Listeria: a Survivor

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 recent years concerning the mechanism which this pathogen is using to invade 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). Preparation technologies, like “Cook Chill”, “sous-vide” and other new processes to extend shelf life have led to increasing problems with Listeria.

L-form of Listeria monocytogenes

Figure 1 L-form of Listeria monocytogenes (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.

Electron micrograph of a Listeria monocytogenes bacterium in tissue

Figure 2 Electron micrograph of a Listeria monocytogenes bacterium in tissue.

Listeria monocytogenes is the infectious agent responsible for the foodborne 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 Culture 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., particularly L. monocytogenes, using 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 α-D-mannopyranoside 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. 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 chromogenic media commercially available, the most important agar is the Listeria agar according to Ottavani and Agosti, which is described in ISO 11290 parts 1 (detection of Listeria monocytogenes and Listeria spp.) and part 2 (enumeration of Listeria monocytogenes and Listeria spp.), in FDA-BAM and in several other country regulations. This agar uses the following systems for differentiation:

  1. Detection of ß-glucosidase activity by inclusion of X-glu = 5-bromo-4-chloro-3-indolyl- ß-D-glucopyranoside as chromogenic substrate). This enzyme is common in all Listeria species and they appear as blue-green colonies on this agar.
  2. Detection of pathogenic Listeria by inclusion of lecithin which is a  phosphatidylinositol specific phospholipase C on a selective media. Listeria monocytogenes hydrolyses the specific purified substrate added to the agar medium producing an opaque halo around the blue-green colonies. Most Listeria ivanovii also produce an opaque halo around colonies after 48 hours. Non-pathogenic Listeria species such as Listeria innocua shows only blue-green colonies.

There is more information about the detection systems of the chromogenic and other confirmation media in table 2c. To give the media selectivity, phenyl ethanol and a high concentration of lithium chloride and sodium chloride are added to the media. As well as 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.

Figure 3 ISO Protocol (ISO 11290-1:2017 and ISO 11290-2:2017) for detection and enumeration of Listeria monocytogenes and Listeria spp.

Rapid Methods

In addition to the classical method, we can also offer a range of rapid methods which also meet various regulatory requirements. These include immunological methods such as the Assurance® EIA and TRANSIA® PLATE screens, or molecular methods such as the Assurance GDS® system.

Media and Tests

Through the innovations of its chemists, we have 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-c and 3.

Compliance to revised ISO 11290:2017

We have implemented all the requirements described in both part 1 and part 2 of the revised
EN ISO 11290:2017. Our GranuCult® granulated media, Readybag® pre-weighed granulated media in ready-to-use bags, ReadyPlate™, and ReadyTube® ready-to-use media are all compliant with EN ISO 11133:2014. All information regarding compliance with additional reference standards are displayed on the product label and in the technical information available on our website.

The following culture media and accessories described in the revised EN ISO 11290:2017 are available:

Pre-enrichment and Selective Enrichment Media


 

Description Cat. No.
Half FRASER (Demi FRASER) broth (base) with antibiotics 100025
FRASER Listeria Selective Supplement 100093
Half FRASER (Demi FRASER) broth with supplements 102449
Half FRASER (Demi FRASER) broth with supplements 101865
ReadyTube™225 Half Fraser ISO 11290 146476
FRASER broth (base) 110398
ReadyTube™1000 BPW ISO 6579,6887,21528 146403

 

Fraser Broth

Figure 4 Fraser Broth (69198)

Plating Out Identification Media

Table 2b: Identification Media



Description  
Listeria agar (base) acc. OTTAVIANI and AGOSTI 100427
ChromoCult® Listeria Agar Enrichment-Supplement 100439
ChromoCult® Listeria Agar Selective-Supplement 100432
PALCAM Listeria-Selective agar (Base) 111755
PALCAM Listeria Selective-Supplement acc. to van Netten et al. 112122
Oxford-Listeria-Selective-Agar (Base) 107004
Oxford-Listeria-selective supplement 107006

 

Confirmation Media (for Differentiation)

Table 2c: Confirmation Media


 

Cat. No. Description Features Package Size
B1676 Blood Agar Base No. 2 Lysis test (ß-hemolysis) 500g
77408 Listeria mono Differential Agar, Base (Agar Listeria Ottaviani & Agosti, chromogenic media acc. ISO, see Figure 6) Presence of ß-glucosidase and phosphatidylinositol specific phospholipase C , selective media 500g
03708 Listeria mono Enrichment Supplement I (Use with 77408) 5 vials
92301 Listeria mono Selective Supplement I (Use with 77408) 5 vials
91603 Listeria mono Selective Supplement II (Use with 77408) 5 vials
55265 Listeria Motility Medium Motility test 500g
80547 Rhamnose Broth Rhamnose fermentation 500g
107228 Buffered Peptone Water    
105450 Nutrient agar    
105458 Tryptic Soy agar    
112535 Maximum Recovery Diluent    
110328 Blood agar (base) no. 2    

 

Biochemical Tests 

Table 3: Biochemical tests


 

Cat. No. Description Testing features Package Size
29333 Barritt's Reagent A Detection of acetoin production 100mL
39442 Barritt’s Reagent B Detection of acetoin production 100mL
88597 Catalase Test (H2O2, 3% solution) Presence of catalase 100mL
77730 Gram Staining Kit Cell wall properties 1Kit
40405 Hippurate Disks Hydrolysis of hippuric acid 25Disks
01869 Hippurate Strips Kit Hydrolysis of hippuric acid 50Strips
94438 Mannitol disks Fermentation abilities 10 x 25 Disks
07345 Oxidase Reagent acc. Gaby-Hadley A Presence of oxidase 100mL
07817 Oxidase Reagent acc. Gaby-Hadley B Presence of oxidase 100mL
18502 Oxidase Reagent acc. Gordon-McLeod Presence of oxidase 100mL
40560 Oxidase Strips Presence of oxidase 100Strips
70439 Oxidase Test Presence of oxidase 50Disks
93999 Rhamnose disks Fermentation abilities 10 x 25 Disks
07411 Xylose disks Fermentation abilities 10 x 25 Disks

 

Listeria mono Differential Agar

Figure 6 Listeria mono Differential Agar (Agar Listeria Ottaviani & Agosti, 77408)

 

Schematic of biochemical identification for Listeria spp

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

Certified Reference Material

For testing the performance of the media and tests. Using Vitroids™ and LENTICULE® discs is time saving because it removes the need for preparing stock cultures. The organisms need no recovery time and no pre-enrichment step. In addition the product concentrations are designed in a range where no or only minimal dilutions are needed. The discs readily dissolve in liquid media and automatically on agar plates resulting in easy handling and a very economical solution. Utilization of new technology has allowed us to make major improvements in the field of Microbiological Reference Materials. The main areas of development are stability, temperature resistance, adjusting the narrow defined CFU range, rehydration time and better within batch reproducibility. In addition, each disc is produced under ISO 17034 (production of reference materials) and certified according to ISO/IEC 17025.

Vitroids™ and LENTICULE® discs

 

 

Cat. No. Description CFU Range (mean value) Package Size
CRM11288L Listeria innocua NCTC 11288 Lenticule® discs 30-120 10 vials
VT001093 Listeria monocytogenes 1/2a WDCM 00109 Vitroids™ 80-130 10 vials
CRM11994L Listeria monocytogenes NCTC 11994 Lenticule® discs 30-120 10 vials
CRM11994M Listeria monocytogenes NCTC 11994 Lenticule® discs 500-50000 10 vials

 

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)