The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L. monocytogenes and L. innocua. The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution. Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L. monocytogenes and L. ivanovii. Isolates of L. monocytogenes with atypical properties require tools of molecular biology for final identification. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria. Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.
Listeria monocytogenes, the causative agent of listeriosis, was discovered more than 70 years ago by E.G.D. Murray and J. Pirie, who were working independently of each other . The first documented human listeriosis case involved a soldier who suffered from meningitis at the end of World War I . Since then, listeriosis has emerged as an atypical foodborne illness of major public health concern because of the severity of the disease (meningitis, septicemia, and abortion), the high case fatality rate (20–30% of cases), the long incubation time, and the predilection for individuals who have an underlying condition which leads to impairment of T-cell-mediated immunity.
The genus Listeria belongs to the Clostridium subbranch, together with Staphylococcus, Streptococcus, Lactobacillus, and Bronchothrix. This phylogenetic position of Listeria is consistent with its low G+C DNA content (36–42%).
L. monocytogenes is one of six species in the genus Listeria. The other species are L. seeligeri, L. ivanovii, L. innocua, L. welshimeri, and L. grayi. Two subspecies of L. ivanovii have been describes: L. ivanovii subsp. ivanovii and L. ivanovii subsp. londoniensis. L. murrayi, which was a separate species in the genus Listeria, is now included in the species L. grayi. Based on results of DNA–DNA hybridization, multilocus enzyme analysis, and 16S rRNA sequencing, the six species in the genus Listeria are divided on two lines of descent: (i) L. monocytogenes and its closely related species, L. innocua, L. ivanovii, L. welshimeri, and L. seeligeri, and (ii) L. grayi. All of these species are widespread in the environment, but only L. monocytogenes is considered to be a significant human and animal pathogen. However, occasional human infections due to L. ivanovii and L. seeligeri have also been reported . L. ivanovii is nevertheless mainly responsible for abortion in sheep.
The identification of Listeria species is based on a limited number of biochemical markers, among which hemolysis is used to differentiate between L. monocytogenes and L. innocua, the most frequently encountered non-pathogenic Listeria species. Hemolysis, a major differential characteristic of Listeria species, may be, in some cases (especially for environmental and food isolates), difficult to read on blood agar. The API-Listeria test (bioMérieux, Marcy-l'Etoile, France) was specifically designed for the genus Listeria and includes 10 biochemical differentiation tests in a microtube format (Fig. 1). It includes a patented ‘DIM’ test, based on the absence or presence of arylamidase, which distinguishes between L. monocytogenes and L. innocua without the need for further tests for hemolytic activity . Amino acid peptidase activity against alanyl as well as glycyl (as in the DIM test) gives the same reactions and can be established in a laboratory without using the API test kit [7,8]. The optimum growth temperature is between 30 and 37°C, but growth occurs at 4°C within a few days. Listeria spp. are facultatively anaerobic. Catalase is produced except in a few strains, and the oxidase test is negative .
The biochemical tests useful for discriminating between the species are acid production from d-xylose, l-rhamnose, α-methyl-d-mannoside, and d-mannitol. The scheme for biochemical identification of Listeria species is shown in Table 1. Without using the DIM test (see above) assessment of hemolysis is essential to differentiating between L. monocytogenes and L. innocua, the most frequently isolated non-pathogenic Listeria species.
|L. monocytogenes||L. seeligeri||L. ivanovii||L. innocua||L. welshimeri||L. grayi|
|L. monocytogenes||L. seeligeri||L. ivanovii||L. innocua||L. welshimeri||L. grayi|
+: positive; −: negative; V: variable.
Members of the genus Listeria are generally motile at 20–28°C by means of one to five peritrichous flagella. For demonstration of tumbling motility a hanging drop preparation is made from a young broth culture (e.g. tryptone soya yeast extract broth) incubated at room temperature for 8–24 h. In addition, semi-solid motility agar containing 0.2–0.4% agar is stabbed (about 1 cm). At 20–28°C, listeriae inoculated into the agar column swarm through the ‘motility medium’ and produce cloudiness. This can easily be observed by the naked eye. About 0.5 cm below the surface of the agar a layer of increased growth is found, like an umbrella. In this zone of reduced oxygen tension Listeria shows a better development than under aerobic or strictly anaerobic conditions.
Only three species, L. monocytogenes, L. seeligeri, and L. ivanovii, are hemolytic. They lyse red blood cells from most mammalian animals. The hemolysing activity is most regularly demonstrated using horse or sheep blood-containing agar plates. L. ivanovii exhibits a wide zone of hemolysis, sometimes even multiple zones. Hemolysis of L. monocytogenes resembles that of Streptococcus agalactiae (group B streptococci): the zone of hemolysis is narrow, frequently not extending much beyond the edge of the colonies. Removal of colonies with weak or equivocal hemolytic reactions from the surface of the agar with an inoculation loop allows the unequivocal recognition of hemolytic activity ‘below’ the bacterial growth. Inoculating the agar plate by piercing into the medium once or twice also can help to find an answer concerning hemolytic properties. L. seeligeri produces even narrower zones of hemolysis.
In order to improve the assessment of hemolysis, various authors recommend the use of the CAMP test . The CAMP (Christie, Atkins, Munch-Petersen) test, a method widely used for identifying S. agalactiae, uses a β-hemolysin-producing Staphylococcus aureus and a Rhodococcus equi strain streaked in one direction on a sheep blood agar plate and test cultures of Listeria spp. streaked at right angles to (but not touching) the S. aureus and R. equi lines (Fig. 2). Hemolysis of L. monocytogenes (and to a lesser extent L. seeligeri) is enhanced in the vicinity of the S. aureus streak, and L. ivanovii hemolysis is enhanced in the vicinity of R. equi (typical picture of a shovel). However, because sometimes a synergistic hemolysis reaction can be observed between L. monocytogenes and R. equi, this CAMP reaction must be interpreted with caution .
RAPID'L.MONO® (Bio-Rad, Marnes la Coquette, France) is a selective agar medium, which grows only Listeria sp. and gives a specific and direct identification of L. monocytogenes within 24–48 h. This medium is based both on the specific chromogenic detection of phosphatidylinositol phospholipase C (PIPLC) of L. monocytogenes and L. ivanovii (PIPLC-positive) and on the xylose fermentation . In this way, the colonies of L. ivanovii appear blue, surrounded by a yellow halo (xylose-positive) whilst the colonies of L. monocytogenes are blue without the halo (xylose-negative) (Fig. 3). The colonies of other Listeria spp. remain white (PIPLC-negative). Rarely seen are isolates of L. monocytogenes that lack PIPLC activity, supposed to be avirulent . There are at least two other chromogenic agars commercially available: CHROMagar® Listeria (Mast Diagnostic, Reinfeld, Germany) and BCM®Listeria monocytogenes plating medium (Biosynth International, Naperville, USA).
DNA probe assay for colony confirmation
A chemiluminescence DNA probe assay named AccuProbe® (Gen-Probe, San Diego, CA, USA) is available for rapid confirmation of L. monocytogenes from colonies on primary isolation plates. This assay can be done within 30 min and proved to be highly specific for L. monocytogenes. GENE-TRAK®Listeria monocytogenes Assay (Gene-Trak systems, Framingham, MA, USA) is another commercially available probe-based assay. Isolates of L. monocytogenes with atypical properties require molecular analysis for final identification .
Sequencing for identification
Sequencing for identification can be done using the MicroSeq 500 16S rDNA bacterial sequencing kit (Applied Biosystems, Foster City, CA, USA), a commercially available 16S ribosomal DNA sequencing kit . Polymerase chain reaction (PCR) primers anneal to DNA extracted from pure bacterial isolates, allowing amplification of a 527-bp product from the 5′ end of the 16S rRNA gene to be used for sequencing. DNA extracts can be made using commercial kits, e.g. the QIAamp® DNA Mini Kit (Qiagen, Hilden, Germany). In our laboratory, double-stranded sequence analysis of the first 527 bp of the 16S rRNA gene is completed by using the 310 DNA sequencer (Perkin-Elmer Biosystems, Foster City, CA, USA). As part of the system, a sequence database is included to determine the genus and species of bacteria. The software allows for exporting of sequences to be compared with other databases. As an additional database we use BLAST, accessible via the internet under http://www.ncbi.nlm.nih.gov.
Listeria strains are divided into serotypes on the basis of somatic (O) and flagellar (H) antigens . Thirteen serotypes are known for L. monocytogenes: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 (Table 2). Serotyping antigens are shared among L. monocytogenes, L. innocua, L. seeligeri, and L. welshimeri. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria and for allowing a first-level subtyping for epidemiological purposes. The introduction of a commercial kit for serotyping Listeria (Denka Seiken, Tokyo, Japan) greatly improved the availability of this method.
Numbers in parentheses=variable.
aSerovars occur in non-L. monocytogenes species; all L. ivanovii are serovar 5.
The National Committee for Clinical Laboratory Standards (NCCLS) has not yet provided specific guidelines for the testing of Listeria. Usually susceptibility testing is performed according to NCCLS guidelines for bacteria that grow aerobically using Mueller–Hinton agar with 5% horse blood . For trimethoprim–sulfamethoxazole, the blood is lysed. The pattern of antimicrobial susceptibility of L. monocytogenes has been relatively stable for many years. In vitro, the organism is susceptible to penicillin, ampicillin, gentamicin, erythromycin, tetracycline, rifampicin, and chloramphenicol, but only moderately susceptible to quinolones [19,20]. Threlfall et al.  reported 1–5% tetracycline resistance in L. monocytogenes from humans and food in the UK. Cephalosporins are generally ineffective and should not be used therapeutically . Antimicrobial susceptibilities of Listeria have not changed markedly over the past 35 years in the UK [19,21].
L. monocytogenes can be readily cultured from clinical specimens obtained from normally sterile sites such as blood, cerebrospinal fluid, amniotic fluid, placenta, or ear swabs from newborns by directly plating the material onto blood agar plates and incubating overnight at 35°C in ambient atmosphere. Stool specimens, environmental specimens, and food specimens should be selectively enriched for Listeria before being plated onto selective agar media. Immunomagnetic separation using Dynabeads® anti-Listeria (Dynal, Oslo, Norway) proved to be a valuable detection tool for samples with high levels of background flora . Media and incubation conditions (time period, temperature, atmosphere) vary strongly between different protocols.
For testing food and feed products, it is highly recommended to apply to the respective standards of the International Organization for Standardization [24,25]. ISO 11290 consists of the following parts, under the general title ‘Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Listeria monocytogenes’: Part 1: Detection method; Part 2: Enumeration method; Annexes A and B form an integral part of this part of ISO 11290; Annex C is for information only. Primary enrichment is done in a selective liquid medium containing one volume of lithium chloride and half a volume of both acriflavine and nalidixic acid (half Fraser broth) at 30°C for 24 h. Secondary enrichment is done with a selective liquid medium with full concentration of selective agents (Fraser broth) for 48 h at 35°C or 37°C, using a culture obtained from primary enrichment as inoculum. Samples of primary and secondary enrichment broths are plated out onto Oxford agar and on PALCAM (polymyxin–acriflavine–lithium chloride–ceftazidime–esculin–mannitol) agar. After incubation for 24 h at 30°C — for products heavily contaminated by a supplementing flora at 35°C or 37°C — the Oxford agar plates are checked for the presence of characteristic colonies; if no colonies are observed plates are incubated for an additional 24 h.
Typical colonies of Listeria spp. grown on Oxford agar for 24 h are small (1 mm) grayish colonies surrounded by black halos. After 48 h colonies become darker, with a possible greenish shine, and are about 2 mm in diameter, with black halos and sunken centers.
PALCAM agar is incubated at 30°C for 48 h under microaerobic conditions (5% oxygen, 7.5% carbon dioxide, 7.5% hydrogen, 80% nitrogen). On PALCAM agar, Listeria colonies appear gray-green, are 1.5–2 mm in diameter, and have black sunken centers; esculin, ferric iron, d-mannitol, and phenol red contribute to this color formation.
Suspect colonies are transferred to blood agar plates and incubated for 18 h at 35°C or 37°C for further work-up. A simplified genus identification is based on Gram stain (revealing Gram-positive, slim, short rods), positive catalase reaction (3% [m/m] hydrogen peroxide solution), and observation of motility. Classical cold enrichment over months is no longer necessary . Also the Henry illumination test is rarely employed. When examined under obliquely transmitted light (Henry illumination) from directly above the plate, colonies of Listeria spp. exhibit a bluish color and a granular surface.
Most commercially available tests for the rapid detection of Listeria species from food samples in selective enrichment broths are based on immunoassays that use monoclonal antibodies and are genus-specific. Vidas-LMO (bioMérieux), Lister test (Vicam, Watertown, MA, USA), and a DNA probe L. monocytogenes assay (Gene-Trak systems) are L. monocytogenes-specific [11,26]. All those kits are not designed for the analysis of clinical specimens.
PCR is the only test utilized for rapid detection of L. monocytogenes in food samples and in clinical specimens. The PCR assay is particularly useful when prior administration of antimicrobial agents compromises culture. Various test protocols were evaluated for cerebrospinal fluid samples and tissue samples (fresh or in paraffin blocks) . For food and environmental specimens also commercial PCR assays are available. Probelia®Listeria monocytogenes (Sanofi Diagnostic Pasteur, Marnes La Coquette, France) is licensed for the detection of L. monocytogenes DNA in food products and employs a peroxidase-labeled probe to detect amplicons bound to microtiter plates. Also the BAX® Screening System (Qualicon, Wilmington, DE, USA) uses PCR to screen food and environmental samples for L. monocytogenes and also for Listeria genus. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.
Virulence testing/animal testing
Many pathogenicity tests have been developed for L. monocytogenes, including a fertilized hen egg test, tissue culture assays, and tests using laboratory animals . Immunocompetent and immunocompromised mice were infected intraperitoneally, intravenously or intragastrically, and virulence was evaluated either by comparing the LD50 or by enumerating bacteria in the spleen or liver . But so far no clear correlation between origin (human, animal, environment, food) or strain characteristics (serotype, phage type, multilocus enzyme type, DNA micro- or macrorestriction patterns) and virulence has been observed. Animal testing is not part of the routine diagnostic armamentarium for Listeria. Presently, all strains of L. monocytogenes should be considered to be potentially capable of causing human disease .
Serologic responses to whole-cell antigens are not diagnostic, because of antigenic cross-reactivity between L. monocytogenes and other Gram-positive bacteria such as staphylococci, enterococci, and Bacillus species . Furthermore, patients with culture-confirmed listeriosis have had undetectable antibody levels . A variety of test systems is commercially available, but additional studies must be performed before any of these serologic tests can be recommended.
National Listeria reference laboratories
Isolates which are considered to be L. monocytogenes, L. ivanovii, or L. seeligeri should be sent to the appropriate national Listeria reference laboratory. The dispatch must be accompanied by all possible information concerning the strain(s). Data from several routine laboratories may be needed to discover and solve outbreaks. It is now common for foodstuffs to be manufactured or harvested at one place and then distributed. This distribution can be within a country, across economic regions (such as the European Union), continents or even world-wide. Based on Decision 2119/98/EC (Community Network for the Epidemiological Surveillance and Control of Communicable Diseases) the various national reference laboratories created a surveillance network that can even react in a real-time manner to international outbreaks of listeriosis .
In the year 2002 traditional microbiological methods are still the ‘gold standard’ exploited by clinical microbiologists for routine detection and identification of Listeria. The usefulness of rapid, molecular-based methods in this area is obvious. Perhaps the most attractive feature is the greatly enhanced time to identification. It is possible with these methods to achieve the appropriate organism identification in a fraction of the time required by traditional methods. There appears to be a bright future for molecular-based methods for the rapid identification of Listeria grown in culture or directly in specimens. Advances in the development of semi-automated platforms and simplified application methods have put molecular-based methods within the reach of most routine laboratories. Nevertheless, the need to obtain viable isolates as a prerequisite for elucidating outbreaks and tracing the source of infection will ensure the survival of the so-called traditional microbiological methods.