Listeria
J. McLauchlin, in Emerging Foodborne Pathogens, 2006
15.6 Growth and isolation of Listeria
Listeria spp. grow well on a wide variety of non-selective laboratory media, hence culture from normally sterile sites such as blood or cerebrospinal fluid does not require special media. For specimens such as faeces, vaginal secretions, food and environmental samples, special selective media are necessary.
Prior to the mid-1980s, cold enrichment, utilising the ability of Listeria to outgrow competing organisms at refrigeration temperatures in non-selective broths, was the main method used for selective isolation [Gray and Killinger 1966]. When growing on transparent media illuminated by oblique transmitted light and viewed at low magnification [Henry illumination technique] all Listeria colonies have a characteristic blue colour with a central ground glass appearance. However, because of the degree of skill required in recognising characteristic colonies, the lack of specificity and the slowness of these methods [some workers subcultured broths for up to six months], the emergence of listeriosis in 1980s resulted in much improved methodologies.
Media have been developed that rely on a number of selective agents, these include: acriflavin, lithium chloride, colistin, ceftazidime, cefotetan, fosfomycin, moxolactam, nalidixic acid, cycloheximide and polymyxin. Such media have resulted in the widespread ability of microbiology laboratories [especially those involved with the examination of foods] to selectively isolate Listeria. Numerous enrichment and selective isolation media have now been developed. Those mentioned here [or modifications of these] are used most frequently for the examination of foods. For selective broths: US Food and Drugs Administration [FDA] method [Lovett et al., 1987], the US Department of Agriculture [USDA] method [McClain and Lee 1988], or the Netherlands Government Food Inspection Service [NGFIS] method described by Van Netten et al. [1989] are most often used. Selective agars most frequently used are those of Curtis et al. [1989]; Oxford formulation or the PALCAM agar [named after an acronym of the ingredients, polymyxin B, acriflavine, lithium chloride, ceftazidime, aesculin and mannitol] of Van Netten et al. [1989]. These media are listed in internationally agreed standard methods [Anon. 1996b], which can also be use for quantification of the levels of Listeria contamination in an individual food [Anon. 1998].
All Listeria species are isolated by these methods and are morphologically indistinguishable from each other. To differentiate L. monocytogenes from other Listeria species on selective agars, substrates have been added to selective media to detect phospholipase [Notermans et al., 1991] or ß-glucosidase and enhanced haemolysis [Beumer et al., 1997]. Selective media, based on lipase and ß-glucosidase activity, which successfully differentiates L.monocytogenes from populations of other Listeria species, are now commercially available [Vlaemynck et al., 2000].
Non-cultural techniques such as those based upon immunoassays and the polymerase chain reaction are used increasingly for the detection of Listeria in enrichment broths for the examination of foods.
L. monocytogenes has been isolated from numerous types of raw, processed, cooked and ready-to-eat foods, usually at levels below 10 organisms per g. As outlined in Section 15.3, the properties favour transmission through food and a wide variety of food and food matrices will support the growth of this bacterium, which, especially towards the end of an extended shelf-life can become very heavily contaminated. Such problem foods types which support the growth of L. monocytogenes include soft cheese, milk, pâté, frankfurters and other sausages, cooked meat and poultry, smoked fish and shellfish, processed vegetables and some cut fruit including melon. Examples of rates of contamination for two of these problem food types examined in the UK are given in Table 15.6, and these are further discussed in Section 15.7. Growth can be localised within specific areas of an individual food, either because of the source of contamination [i.e. within cut or contact surfaces or where raw herbs and spices have been added] or because of the physicochemical properties of the foods such as in the areas of higher pH associated with the rind or with mould growth within a soft cheese.
The unusual tolerance of the bacterium to sodium chloride and sodium nitrite, and the ability to multiply [albeit slowly] at refrigeration temperatures makes L. monocytogenes of particular concern as a post-processing contaminant in long-shelf-life refrigerated foods. The widespread distribution of L. monocytogenes and the ability to survive on dry and moist surfaces favours post-processing contamination of foods from both raw product and factory sites.
Listeria
Siyun Wang, Renato H. Orsi, in Foodborne Infections and Intoxications [Fourth Edition], 2013
Microbiological characteristics of Listeria spp.
Listeria species are short, Gram-positive, non-spore-forming, facultative anaerobic rods [5]. They vary in size [0.40.5 in diameter by 12µm long], have rounded ends, and are not encapsulated. They are motile by means of a few peritrichous flagella, with motility typically manifesting itself at 30 °C but not at 37 °C. Listeria species are able to grow at temperatures ranging from 045 °C [5]. Growth can also occur between pH 6 and pH 9, or in nutrient broth supplemented with up to 10% [w/v] NaCl [5].
At present there are eight recognized species that belong to the genus Listeria, namely L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, L. marthii, and L. rocourtiae[10]. Among them, L. monocytogenes and L. ivanovii have been identified to be pathogens of warm-blooded hosts. Although 13 L. monocytogenes serotypes have been recognized, most cases of human disease involve strains of three serotypes, i.e., serotypes 1/2a, 1/2b, and 4b [11]. DNA sequencing and enzyme electrophoresis studies have elucidated at least four evolutionary lines for L. monocytogenes, namely lineages I, II, III, and IV [Table 11.1]. Lineages I and II represent the majority of isolates involved in human clinical cases [Figure 11.1] whereas lineage III and IV strains have been found less frequently among humans and are more common among animals [17]. This observation suggests a putative difference in the ecological distribution of lineage III and IV strains compared with lineage I and II strains. It is possible that lineages III and IV are best adapted to non-primate mammal hosts. An alternative explanation is that the low frequency of lineage III and IV isolates among humans is due to low prevalence of these isolates in food [16,18], and thus a low exposure of humans to these isolates. This low prevalence of lineage III and IV isolates in foods may be related to the fact that these isolates appear to be more susceptible than lineage I and II isolates to at least some of the stresses experienced in foods and food processing environments [19].
Table 11.1. Summary of L. monocytogenes Lineages
| I | First described in an MLEE study by Piffaretti et al. [13] | 1/2b, 3b, 3c, 4b | Lowest diversity among the lineages; lowest levels of recombination among the lineages | Commonly isolated from various sources; over-represented among human isolates |
| II | First described in an MLEE study by Piffaretti et al. [13] | 1/2a, 1/2c, 3a | Most diverse, highest recombination levels | Commonly isolated from various sources; over-represented among food and food-related as well as natural environments |
| III | First described using partial sequence data analyses by Rasmussen et al. [14] | 4a, 4b, 4c | Very diverse; recombination levels between those for lineage I and lineage II | Most isolates obtained from ruminants |
| IV | First described as IIIB using partial sequence data analyses by Roberts et al. [15]; first reported as lineage IV by Ward et al. [16] | 4a, 4b, 4c | Few isolates analyzed to date | Most isolates obtained from ruminants |
Adapted from Orsi et al.[12].
Figure 11.1. Distribution of L. monocytogenes lineages among different ecological compartments.
The thickness of each arrow represents the proportion of isolates belonging to each lineage among isolates obtained from a given ecological compartment [i.e., outbreak, sporadic cases, animals, foods, and environment]. For sporadic cases, broken arrows indicate the proportion of isolates associated with cases in Northern Europe [N-EUR], where lineage II strains seem to be more common among isolates from sporadic cases than lineage I strains, while solid arrows indicate the proportion of isolates associated with cases in the USA [where lineage I strains seem to be more common among isolates from sporadic cases than lineage II strains]. Lineage III and IV [previously designated IIIA/C and IIIB, respectively] are not represented in this figure, as isolates from these lineages are very rare among all ecological compartments. Lineages III and IV show the highest prevalence among animal isolates, but also have been isolated from human cases.
Adapted from Orsi et al. [84].In most cases, infection by L. monocytogenes appears to be self-limiting, with clinical symptoms of invasive listeriosis appearing predominantly in the immunocompromised, the elderly, pregnant women, and neonates. However, listeriosis has one of the highest case-fatality ratios of all foodborne bacterial infections. The pathogenesis of L. monocytogenes is well studied and key aspects of this research have been summarized in a number of reviews [2025]. Importantly, L. monocytogenes is able to cross the gastrointestinal, placental, and bloodbrain protective barriers. The development of listeriosis is typically initiated by ingestion of the organism, followed by its survival against the non-specific immune system defenses of the gastrointestinal tract. The organism is known to invade the intestinal epithelium or Peyers patches allowing for crossing of the intestinal barrier. From there, the bacteria can enter the draining lymph nodes and disseminate via the bloodstream to the liver and spleen.
The intracellular cycle of L. monocytogenes infection is governed by multiple virulence factors such as internalin A and internalin B [encoded by inlA and inlB; facilitate host cell invasion], hemolysin [encoded by hly; facilitates phagosome lysis], phosphatidylinositol-specific phospholipase C [PI-PLC, encoded by plcA], phosphatidylcholine-specific phospholipase C [PC-PLC, encoded by plcB], and hexose-6-phosphate transporter [Hpt, facilitates intracellular growth], as well as actin polymerization protein encoded by actA, which facilitates cell-to-cell spread. The central transcriptional activator PrfA regulates the expression of many gene products that are required for bacterial virulence. Outside the host, PrfA exists in a low-activity state, with correspondingly low levels of virulence gene expression. Once inside the host, PrfA becomes activated and induces the expression of gene products that are required for infection [26]. Furthermore, there are overlapping and complementary interactions between the transcriptional regulations of PrfA and the alternative general stress response sigma factor, σB. One of the three promoters upstream of prfA [P2prfA] is σB dependent, indicating a direct regulatory link between σB and PrfA [27]. Some virulence genes [e.g., lapB, bsh, inlA, and inlB] are preceded by both PrfA boxes and σB promoters and appear to be co-regulated by PrfA and σB[2729].
In the past few years, the genome sequences of more than 50 Listeria strains have become publicly available and at least 33 of them belong to L. monocytogenes [//www.genomesonline.org and //www.ncbi.nlm.nih.gov]. The analyses of completely sequenced genomes should help resolve aspects of the pathogenesis of L. monocytogenes, i.e., shed further light on the set of genes required for the intracellular replication and cell-to-cell spread inside the host.
Listeria
Elliot T. Ryser, in Foodborne Infections and Intoxications [Fifth Edition], 2021
Detection of Listeria spp. and L. monocytogenes
Traditionally, food laboratories have relied on conventional culture-based microbiological methods for the detection of Listeria spp. and L. monocytogenes. Various chromogenic media such as Agar Listeria according to Ottaviani and Agosti [ALOA] allow for the direct differentiation of L. monocytogenes from other Listeria spp. [36, 37]. Standard enrichment and plating methods used by regulatory agencies for isolating Listeria spp. from food and environmental samples have been previously reviewed [38, 39]. Biochemical identification of Listeria to the species level has been traditionally based on hemolysin production and acid production from various sugars including rhamnose and xylose. However, these conventional culture-based methods for isolation and identification, which remain challenging, laborious, and time-consuming, have now been at least partially supplanted by more rapid means.
A wide range of miniaturized biochemical test kits, as well as more rapid methods including immunoassay-basede.g., enzyme-linked immunosorbent assays [ELISA]and nucleic acid-based assays that probe for specific genes, are now commercially available and in widespread use [40, 41]. These relatively fast, high-throughput tests are relatively simple and easy to interpret and require minimal treatment of the sample. However, immunoassays remain dependent on variably expressed antigens, making them less reliable compared to nucleic acid-based detection methods [38, 4042]. Cross-reactivity can also occur if antigens are shared between closely related species, e.g., L. monocytogenes and L. innocua, leading to false-positive results [4042]; this is one of the reasons why antibody-based assays are less commonly used for the detection of L. monocytogenes.
Compared with conventional and immuno-based methods, nucleic acid-based methods are less time-consuming and far more sensitive. Nucleic acid-based methods, especially PCR-based assays, have been the basis for many commercially available detection systems. Various genes such as hly [the most common target], inlA, inlB, iap, intergenic spacer regions, genes encoding invasion-associated protein p60, aminopeptidase C, and phospholipase C protein have been targeted for the detection of L. monocytogenes [42]. Real-time [quantitative] PCR allows visualization of the accumulating amplicon, in contrast to conventional PCR where the resulting product is observed at the end of the reaction. The progress of real-time PCR is measured by monitoring the change in fluorescence levels, which in turn depends on the amount of accumulated PCR product. Advantages of RT-PCR include increased sensitivity to detect trace amounts of target DNA, automation, and the ability to quantify bacterial load without any post-PCR handling. A wide range of PCR-based detection systems including TaqMan real-time PCR, molecular beacons, and scorpions, has been developed for Listeria spp. and L. monocytogenes. [40, 41, 43]. In one of the earlier examples, a TaqMan real-time quantitative PCR assay targeting iap was able to distinguish L. monocytogenes from other bacterial species in five food matrices, including whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts [44].
MICROBIOLOGICAL ANALYSIS | Standard Methods
A.O. Gill, ... F.M. Nattress, in Encyclopedia of Meat Sciences [Second Edition], 2014
Listeria spp.
Listeriae are psychrotrophic and can grow under microaerobic conditions so that their numbers can increase during refrigerated storage of meats, both anoxically and aerobically packaged. Listeria spp. can be difficult to recover and two enrichment broths are usually used [Table 5]. After enrichment, PCR screening test may be performed and enrichment media is streaked onto at least two selective media to ensure isolation of as many Listeriae as possible. Typical colonies are selected for biochemical and serological characterization. Recovery of injured cells is problematic with Listeria and the use of Listeria Repair Broth has been suggested. Listeria can be confirmed and differentiated using a kit such as the API Listeria kit, observation of hemolysis, and the CAMP test for ß-lysin factor.
Pathogenicity Islands
Kelly N. Hallstrom, Beth A. McCormick, in Molecular Medical Microbiology [Second Edition], 2015
Listeria spp.
Listeria spp. include Listeria monocytogenes, a cause of foodborne illness. The infection [listeriosis] typically causes gastroenteritis, but can spread beyond the intestines. Most healthy people who are exposed to L. monocytogenes will only present with mild symptoms; however, listeriosis can become a serious infection in the young, elderly and immunocompromised. Further, pregnant women who become infected are at risk of miscarriage, stillbirth and premature labour.
Listeria spp. contain the Listeria pathogenicity island 1 [LIPI-1], a PAI of about 9kb that contains several genes that promote pathogenesis. These genes include plcA and prfA, which together form an operon. prfA is a transcription activator that regulates the expression of various Listeria virulence genes, and plcA is a phospholipase C with phosphatidylinositol activity. LIPI-1 also contains the lecithinase operon, which contains mpl, actA and plcB. actA encodes an actin-polymerizing protein that recruits and polymerizes actin filaments to Listeria once it is inside the cell to promote its intracellular motility [67,68]. plcB encodes a phospholipase C with lecithinase activity, and mpl encodes a metalloprotease that processes PlcB into a mature form. PlcB assists with Listeria escape from phagosomes, thus promoting the spread of Listeria to other cells [69].
LIPI-1 also contains Hly, which encodes listeriolysin O [LLO], which lyses erythrocytes and other cells, but also lysis vacuoles of eukaryotic cells allowing Listeria to spread through the cytoplasm. Deletion of hly leads to avirulence, demonstrating the importance of LLO in Listeria pathogenesis.
Listeria ivanovii [a pathogen of ruminating animals] also contains LIPI-2 and the inlCD gene cluster. Both of these PAIs encode small internalins, which contribute to internalization and host cell specificity.
LISTERIA | Introduction
C.A. Batt, in Encyclopedia of Food Microbiology [Second Edition], 2014
Characteristics of the Species
Listeria is a Gram-positive rod that is typically of 0.52 μm in length. It is non-spore-forming and is not encapsulated. Listeria can appear coccoid and motile depending upon the growth temperature. They have an optimum growth temperature of 3037 °C, and some species, most notably Listeria monocytogenes, can grow at temperatures as low as 4 °C. As such, these species are a particular foodborne hazard because of their ability to replicate, albeit slowly, at refrigerated temperatures. At 2025 °C, they form flagella [and other antigens as well as virulence factors] and are therefore motile, whereas at 37 °C they are not. Listeria is a facultative anaerobe and grows vigorously on a variety of complex media.
The genus Listeria is characterized by its catalase activity, its lack of hydrogen sulfide production, and its production of acid from glucose. It has a positive methyl red reaction and a positive VogesProskauer reaction. It does not produce indole, utilize citrate, or possess urease activity. At one time, there was only a single species, L. monocytogenes in the genus Listeria. Subsequently, Listeria denitrificans, Listeria grayi, Listeria murrayi, Listeria innocua, Listeria ivanovii, Listeria welshimeri, and finally Listeria seeligeri were added. Listeria denitrificans subsequently was reclassified as Jonesia denitrificans. Finally, it has been suggested based on rRNA sequences that L. murrayi and L. grayi are a single species. Multilocus enzyme electrophoresis [MEE] reveals that L. monocytogenes, L. ivanovii, L. welshimeri, and L. seeligeri all form distinct clusters with no overlap. 16S rRNA sequences help to form two groups: one consists of L. grayi and the other consists of L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, and L. seeligeri. From this latter group, a further division that clusters L. monocytogenes and L. innocua appears distinct from L. ivanovii, L. seeligeri, and L. welshimeri. This next stage of distinction is curious as only L. monocytogenes and to a lesser extent L. ivanovii are considered to be virulent. Among the various Listeria species, the most studied is L. monocytogenes. Listeria monocytogenes is covered in detail elsewhere. Among the other Listeria species, none are considered to be highly virulent, and apart from L. monocytogenes, only L. ivanovii has been associated with disease in animals. There are rare reports of human disease caused by L. ivanovii, but these may be compromised by difficulty in accurately identifying the organism to the species level.
Virulence in Listeria is mediated by a number of factors, some of which are unique to L. monocytogenes, whereas a number also are shared by the non-L. monocytogenes species, including L. ivanovii and L. welshimeri. Table 1 presents a list of a selected group of virulence factors. Most of these virulence genes, including prfA, plcA, hlyA, and actaA, are clustered into a single operon.
Table 1. Selected virulence genes found in Listeria
| PrfA | prfA | Regulatory protein for operon |
| PI-PLC | plcA | Phospholipase |
| LLO | hylA | Hemolysis |
| ActA | actA | Actin polymerization |
| InlA | InlA | Internalin needed for cell entry |
A Positive Test Result
Listeria spp. may be an indicator of the presence of L. monocytogenes. Surveys of foods, processing plants, and other environments document that non-L. monocytogenes often are found in samples that contain L. monocytogenes. For example, whereas a total of 12.5% of fresh chicken wings tested positive for L. monocytogenes, more than 42% tested positive for all Listeria species. Testing for Listeria spp. in environmental samples of food production environments has been recognized widely as an effective control for the pathogen. It serves as an indicator whose presence is correlated to the presence of L. monocytogenes. Although the ecology of Listeria species is not completely understood nor is the overlap in the ecology of non-L. monocytogenes versus L. monocytogenes known, surveys for all Listeria might be a useful indicator of the presence of L. monocytogenes.
MICROBIOLOGICAL SAFETY OF MEAT | Listeria monocytogenes
R.A. Holley, R.P. Cordeiro, in Encyclopedia of Meat Sciences [Second Edition], 2014
Morphology, Culture, and Metabolism
Listeriae are Gram-positive, short rods [diameter 0.5µm, length 0.52µm] with rounded or sometimes coccoidal ends [Figure 1]. They do not form spores or capsules. Listeria are motile with peritrichous flagella at 2025°C, but are not motile, or less noticeably motile at 37°C. All listeriae show characteristic tumbling motility when viewed microscopically in hanging-drop fresh broth cultures and, owing to their microaerophilic nature, grow in umbrella forms approximately 0.5-cm below the surface of stabbed semisolid media.
Figure 1. Photomicrograph of Listeria monocytogenes; bar=1µm.
Courtesy of JA Vazquez-Boland. Copyright © JA Vazquez-Boland.Listeriae are fastidious, and grow on common bacteriological media. After overnight incubation, the colonies are smooth, bluish-gray, translucent, and 0.20.8mm in diameter, but after prolonged incubation can be much larger. Rough colonies are sometimes observed. Colonies on a clear medium, which when viewed under a microscope with obliquely transmitted light, typically appear sparkling blue-green, a characteristic useful for distinguishing them from colonies of other microorganisms.
Listeria utilize glucose, forming lactate, acetate, and acetoin under aerobic conditions, but acetoin is not produced anaerobically. They are oxidase-negative and catalase-positive; hydrolyze aesculin but not urea, gelatin, or casein; and are methyl red- and VogesProskauer-positive. Listeria are facultatively anaerobic but prefer microaerophilic atmospheres when growing aerobically. Listeria require cystine, leucine, isoleucine, arginine, valine, cysteine, riboflavin, biotin, thiamin, and thioctic acid for growth. Listeria are able to grow at temperature, pH, and water activity [aw] ranging from 0 to 45°C, 5.6 to 9.6, and 0.90 to >0.97, respectively.
MICROBIOLOGICAL SAFETY OF MEAT | Listeria monocytogenes
S. Buncic, S.M. Avery, in Encyclopedia of Meat Sciences, 2004
Morphology, Culture Growth and Metabolism
Listeriae are Gram-positive, short rods [diameter 0.5 μm, length 0.52 μm] with rounded ends or sometimes coccoidal [Figure 1 ]. They do not form spores or capsules. Listeria are motile with peritrichous flagella at 20 °C25 °C but are not motile [or only very weakly] at 37 °C. At 37 °C, L. innocua is more motile than L. monocytogenes. All listeriae show characteristic tumbling motility when viewed microscopically in hanging-drop fresh broth cultures, and grow in umbrella forms about 0.5 cm below the surface of stabbed semi-solid media owing to their microaerophilic nature.
Figure 1. Photomicrograph of Listeria monocytogenes; BAR = 1 μm. [Courtesy of JA Vazquez-Boland. Copyright © JA Vazquez-Boland.]
Listeriae are fastidious, and grow on common bacteriological media. After overnight incubation, the colonies are smooth, bluish-grey, translucent and 0.20.8 mm in diameter, but after prolonged incubation can be much larger. Rough colonies are sometimes observed. Colonies on a clear medium under a microscope with obliquely transmitted light typically appear as sparkling blue-green, a characteristic useful for distinguishing them from colonies of other microorganisms.
Listeria utilizes glucose, forming lactate, acetate and acetoin under aerobic conditions, but acetoin is not produced anaerobically. It is oxidase-negative and catalase-positive, hydrolyses aesculin but not urea, gelatin or casein and is methyl red-positive and VogesProskauer-positive. Listeria is facultatively anaerobic but prefers microaerophilic atmospheres when growing in oxygen. Listeria requires growth factors: cystine, leucine, isoleucine, arginine, valine, cysteine, riboflavin, biotin, thiamin and thioctic acid.
Clinical Bacteriology
Jeffrey K. Actor PhD, in Elsevier's Integrated Review Immunology and Microbiology [Second Edition], 2012
Listeria
Listeria is a gram-positive, catalase-positive rod [diphtheroid] that is not capable of forming endospores. Two species are of human pathogenic significance: L. monocytogenes and L. ivanovii. In particular, L. monocytogenes causes meningitis and sepsis in newborns and accounts for 10% of community-acquired bacterial meningitis in adults. While host monocytes are critical for control and containment of Listeria, they also are involved in disseminating infection to other areas of the body. Listeria is also diarrheagenic in humans, with those infected having vomiting, nausea, and diarrhea. Ingestion of Listeria from unpasteurized milk products can lead to bacteremia and septicemia with meningoencephalitis. When transmitted across the placenta to the fetus, infection can lead to placentitis, neonatal septicemia, and possible abortion. Individuals at particular risk for listeriosis include newborns, pregnant women and their fetuses, the elderly, and persons lacking a healthy immune system. The bacterium usually causes septicemia and meningitis in patients with suppressed immune function. Antibiotics are recommended for treatment of infection because most strains of Listeria are sensitive to ampicillin plus an aminoglycoside. Identification uses β-hemolysis on blood agar plates.