Listeria Monocytogenes: Clinical Features and Identification

In this article we will discuss about:- 1. Introduction to Listeria Monocytogenes 2. The Organism of Listeria Monocytogenes and its Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.

Contents:

1. Introduction to Listeria Monocytogenes
2. The Organism of Listeria Monocytogenes and its Characteristics
3. Pathogenesis and Clinical Features of Listeria Monocytogenes
4. Isolation and Identification of Listeria Monocytogenes
5. Listeria Monocytogenes Association with Foods

---

1. Introduction to Listeria Monocytogenes:

Listeria Monocytogenes is the only important human pathogen among the seven species currently recognized within the genus Listeria, although L. seeligeri, L. welshimeri, and L. ivanovii have occasionally been associated with human illness.

It was first described by Murray in 1926 as Bacterium monocytogenes, the cause of an infection of laboratory rabbits where it was associated with peripheral blood monocytes as an intracellular pathogen, and it has since been established as both an animal and human pathogen.

As an important veterinary problem, it causes two main forms of disease: a meningoencephalitis most common in adult ruminants such as sheep and cattle, and a visceral form more common in monogastrics and young ruminants which attacks organs other than the braimzausing stillbirth, abortion and septicaemia. Listeriosis in sheep increased in Britain from 86 recorded incidents in 1979 to 423 in 1988.

This was partly due to the increased size of the national flock over that period but has also been attributed to changes in silage-making techniques towards greater use of big-bale ensilage. In big-bale ensilage, the silage is made in large plastic bags rather than in a single large clamp.

Rupture of the bag or inadequate sealing at its neck can allow mould growth to occur on the lactic acid present increasing the silage pH to a value at which Listeria Monocytogenes can flourish.

The recent widespread concern it has caused is largely attributable to the realization that food is a major source of the infection (a possibility first suggested as long ago as 1927), the psychrotrophic character of the organism, and the high mortality rate of the illness.

Reported incidence of human listeriosis increased in several countries during the 1980s, but remains generally low when compared to other foodborne infections such as salmonellosis. For example, in England and Wales reported cases of listeriosis peaked in the late 1980s at around 300 per year while reports of Salmonella and Campylobacter infections numbered nearly 27 500 and 29 000 respectively.

Reported cases of listeriosis dropped in 1990 and 1991 to 118 and 131 respectively; a decrease attributed to the effect of Department of Health advice to the immuno-compromised and pregnant to avoid soft cheeses and to reheat certain chilled foods adequately and to withdrawal of contaminated pate from a single manufacturer.

In the United States, the Center for Disease Control (CDC) has estimated an annual incidence of around 1700 cases resulting in 450 adult deaths and 100 foetal and postnatal deaths.

---

2. The Organism of Listeria Monocytogenes and its Characteristics:

Listeria Monocytogenes is a Gram-positive, facultatively anaerobic, catalase-positive, oxidase- negative, non-spore former. The coccoid to rod shaped cells (0.4-0.5 µm x 0.5-2.0 µm) cultured at 20-25 °C possess peritrichous flagella and exhibit a characteristic tumbling motility. Colonies on tryptose agar viewed under oblique illumination have a characteristic blue-green sheen.

Listeria Monocytogenes elaborates a 58 kDa β-haemolysin, listerolysin O, which acts synergistically with the haemolysin produced by Staphylococcus aureus to give enhanced haemolysis on blood agar.

This reaction forms the basis of a useful diagnostic test to distinguish Listeria Monocytogenes from L. innocua, and is known as the CAMP test after Christie, Atkins, and Munch-Peterson who first described the phenomenon with group B streptococci.

Listeria Monocytogenes will grow over a wide range of temperature from 0-42 °C with an optimum between 30 and 35 °C. Below about 5 °C growth is extremely slow with lag times of 1 to 33 days and generation times from 13 to more than 130 h being recorded.

The thermal survival characteristics of Listeria Monocytogenes have received considerable attention following an outbreak in the United States associated with pasteurized milk and the suggestion that the organism could survive commercial pasteurization conditions.

Despite some conflicting data in the literature, it appears that the heat resistance of Listeria Monocytogenes is similar to that of other non-spore forming Gram-positives with a typical D₆₀ of a few min and a D₇₀ of a fews.

It was proposed that Listeria Monocytogenes cells in contaminated milk are protected from heat by their intracellular location within milk leucocytes but subsequent studies have failed to demonstrate any significant effect.

Models of the thermal inactivation of Listeria Monocytogenes in milk have indicated that conventional HTST pasteurization achieves a reduction of 5.2 log cycles in the number of survivors; an acceptable safety margin assuming low numbers of the organism present on the incoming milk.

Growth of all strains is inhibited at pH values below 5.5 but the minimum growth pH is dependent on both strain and acidulant and has been variously reported as between 5.6 and 4.4. Listeria Monocytogenes is also quite salt tolerant being able to grow in 10% sodium chloride and survive for a year in 16% NaCl at pH 6.0.

The organism is ubiquitous in the environment. It has been isolated from fresh and salt water, soil, sewage sludge, decaying vegetation, and silage. Its prolonged survival in the environment has been demonstrated in one study where the level of Listeria Monocytogenes in sewage sludge sprayed on to agricultural land remained unchanged for more than 8 weeks.

Asymptomatic human and animal carriage is also common with reports of isolation of the organism from the faeces of, among others, cattle, pigs, sheep, chickens, turkeys, ducks, crustaceans, flies and ticks.

In a study of faecal carriage in human population groups, it was isolated from 4.8% of healthy slaughterhouse workers, 1.2% of hospitalized adults, 1% of patients with diarrhoea, and 26% of household contacts of listeriosis patients.

---

3. Pathogenesis and Clinical Features of Listeria Monocytogenes:

Its ubiquity in the environment suggests that human exposure to Listeria Monocytogenes must be frequent. Incidence of infection is however low since invasive infection will result only if a susceptible individual is exposed to a sufficiently high dose of a virulent strain.

Incubation periods for the disease have varied from 1 day to as long as 90 days with a typical incubation period of a few weeks; a situation which makes the identification of food vehicles difficult if not often impossible.

Symptoms of the disease, which is most likely to develop in pregnant women, the very young or elderly and the immunocompromised, can vary from a mild, flu-like illness to meningitis and meningoencephalitis.

In pregnant women, it most commonly features as an influenza-like illness with fever, headache and occasional gastrointestinal symptoms, but there may be an associated trans placental foetal infection which can result in abortion, stillbirth, or premature labour.

Listeriosis in the newborn can be an early-onset syndrome, which occurs at birth or shortly afterwards, or a late-onset disease appearing several days to weeks after birth. Early-onset illness results from in utero infection, possibly through the aspiration of infected amniotic fluid, and is characterized by pneumonia, septicaemia and widely disseminated granulomas (abscesses). Meningitis is rare.

In the late-onset syndrome, meningitis is more common, 93% (39 of 42) of late- onset cases in Britain between 1967 and 1985 had evidence of infection of the central nervous system. Infection may occur from the mother during passage through the birth canal, but some may also be acquired after delivery. A study in the UK found a lower mortality rate for late-onset disease (26%) than for early- onset listeriosis (38%).

Listeriosis in non-pregnant adults is usually characterized by septicaemia, meningitis and meningoencephalitis, but can also include endocarditis. It is particularly associated with those with an underlying condition which leads to suppression of their T cell mediated immunity, so that malignancies or immunosuppression (after renal transplantation, for example) are often predisposing factors.

Although not a common infection in AIDS patients, its incidence is around 300 times that in the general population. Other conditions such as alcoholism, diabetes and cirrhosis can act as predisposing factors, but illness does often occur in otherwise healthy individuals who only account for about 18% of adult cases in England and Wales.

Adult listeriosis has a high mortality rate, figures calculated using data for 1989 gave values around the world of between 13 and 34%. Early treatment with antibiotics, normally ampicillin, with or without an aminoglycoside, or chloramphe­nicol, is essential but in the most severe forms, the prognosis remains poor.

The pathogenesis of listeriosis is not well understood. Much of the information we have comes from studies in mice and it is not yet clear how readily this can be extrapolated to human cases. Listeria Monocytogenes is a facultative intracellular pathogen which like Mycobacterium, Brucella, and others can survive and multiply in cells of the monocyte-macrophage system.

The organism attaches to intestinal cells and induces its endocytosis; processes promoted by the virulence factor p60, a 60 kDa protein.

Once inside the phagocytic vacuole some protection from toxic superoxide anion is given by production of high levels of the enzymes superoxide dismutase and catalase, but in order to multiply intracellularly the organism must escape from the vacuole into the more conducive environment of the cytoplasm.

Production of the haemolysin, listeriolysin O, is essential to this process. Recovery follows inactivation of the listeria by macrophages activated by listeria-sensitized T cells.

---

4. Isolation and Identification of Listeria Monocytogenes:

Low-temperature enrichment at 4 °C is the traditional technique for isolating Listeria Monocytogenes from environmental samples, but the increased interest in routine isolation of the organism from foods has led to its replacement by more rapid, selective enrichment procedures based on antibiotic cocktails as selective agents and incubation at near-optimal growth temperatures.

Selective agars have likewise relied on a combination of selective agents such as lithium chloride, phenyl ethanol and glycine anhydride and antibiotics. Identification of presumptive Listeria colonies was based on microscopic examination of plates illuminated from below at an incident angle of 45° (Henry illumination), when they appear blue-grey to blue-green.

Some media avoid the use of this technique by incorporating aesculin and ferric ammonium citrate so that Listeria colonies appear dark brown or black as a result of their ability to hydrolyse aesculin.

Confirmation of Listeria Monocytogenes requires further biochemical testing including sugar-fermentation tests to distinguish it from other Listeria species and, in particular, the CAMP test to differentiate Listeria Monocytogenes from L. innocua.

Specific miniaturized test kits have been produced to simplify this procedure including one which replaces the CAMP test, which is not always easy for the inexperienced to interpret, with one for acrylamidase activity (Listeria Monocytogenes, negative; L. innocua, positive). Enzyme-linked immunosorbent assay (ELISA) and gene probe kits are also available.

Listeria Monocytogenes may be serotyped according to a scheme based on somatic and flagellar antigens. This is of limited epidemiological value since the majority of human cases of listeriosis are caused by just three of the thirteen serotypes identified (1/2a, 1 /2b, and 4b).

---

ADVERTISEMENTS:

5. Listeria Monocytogenes Association with Foods:

Its widespread distribution in the environment and its ability to grow on most non-acid foods offer Listeria Monocytogenes plenty of opportunity to enter the food chain and multiply. The transmission of listeriosis by food was first convincingly demonstrated in an outbreak that occurred in the Maritime Provinces of Canada in 1981.

The outbreak involved 41 cases in all. Of the 34 perinatal cases, there were 9 stillbirths, 23 neonatal cases with a mortality rate of 27%, and 2 live births of healthy infants. The mortality rate in adult cases was 28.6%.

Coleslaw was implicated as the result of a case control study and Listeria Monocytogenes serotype 4b (the outbreak strain) was isolated from a sample of coleslaw in a patient’s refrigerator. It was not possible to isolate the organism at the manufacturer’s plant but it transpired that a farmer who supplied cabbages to the manufacturer also kept sheep, two of whom had died of listeriosis.

The cabbage had been grown in fields fertilized by fresh and composted manure from the sheep and the harvested cabbages had been stored in a large shed through the winter – factors thought to account for the introduction of the organism and its multiplication to dangerous levels.

Raw vegetables, in the form of a garnish containing celery, tomatoes and lettuce, were also implicated on epidemiological grounds in an outbreak that occurred in eight Boston hospitals in 1979.

Surveys in the UK, the United States, Australia and elsewhere have reported a high frequency of isolation of Listeria Monocytogenes from meats and meat products, where serotype 1 generally predominates. A number of sporadic cases of listeriosis have been associated with products such as pork sausage, turkey frankfurters, cook-chill chicken, and chicken nuggets.

Listeria Monocytogenes is relatively resistant to curing ingredients and has been found in a range of delicatessen meats such as salami, ham, corned beef, brawn, and pate.

In an Australian survey 13.2% of samples were found to be positive, largely as a result of cross-contamination in the shop. In Britain in 1989/90, high levels on vacuum- packed ham and on pate, from which serotype 4b was isolated, prompted the recall of both products from the market.

Dairy products such as raw and pasteurized milk and soft cheeses have been associated with a number of major outbreaks of listeriosis. The overall incidence of Listeria Monocytogenes in raw milk derived from surveys in Australasia, Europe and the United States averages at around 2.2%, although one Spanish study reported an incidence in excess of 45%.

Pasteurized milk was responsible for an outbreak in Massachusetts in 1983 involving 42 adult and 7 perinatal cases with an overall mortality rate of 29%. The milk had come from farms where bovine listeriosis is known to have occurred at the time of the outbreak.

It was the absence of evidence of improper pasteurization at the dairy that gave rise to the concern that Listeria Monocytogenes might display marked heat resistance in some instances.

Soft cheeses are also frequently contaminated with Listeria Monocytogenes. In 1985 there was an outbreak in California in which a Mexican-style soft cheese which had been contaminated with raw milk was the vehicle. One hundred and forty-two cases were recorded comprising 93 perinatal and 49 adult cases with an overall mortality rate of 34%.

This outbreak served to focus attention on soft cheeses and there have since been other incidents identified in which they have been implicated, including a major outbreak covering the period 1983-87 with 122 cases and 31 deaths associated with the Swiss cheese Vacherin Mont d’Or.

This association with soft cheeses appears to be due to the cheese ripening process. Listeria Monocytogenes survives poorly in un-ripened soft cheeses such as cottage cheese but well in products such a Camembert and Brie. During the ripening process, microbial utilization of lactate and release of amines increase the surface pH allowing Listeria to multiply to dangerous levels.