Listeria

HISTORY:

Listeria monocytogenes, type species of the genus Listeria (named after the English surgeon Lord Lister) was described in 1926 by Murray; The bacterium was isolated during an epizootic reaching rabbits and guinea pigs showed strong increase in circulating monocytes and hepatic necrosis lesions. This bacterium was then isolated from various domestic and wild animals.

In humans, it was originally isolated during a meningitis in adults and in various pathological conditions until 1933 Burn shows its role in infection in the neonatal period. Seeliger’s work has subsequently emphasized the important role of L. monocytogenes in human pathology.

I – GENERAL CHARACTERISTICS OF GENRE:

The genus Listeria comprises small Gram positive bacilli regular shape, short, sometimes curved to rounded tips (0.4-0.5 x 0.5-2 pm) singly or in short chains with a palisade arrangement and letters as corynebacteria with sometimes filamentous forms (6-20 microns) in old cultures. They are non-spore forming, non-encapsulated and mobile at 20-25 ° C by peritrichous flagella. These are airports facultative anaerobic bacteria. The optimal cultivation temperature is between 30 and 37 ° C, but the cultivation can be 1 to 45 ° C.

On blood agar they give hemolytic colonies smooth, translucent, gray-blue (pathogenic species).

They have the following characteristics: catalase positive, oxidase negative; glucose fermented with lactic acid production; esculin and hippurate hydrolysis, methyl red positive reactions and Voges-Proskauer; no production of indole, or HS, no hydrolysis of urea. There antigenic types defined by antigens and H antigens 0

II – POSITION TAXONOMY AND NOMENCLATURE:

The taxonomic position of the genus Listeria is not definitively established. This is a distinct genus unrelated Corynebacteria, but closer to the data of the numerical taxonomy, serology, biochemistry and the study of nucleic acids of Bacillus, Erysipelothrix, Lactobacillus and Streptococcus ( Bergey’s Manual of Systematic Bacteriology, Vol. 2, 1986). Recent taxonomic studies, biochemical and DNA-DNA hybridization have clarified the role of different species in the genus, which has been enriched with new species.

The Listeria genus includes species pathogenic to humans or animals and non-pathogenic species. L.monocytogenes is the only species pathogenic for both man and animals.

The other species are: L. ivanovii (eg 5 serovar L. mocytogenes, pathogenic for animals but rarely found in humans);L. innocua (nonpathogenic) L. welshimeri (nonpathogenic) L. seeligeri (nonpathogenic).

Two species L. grayi (isolated in the chinchilla) and L. murrayi (in the soil) of uncertain classification, have been proposed to form the kind “Murraya” with two species “Mr. grayi subsp. grayi “and” Mr. grayi subsp. murrayi “.

The species L. denitrificans is excluded from the genus Listeria to be transferred to the Jonesia group.

III – HABITAT AND EPIDEMIOLOGY:

L. monocytogenes is a ubiquitous saprophyte bacteria. Widespread in nature, it has been isolated from soil, water, plants (silage) but also in milk, chicken meat, vegetables (cabbage) and in the feces of healthy subjects (man and Many animal species). The bacterium survives in the natural environment, resists harsh conditions and can multiply there even at low temperatures (property used as enrichment method).

Infection with L. monocytogenes and listeriosis common disease to humans and animals, is a saprozoonose. Human contamination is most often performed by the digestive tract and occasionally ocular, respiratory or skin.

The contamination is rarely direct contact with infected animals and then concerns the exposed (farmers, veterinarians); cases of human transmission in hospitals (maternity) were sometimes described as small outbreaks.

Indirect contamination is the most common mode of transmission.

Man comes into contact with the bacteria present in the environment (soil, water, animal excretions) or in contaminated food of animal origin (milk, meat, poultry, meats, cheeses …) or vegetable (crudités , cabbage, …).

Cases of human listeriosis usually occur as sporadic cases with seasonal variations. When contamination by ingestion of contaminated food, real epidemics were observed.

This was the case in Anjou (156 cases between 1975 and 1978), Nova Scotia – due to coleslaw – (41 cases), Boston (49 cases) following ingestion of pasteurized milk; of soft cheeses were responsible for the epidemic in Los Angeles in 1985 (142 cases) or that of Switzerland in 1987 (122 cases). Note that L. monocytogenes was isolated from 4% of samples of raw milk and cheese, 30% of samples of meat products, 26% of seafood or 5% of prepackaged salads and vegetables. Counts can reach 100 to 1000 CFU / g of meat and even 100 000 10 000 000 CFU / g cheese!

Human disease can be observed at all ages but with a distribution of cases by age groups, which shows three peaks: the first peak occurs before the age of 1 year and corresponds to the neo listeriosis home; a peak between 20 and 40 years, which corresponds mainly to cases of maternal-fetal infection Matemo; finally a third peak between 60 and 80 years, highlighting the role of the field.

Animal disease is more common during cold seasons (winter, early spring). In human cases appear more frequent in autumn and spring.

IV – PATHOGENICITY NATURAL:

L. monocytogenes is a pathogenic bacterium to humans and many animal species.

A – In the pet:

This bacterium is responsible for different clinical manifestations occurring in winter and spring: abortion and stillbirth in cattle and sheep; septicemia lamb before weaning; encephalitis in different ruminant species; purulent conjunctivitis and keratitis; mastitis. Some of these locations promote human contamination.

B – In men:

The number of cases of listeriosis reported has increased significantly since the 1960s Is this a better knowledge of this bacterial species or a change in the ecological conditions that favored its spread and increased contact with humans?

In France, the impact would be of the order of 11-15 cases per million inhabitants.

1. The maternal-fetal listeriosis:

Neonatal listeriosis can exist in two major clinical forms:

– Early infection manifesting in the first days of life (early hours, first 5 days). This form corresponds to a generalized infection sepsis occurred before birth.

The infection in an infant born prematurely or low weight manifests itself in a serious condition with generalized septicemic forms, with or without meningeal damage, lung infections or local conjunctival being rarer.

The major form is associated with multiple granulomatous disease outbreaks (hence the name: Granulomatosis infantiseptica) Mortality is important..

– Late-onset meningitis is a form comparable to that of the adult whose prognosis is less grim.

Pregnant women the disease has been mild or discrete, isolated fever or flu-like symptoms go unnoticed. When it occurs in early pregnancy, it causes an abortion; a later infection causes premature delivery.

2. Listeriosis in adults:

Forms of adults usually concern, but not exclusively, subjects with impaired immunity, especially in cellular immunity, in hematological malignancies, cancers, or during immunosuppressive therapy, in diabetes and cirrhosis, as well as during pregnancy. L. monocytogenes in these cases may be regarded as an opportunistic bacterium.

This is most often neuro-meningeal forms: meningitis, meningoencephalitis or encephalitis. The appearance of the cerebrospinal fluid is highly variable and can be misleading: cloudy with a variegated form with cytology usually moderate (between 100 and 500 parts per mm9), but also clear liquid resembling a viral meningitis or tuberculous meningitis justifying cultivation of all CSF and blood cultures systematic practice. The septicemic forms are less frequent with or without metastases. There are also rare localized forms: eye, skin, bone, lung encountered … especially in immunocompromised patients, or for cutaneous forms without systemic involvement among people in contact with infected animals.

The mortality of listeriosis in France is about 30%, it was the same in the recent outbreaks observed in the world.

V – Pathophysiology – FACTORS VIRULENCE:

L. monocytogenes is a bacterium in intracellular multiplication and virulence is related to its ability to multiply in macrophages. The bacterium produces a hemolysin, extracellular protein toxin having antigenic relationship with streptolysin 0. The hemolysin binds to cholesterol and is responsible for in vitro cytotoxic activity on various eukaryotic cells in culture and in vivo system lesions reticuloendothelial and lethal to laboratory animals. The role of hemolysin in virulence of L. monocytogenes was suspected because of the lack of virulence of the nonhemolytic strains.

Recent studies have shown that the hemolysin and listeriolysin is a major virulence factor involved in the intracellular growth of L. monocytogenes.

Hemolysin production in vacuoles after phagocytosis causes the destruction of cell membranes, the lysosomes, the vacuole membrane resulting in the release of iron whose presence stimulates bacterial growth. Other products may play a role in virulence as phospholipase C and superoxide dismutase activity. The intemaline is a protein expressed on the surface of the bacterium which promotes its phagocytosis.

To date, only two species: L. monocytogenes and L. ivanovii are naturally (L.ivanovii for animals) and experimentally pathogens.

VI – BACTERIOLOGICAL DIAGNOSIS:

A – pathological products:

The sampling nature varies according to clinical disease and age of the patient.

In the newborn the bacteria can be sought in the gastric fluid, meconium, CSF, blood sampling devices; maternal concern withdrawals placenta, lochia, amniotic fluid.

Blood culture is indicated when unexplained infection in pregnant women. In other cases, research is carried out at the blood, CSF, skin lesions. Search in feces can detect carriers.

Immediate culturing is desirable, but if necessary conservation is possible at room temperature or at 4 ° C due to the resistance of the bacteria. Incubation at 4 ° C can be used to enrich the medium in Listeria (saddles, epidemiological surveys).

Rapid diagnostic attempts search of soluble antigens were conducted by immunoelectrophoresis against or ELISA;the results are still disappointing: ELISA for the type 4b if the specificity was 100%, the sensitivity is insufficient because the antigen is detected in only a quarter of the cases.

B – Direct examination:

L. monocytogenes is a small gram-positive bacillus 0.5 x 1-4 ​​microns. In pathological products, L. monocytogenes is usually as short bacillus and forms exist coccobacillary short chains. In the cerebrospinal fluid where the bacteria are usually scarce L. monocytogenes is intra- or extracellular. Confusion risks are possible on direct examination but should be avoided (the most common errors are: Corynebacterium, Streptococcus, Haemophilus even if the discoloration was too large).

After liquid culture the bacteria can be longer and arranged in palisade.

C – cropping characters:

L. monocytogenes grows on usual media; Growth is promoted by the presence of glucose (0.1%) serum (1%) or of blood (5%). The optimum pH is 7 to 7.4. Growth is achieved by aerobic or microaerophilic. The optimal cultivation temperature is between 30 and 37 ° C, the growth temperature limit is 1 ° C and 45 ° C. A + 4 ° C, L. monocytogeneshas the property of ultiplier faster than many species and this property is used as an enrichment method for contaminated or multi-microbial products (method of Gray). Culture is possible hostile environments (high salt, bile, potassium tellurite) on MacConkey agar, but not in the presence of sodium azide.

On nutrient agar (tryptose agar), after 18 hours at 37 ° C, colonies are small, rounded, translucent bluish gray. They have a blue-green iridescence characteristic at an oblique light examination (for identifying colonies in culture polymicrobial products).

On blood agar (sheep, horse, rabbit), the colonies have the same appearance and are surrounded by a narrow zone of hemolysis sometimes only barely above the edge of the colony and made visible by moving the colony. CAMP-test, used for group B streptococci, allows the study of Listeria strains with low or questionable hemolysis;accentuation of hemolysis of S. aureus was observed for L. monocytogenes and L. seeligeri and that ofRhodococcus equi for L. ivanovii (see Table I). L. ivanovii usually produces a wide zone or multiple zones of hemolysis. Non-hemolytic strains are considered non-pathogenic.

Selective media have been proposed for culture from the environment or multi-microbial products; they contain antibiotics such as colistin or nalidixic acid (40 mg / 1).

Different Listeria species are motile bacteria. Mobility is expressed at 25 ° C, but very low or zero at 37 ° C. It is pursued in liquid medium (flips and rotations on microscopic examination) or agar (umbrella away from the surface) after culture at 25 ° C.

D – Identification Characters and experimental pathogenicity:

The main characteristics of L. monocytogenes identification are the morphology of the bacteria and the appearance of the colonies; mobility at 25 ° C; catalase positive; hydrolysis of esculin (fast positive feedback, resulting in some important hours for rapid diagnosis of suspected); Acidification of glucose and salicin, rhamnose; no acidification mannitol, xylose and arabinose (Table I).

The enzyme electrophoretic analysis showed that epidemics were well due to the same clone. Furthermore, a probe corresponding to the DNA fragment encoding the p hemolysin gene appeared to be strictly specific for L.monocytogenes (Datta).

L. monocytogenes and L. ivanovii are pathogenic to the mouse intraperitoneally.

The Anton test can identify pathogenic strains of L. monocytogenes.

The introduction into the conjunctival sac of the rabbit eye or guinea pig 2-3 drops of bacterial suspension (prepared from a culture of 18 hours, 3 drops into 5 ml distilled water) causes purulent conjunctivitis in 2 to 5 days cured by local administration of ampicillin.

E – Differential diagnosis:

The differential diagnosis of different Listeria species are presented in Table I.

The principal characteristics to differentiate Listeria related species are presented in Table II. These characteristics relate to gram-positive bacteria (excluding morphology). It should be considered in the differential diagnosis of isolated circumstances. The most frequent confusions are those with a streptococcus, a corynebacterium possiblyHaemophilus.

F – Classification: serotypes, phage types:

There exist Ag 0 and Ag H for defining serovars. One serovar may be found in two different species (see Table 1 and bacteriological diagnosis).

Somatic antigens (Ag 15 0: 1 to XV) and flageiïaires (5 Ag H: A-E) are used to define 16 serovars or serotypes (Paterson, Seeliger, Donker-Voet) in the genus Listeria These serovars presented in. Table III, are: serovar 1/2 (a, b and c); serotype 3 (a, b and c); serovar 4 (a, ab, b, c, d, e, f and g); serovars 5, 6 and 7. With the exception of serotype 5 strains belonging to the species L. ivanovii, there is no correlation between the species and serovar. L. speciesgrayi and L. murrayi are related to each other but have no kinship with other species of the genus Listeria, also is it proposed to classify them into a new genre: Murraya.

Serovars l / 2a, l / 2b and 4b above (65 to 70% of strains) are most often encountered in France in humans. These same serotypes were found in animals and other serovars rarer.

In practice, the typing additional isolation and identification is performed by slide agglutination with specific antisera.

Phage typing of L. monocytogenes is possible with lots of phages that allow typing 54% of serovar 1/2 strains and 77% of strains of serovar 4. phages specific lytic activity serovar whose determination must precede any phage.Phage typing, expanded to five species of Listeria with new phages, did not establish geographic phagovar serovar-origin-host relationship.

G – Indirect diagnosis:

Looking for antibodies against the antigens 0 and H types 1 and 4b is possible by slow tube agglutination. The reaction is similar to that used for the Salmonella: 0 agglutination granular agglutination H flaky.

The interpretation of the results should always consider the clinical and epidemiological context because there are antigenic communities between Listeria, Staphylococcus and Enterococcus. The rise in agglutinating antibodies is often late and even a high (> 1/320) was not significant, high levels being found in patients without a history of listeriosis.

More encouraging results have been obtained by measuring antibodies against listeriolysin 0 (OLA), although this enzyme has a cross antigenicity with streptolysin 0, pneumolysin and perfringolysin; in the experience of Berche: by dot blot titration, anti LLO antibodies are found in some capacity> 1/100 in 96% of patients and 12 to 16% of controls.

Despite recent progress, the isolation of Listeria remains the best means of diagnosis.

VII – OTHER SPECIES:

The other Listeria species are also (except L. grayi isolated from the single chinchilla) widely distributed in nature. L.ivanovii is hemolytic pathogenic in animals (sheep) and was rarely seen in humans.

L. innocua and L. welshimeri are haemolytic and have no known pathogenicity. L. seeligeri is weakly hemolytic and devoid of experimental pathogenicity, but a case of meningitis in a human subject without immune deficiency has been described. These species are shown in Table I.

VIII – TREATMENT AND PREVENTION:

L. monocytogenes is a species susceptible to the antibiotics effective against Gram-positive bacteria. The main active antibiotics are beta-lactam antibiotics with good activity of penicillins, especially ampicillin, and poor activity in particular those of third-generation cephalosporins. Are also active, aminoglycosides (gentamicin and tobramycin above), tetracyclines, erythromycin, chloramphenicol. Second generation quinolones have reduced activity on L. . monocytogenes resistors were encountered vis-à-vis penicillin, erythromycin, rifampicin and sulfamethoxazole – trimethoprim.

The treatment of choice is ampicillin + aminoglycoside which allows for the best in vitro bactericidal effect.

There is no vaccine to confer protection both in humans and in animals. As prevention is mainly based on individual measures to direct contamination and monitoring of food quality, all measures difficult to observe because of the distribution of the bacteria in nature and strength.

It is recommended that pregnant women and immunocompromised avoid the ingestion of raw vegetables, raw milk or improperly pasteurized and fresh cheese or soft cheese.