeria Selective Enrichment Broth, modified 500g
 
Table 2a: Selective Enrichment Media

 

Figure 4 Fraser Broth (69198)

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

 

Identification Media

Cat. No. Brand Description Features Package Size
62355 Sigma-Aldrich Listeria Selective Agar Selective media 500g
62653 Sigma-Aldrich LPM Agar Selective media 500g
43963 Sigma-Aldrich Moxalactam Supplement   5 vials
75805 Sigma-Aldrich Oxford Agar esculin hydrolysis, selective media 500g
51352 Sigma-Aldrich Oxford-Listeria Selective Supplement (uses with 75805) 10 vials
75977 Sigma-Aldrich PALCAM Listeria Selective Agar esculin hydrolysis, selective media 500g
15776 Sigma-Aldrich PALCAM Listeria Selective Agar, Vegitone esculin hydrolysis, selective media 500g
91986 Sigma-Aldrich PALCAM Listeria Selective Supplement according to Van Netten et al. (uses with 75977 and 15776) 10 vials
 
Table 2b: Identification Media

 

Identification Media

Cat. No. Brand Description Package Size
93395 Sigma-Aldrich Tryptone Soya Yeast Extract Agar 500g
 
Table 2c: Purification Media

 

Confirmation Media (for Differentiation)

Cat. No. Brand Description Features Package Size
B1676 Sigma Blood Agar Base No. 2 Lysis test (ß-hemolysis) 500g
B1676 Sigma-Aldrich Carbohydrate Consumption Broth Fermentation ability 500g
53707 Sigma-Aldrich HiCrome™ Listeria Agar Base, modified (chromogenic media) ß-glucosidase activity, rhamnose fermentation, selective media 250g
59688 Sigma-Aldrich HiCrome™ Listeria Selective Supplement (use with 53707) 5 vials
92302 Sigma-Aldrich Listeria mono Confirmatory Agar, Base (see Figure 5) Presence of phosphatidylinositol specific phospholipase C and fermentation of a-methyl D-mannoside, selective media 38.5g, 500g
15895 Sigma-Aldrich Listeria mono Enrichment Supplement II (Use with 92302) 5 vials
92301 Sigma-Aldrich Listeria mono Selective Supplement I (Use with 92302) 5 vials
91603 Sigma-Aldrich Listeria mono Selective Supplement II (Use with 92302) 5 vials
77408 Sigma-Aldrich Listeria mono Differential Agar, Base (ALOA, chromogenic media acc. ISO, see Figure 6) Presence of ß-glucosidase and phosphatidylinositol specific phospholipase C , selective media 500g
03708 Sigma-Aldrich Listeria mono Enrichment Supplement I (Use with 03708) 5 vials
92301 Sigma-Aldrich Listeria mono Selective Supplement I (Use with 03708) 5 vials
91603 Sigma-Aldrich Listeria mono Selective Supplement II (Use with 03708) 5 vials
55265 Sigma-Aldrich Listeria Motility Medium Motility test 500g
80547 Sigma-Aldrich Rhamnose Broth / Methyl a-D-mannopyranoside Broth Rhamnose and methyl a-D-mannopyranoside fermentation 500g
80301 Sigma-Aldrich Rhamnose Broth Supplement (uses with 80547) 25mL
02046 Sigma-Aldrich Methyl a-D-mannopyranoside Supplement (uses with 80547) 5mL
 
Table 2d: Confirmation Media

 

Cat. No. Brand Description Testing features Package Size
88597 Sigma-Aldrich Catalase Test (H2O2, 3% solution) Presence of catalase 100mL
77730 Sigma-Aldrich Gram Staining Kit Cell wall properties 1Kit
40405 Sigma-Aldrich Hippurate Disks Hydrolysis of hippuric acid 25Disks
01869 Sigma-Aldrich Hippurate Strips Kit Hydrolysis of hippuric acid 50Strips
94438 Sigma-Aldrich Mannitol disks Fermentation abilities 10 x 25 Disks
07345 Sigma-Aldrich Oxidase Reagent acc. Gaby-Hadley A Presence of oxidase 100mL
07817 Sigma-Aldrich Oxidase Reagent acc. Gaby-Hadley B Presence of oxidase 100mL
18502 Sigma-Aldrich Oxidase Reagent acc. Gordon-McLeod Presence of oxidase 100mL
40560 Sigma-Aldrich Oxidase Strips Presence of oxidase 100Strips
70439 Sigma-Aldrich Oxidase Test Presence of oxidase 50Disks
93999 Sigma-Aldrich Rhamnose disks Fermentation abilities 10 x 25 Disks
07411 Sigma-Aldrich Xylose disks Fermentation abilities 10 x 25 Disks
 
Table 3: Biochemical tests

 

Figure 6 Listeria mono Differential Agar (ALOA, 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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