MICROBIOLOGICAL QUALITY AND SAFETY IN DAIRY INDUSTRY

Lesson 26. ENUMERATION OF LISTERIA MONOCYTOGENES

Module 5. Microbiological quality and safety evaluation of milk and milk products

Lesson 26
ENUMERATION OF LISTERIA MONOCYTOGENES

26.1 Introduction

Majority of illnesses and death each year affecting millions of people throughout the world are due to foodborne diseases. Pathogenic bacillus Listeria monocytogenes not only causes a severe human foodborne disease, but also has been linked to infections in more than 20 different animal species. Animal listeriosis has particularly been reported in sheep cattle, goats, and; symptoms include meningitis, abortions, and septicaemia as well as, less commonly, non-systemic infections such as uveitis and mastitis. Human listeriosis outbreaks have been traced to a variety of different dairy products (including pasteurized milk, butter, chocolate milk, Hispanic style cheeses) have been reported. According to the USDA/ FDA Listeria risk assessment, Hispanic style fresh cheeses represent a particular high risk food for acquiring listeriosis. Ability to survive outside a mammalian host and under a variety of stress conditions makes L. monocytogenes strains a particular concern for the dairy industry. Listeria monocytogenes is an intracellular pathogen and affects healthy as well as immuno-suppressed populations. In healthy individuals, this organism can cause gastroenteritis and fever. Overall, mortality rate due to infection is about 20–30%, with an annual death rate of about 2500 people. The infective dose for this pathogen is not known; however, it is estimated to be about 100 to 106 cells depending on the immunological status of the host. It causes three forms of disease gastrointestinal form, systemic listeriosis, and abortion and neonatal listeriosis. While L. monocytogenes has been found in raw milk, current pasteurization time-temperature combinations effectively inactivate L. monocytogenes. Post-processing contamination from plant environments probably represents the most common source of L. monocytogenes contamination of pasteurized dairy products.

26.2 Some Characteristics of the Genus Listeria

All the seven species in the Gram-positive non-sporing genus Listeria are rods about 0.5 μm × 0.5-2.0 μm, produce catalase and are positive in the methyl red and Voges-Proskauer reactions. They are indole, oxidase, H2S, urease, nitratase and gelatinase negative. The non-capsular facultative anaerobic Listeria spp. grows well at water activity (aw) of >0.95 but can multiply at aw of 0.90. They also grow well at 10% salt concentration but survive at 25.5%. L. monocytogenes can tolerate lower pH when kept at near refrigeration temperatures. Two species L. monocytogenes and L. ivanovii are clearly ß-haemolytic and this ability has been linked to their pathogenicity. Some reactions useful in differentiating the pathogenic Listeria species are listed in Table 26.1.

Table 26.1 Differentiation between the two important pathogenic Listeria species - L. monocytogenes and L. ivanovii.

Note

^a Sheep blood agar stab.
^b Mouse test.
^c Ribose fermenting strains are classified as L. ivanovii subsp. ivanovii and ribose non-fermenters as L. ivanovii subsp. londiniensis.

The CAMP test is considered by many to be the most important test for confirming pathogenic species. This test is performed by streaking a ß-haemolytic Staphylococcus aureus and a Rhodococcus equi culture in parallel and diametrically opposite each other on a sheep blood agar plate. Listeria test cultures are then streaked at right angles to the two other cultures. After 48 hours of incubation at 35-37°C, ß-haemolysis by L. monocytogenes and L. seeligeri is enhanced near the S. aureus streak, whereas for L. ivanovii it becomes enhanced near the R. equi streak. The other Listeria species remain non-haemolytic.

26.3 Isolation and Enumeration Principle of Listeria Species from Dairy Products

Although Listeria grows rapidly on ordinary bacteriological media, direct isolation of the organism from infected and contaminated material is often unsuccessful. Difficulties in isolating Listeria date back to 1926 when L. monocytogenes was first described by Murray et al. Even when the presence of the pathogen had been established, the re-isolation of the organism by direct plating on nutritious agars frequently failed. It was soon clear that the reliability of any isolation method for Listeria intended for food analysis would depend on its ability to cope with low-level contamination (<10² cells per ml), among samples containing high-level competitive microflora, coupled with the ability to recover sub-lethally injured cells.

26.3.1 Enumeration principle

1. Enumeration of Listeria spp. is based on the principle that the organism show tolerance to the selective agent used in the isolation procedure listed in Table 26.2.
2. Ability to hydrolyse esculin by the enzyme ß-glucosidase
3. Organism show weak ß-haemolysis
4. Ability to produce phosphatidylinositol-specific phospholipase-C (PI-PLC)
5. Ability to ferment rhamnose

So based on various formulation and combination different chromogenic and fluorogenic culture media have been developed.

26.4 Conventional Method for Isolation of Listeria spp. From Dairy Products

26.4.1 Cold enrichment method

For many years the only method available was the cold enrichment technique first advocated by Gray et al. (1948) in which sample material was inoculated into a tryptose broth without selective agents and held at 4°C for long periods and has been considered a standard method for nearly 40 years. Cold enrichment is still an excellent isolation technique; however the need for prolonged incubation (up to several months or even a year) is a serious disadvantage. As shown in Figure 26.1, numerous methods have been developed over the years for isolating and detecting Listeria in food, clinical and environmental samples. Three general types can be distinguished:

1) Direct plating
2) Enrichment followed by plating
3) Enrichment followed by a rapid method

Fig. 26.1 (2) Scheme for isolation and detection methods of Listeria. The size of arrows denotes an estimated popularity of various methods

The first and least used is the direct plating of a food suspension onto a selective solid agar medium such as the LiCl-phenylethanol-moxalactam (LPM), PALCAM containing Polymyxin B, Acriflavin, Lithium chloride, Ceftazidime, phenylethanol, moxalactam medium with or without esculin and modified Oxford (MX) agar. Direct plating offers the advantage of allowing the analyst to quantify the populations of the listeriae cells in the food directly. Unfortunately, direct plating is rather insensitive and can detect only 100 CFU of Listeria per gram food. Thus, for smaller numbers of listeriae, some type of enrichment procedure must be employed, in order to resuscitate injured listeriae cells and to increase their numbers relative the background flora. The most widely used approach for isolating and detecting Listeria spp. uses one or two warm enrichment steps in nutrious broth, followed by plating onto a selective agar. Warm enrichment is based on the accelerated growth of listeriae at increased incubation temperature (30-37°C), with the addition of selective compounds to inhibit growth of competitive microflora. Table-26.2 summarise the most commonly used selective agent for isolation of Listeria spp.

Although the analysis time for these methods is dramatically reduced when compared to cold enrichment/ plating methods (3-7 days versus several weeks), they are still too slow and laborious to be suited for routine applications. This has created a tremendous stimulus for developing alternative so-called rapid methods. Generally, these utilize a simplified warm enrichment procedure in combination with the latest developments in chromogenic and fluorogenic substrates, DNA and immuno-technology to reduce isolation and identifications times.

Table 26.2 Example of antimicrobial agents that are commonly used in Listeria selective broth and agar media

26.4.2 Procedures for the isolation of listeria spp. from dairy products

The FDA method employs a single enrichment step using FDA enrichment broth at 30°C.32 After 24 and 48 h the culture is streaked onto selective LiCl-phenylethanol-moxalactam (LPM) and Oxford/ PALCAM agars. The plates are then incubated at 30/35°C (LPM/ Oxford/ PALCAM) for 24-48 hours. Suspected plates are examined by using beamed white light striking the plate bottom at 45° angle. This technique is known as Henry’s Light technique and shows Listeria colonies as sparkling blue or white. In the PALCAM agar, which contains the esculin/ ferric iron indicator system, Listeria colonies show up as black with a black halo under ordinary viewing and appears as light green colonies. Five or more typical Listeria colonies are picked and transferred to trypticase soy agar and incubated at 30 °C for 24-48 hours. Further identification is carried out by conventional morphological examination and biochemical tests.

The USDA method is a two-stage enrichment procedure involving primary enrichment in Listeria enrichment broth I (LEB I), secondary enrichment in Fraser broth and subsequent plating on modified Oxford agar (MXA). Typical Listeria colonies exhibit black halos from esculin hydrolysis. The ISO/ AFNOR method is similar to the USDA method, but uses 1/2 Fraser broth (with half the amount of selective agents) as the first enrichment step and complete Fraser broth as a second enrichment step. Both steps are followed by selective plating on PALCAM and/ or Oxford agars. Typical colonies on the Oxford agar show up as black due to esculin hydrolysis. The PALCAM agar contains two indicator systems: esculin/ ferric iron and D-mannitol with phenol red. This double diagnostic system allows for more easy distinction of Listeria from possible contaminating enterococci and staphylococci. On PALCAM agar Listeria colonies are green with black haloes against a pink-purple background. A summary of the ISO/ AFNOR method is presented in Figure 26.2.

26.4.3 Rapid enumeration of listeria spp. using chromogenic media

Chromogenic and fluorogenic media are microbiological growth media that contain enzyme substrates linked to a chromogen (color reaction), fluorogen (light reaction) or a combination of both. The enzyme substrates, e.g. 5-Bromo-4-chloro3-indolyl-β-D-glucoside, 5-Bromo-4-chloro-3-indoxyl myo-inositol-1 phosphate, ammonium salt, 4-Methylumbelliferyl myo-inositol-1-phosphate N-methyl-morpholine salt, are most commonly used chromogenic and fluorogenic substrate for detection of L. monocytogenes. The target population are characterized by enzyme systems that metabolize the substrate to release the chromogen/ fluorogen. This results in a colour change in the medium and/ or fluorescence under long wave UV light

Fig. 26.2 Conventional enrichment method for the isolation and detection of Listeria according to the NF ISO 11290-1/ AFNOR standard

26.4.4 PALCAM agar base

26.4.4.1 Enumeration principle and interpretation

Differentiation on PALCAM Agar Base is based on esculin hydrolysis and mannitol fermentation. Listeria spp. hydrolyzes esculin, which appears as blackening in the medium. The blackening by esculin-hydrolyzing bacteria results from the formation of 6,7-dihydroxycoumarin, which reacts with ferric ions that are present in the medium as ferric ammonium citrate. Mannitol and the pH indicator, phenol red, have been added to differentiate mannitol-fermenting strains of possible contaminants, including enterococci and staphylococci. Listeria spp do not ferment mannitol, which is demonstrated by a color change in the colony and/or the surrounding medium from red or grey to yellow based on the production of acidic end products. Polymyixin B, acriflavin, ceftazidime, and lithium chloride are selective agents used to suppress Gram-negative and certain Gram-positive bacteria.
Reaction catalysed by the enzyme beta-glucosidase which results in the formation of black color compound for detection of Listeria species

26.4.4.1.1 Colony characteristics of listeria species on PALCAM agar

Listeria spp. are presumptively indicated by grey-green colonies with a black precipitate following incubation for 24 - 48 hours at 35°C on PALCAM Agar Base

Fig. 26.3 PALCAM agar

26.4.5 Chromocult listeria selective agar base

26.4.5.1 Enumeration principle and interpretation

The rich basis of Chromocult Listeria Selective Agar is the addition of 5-bromo-4-chloro-3-indolyl-ß-D-glucopyranoside which makes it possible to differentiate between ß-D-glucosidase positive and negative bacteria. Listeria is ß-D-glucosidase-positive and grows on the medium in the form of blue-green colonies. The addition of inhibitors results in a marked reduction in the growth of the majority of concomitant Gram-positive and Gram-negative pathogens, as well as of yeasts and fungi. To detect L. monocytogenes L-ß - phosphatidylinositol is added to the medium. L. monocytogenes has the enzyme phosphatidylinositol phospholipase C (PI-PLC) described as a virulence factor. This phospholipase activity results in the formation of opaque haloes around L. monocytogenes colonies. Apart from L. monocytogenes, only L. ivanovii among the listeriae shows phospholipase C activity.

Beta-glucosidase forms indigo blur color by hydrolysing chromogenic substrate

26.4.5.1.1 Colony characteristics chromocult listeria selective agar base

All colonies which appear blue-green with an opaque halo on the medium are counted as suspect L. monocytogenes colonies (typical colonies) (Fig. 26.4).

Fig. 26.4 Colony characteristics of Listeria spp. on chromocult listeria selective agar base

26.4.6 HiCrome listeria agar base, modified

26.4.6.1 Enumeration principle and interpretation

HiCrome Listeria Agar Base, allows growth of only Listeria species and gives a specific and direct identification of L. monocytogenes within 24-48 hours after pre-enrichment. This medium is based on both, the specific beta-glucosidase activity detection and the rhamnose fermentation. The colonies of L. ivanovii appear blue without a yellow halo (rhamnose -ve) while the colonies of L. monocytogenes and L. innocua are blue with a yellow halo (rhamnose +ve). Peptone, yeast extract and meat extract provide nitrogenous substances, vitamin B complex and other essential growth nutrients. Rhamnose is the fermentable carbohydrate with phenol red as an indicator. Sodium chloride maintains the osmotic equilibrium. The added lithium chloride and Hi-Crome Listeria Selective Supplement inhibit growth of most Gram positive bacteria, Gram negative bacteria, yeasts and moulds.

26.4.6.1.1 Colony characteristics

The colonies of L. ivanovii appear blue without a yellow halo (rhamnose -ve) while the colonies of L. monocytogenes and L. innocua are blue with a yellow halo (rhamnose +ve) (Fig. 26.5).

Fig. 26.5 Listeria species on hiCrome listeria agar base

26.4.7 ALOA chromogenic agar

26.4.7.1 Enumeration principle and interpretation

To minimise the growth of contaminating organisms, lithium chloride and a balanced antimicrobial mixture is employed. The incorporation of the chromogenic substrate X-glucoside for the detection of β-glucosidase demonstrates the presence of Listeria spp., whilst the detection of a specific phospholipase C enzyme produced by pathogenic Listeria spp. including L. monocytogenes is also achieved. Listeria spp. grow on this medium producing blue/ green colonies, with pathogenic species producing similar coloured colonies surrounded by a characteristic opaque halo after 24 hours incubation at 37^oC. Non-Listeria spp. produces white colonies.

26.4.7.1.1 Colony characteristics

All Listeria spp. Examined produced typical green – blue coloured colonies 1.0 – 2.0 mm in diameter after 24 hours incubation, with all L. monocytogenes producing a distinctive opaque halo. Strains of L. ivanovii also showed an opaque halo.

Fig. 26.6 Listeria species on ALOA chromogenic agar

26.4.8 3M™ petrifilm™ environmental listeria plates

The 3M Petrifilm Environmental Listeria Plate is a sample-ready culture medium used for the detection and/or enumeration of Listeria in environmental samples. Petrifilm Environmental Listeria Plate detects the following Listeria species as red-violet colonies: Listeria monocytogenes, Listeria innocua and Listeria welshimeri, but does not differentiate these organisms from one another. A repair broth (5 mL of buffered peptone water) is added to the collected environmental sample and allowed to remain at room temperature for 1 hour. This short repair step is not an enrichment step. Three millilitres of the sample is pipetted onto the 3M Petrifilm Environmental Listeria Plate and the plate is then incubated for 26-30 hours. The 3M Petrifilm Environmental Listeria Plate methods may be used as a qualitative, quantitative or semi-quantitative test:

• A qualitative interpretation may be desired if a yes/ no answer is sufficient and appropriate
• A quantitative interpretation may be desired if different actions will be taken based upon the number present
• A semi-quantitative interpretation may be desired if different actions will be taken depending on the relative level present, and/ or if recording actual numbers is not desirable.

26.4.8.1 Colony characteristics

Following Listeria species as red-violet colonies: Listeria monocytogenes, Listeria innocua and Listeria welshimeri

Fig 26.7 Colony characteristics of listeria species on 3M™ petrifilm™

26.5 Enumeration Procedure