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Review |
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, PO Box 6100, MS 39762-6100, USA
Correspondence
liu{at}cvm.msstate.edu
Introduction
The genus Listeria represents a group of closely related, Gram-positive, facultative anaerobic, non-spore-forming, rod-shaped bacteria 0.5 µm in width and 1–1.5 µm in length, and with a low G+C content. Taxonomically, it is divided into six species (i.e. Listeria monocytogenes, Listeria ivanovii, Listeria seeligeri, Listeria innocua, Listeria welshimeri and Listeria grayi), of which only L. monocytogenes and L. ivanovii are pathogenic (Robinson et al., 2000). While L. monocytogenes infects both man and animals, L. ivanovii (previously known as L. monocytogenes serotype 5) is principally an animal pathogen that rarely occurs in man (Low & Donachie, 1997). Being tolerant to extreme pH, temperature and salt conditions (Sleator et al., 2003; Liu et al., 2005a), Listeria species are present in a variety of environments, including soil, water, effluents and foods. With manufactured ready-to-eat foods being consumed in increasing quantities, it is no surprise that L. monocytogenes has become recognized as an important opportunistic human foodborne pathogen.
Although L. monocytogenes is infective to all human population groups, it has a propensity to cause especially severe problems in pregnant women, neonates, the elderly, and immunosuppressed individuals. During the early stages of infection, human listeriosis often displays non-specific flu-like symptoms (e.g. chills, fatigue, headache, and muscular and joint pain) and gastroenteritis. However, without appropriate antibiotic treatment, it can develop into septicaemia, meningitis, encephalitis, abortion and, in some cases, death (Vazquez-Boland et al., 2001). Indeed, with mortality rates on average approaching 30 %, L. monocytogenes far exceeds other common foodborne pathogens, such as Salmonella enteritidis (with a mortality of 0.38 %), Campylobacter species (0.02–0.1 %) and Vibrio species (0.005–0.01 %) in terms of disease severity (Altekruse et al., 1997; Mead et al., 1999).
Given the close morphological and biochemical resemblances of L. monocytogenes to other Listeria species, and the non-specific clinical manifestations of listeriosis (Vazquez-Boland et al., 2001), the availability of rapid, specific and sensitive diagnostic tests capable of distinguishing L. monocytogenes from other Listeria species is essential for the effective control of the disease. In addition, with L. monocytogenes comprising a diversity of strains (Kathariou, 2002), the development of subtyping procedures is critical to the epidemiologic investigation of listeriosis outbreaks. Furthermore, since L. monocytogenes demonstrates strain variations in virulence and pathogenicity (Liu et al., 2003a; Roche et al., 2003), the ability to determine accurately and rapidly the pathogenic potential of L. monocytogenes isolates is essential to limit the spread of listeriosis and reduce unnecessary recalls of food products.
Extensive research in recent decades has led to significant insights regarding Listeria species and listeriosis (Vazquez-Boland et al., 2001). The establishment of animal models and in vitro cell culture systems for listeriosis has helped the delineation of key stages in L. monocytogenes infection and pathogenesis. The application of molecular techniques has facilitated the identification and characterization of major virulence-associated genes and proteins in L. monocytogenes. The development of serological and nucleic-acid-based detection procedures has enhanced the laboratory detection and differentiation of Listeria species. Since in-depth reviews on the epidemiology, pathogenesis and virulence determinants of L. monocytogenes are available (Low & Donachie, 1997; Vazquez-Boland et al., 2001; Kathariou, 2002), this communication focuses on recent progress in the species-specific identification, subtyping and virulence determination of L. monocytogenes, and discusses future research needs in these areas.
Molecular characteristics
L. monocytogenes is a remarkable bacterium that has evolved over a long period to acquire a diverse collection of molecules, each with unique properties and functions, and each contributing to the success of L. monocytogenes as an intracellular pathogen. Upon ingestion by the host via contaminated food, L. monocytogenes withstands exposure to host proteolytic enzymes, the acidic stomach environment (pH 2.0), bile salts and non-specific inflammatory attacks, largely through the actions of several stress-response genes (opuCA, lmo1421 and bsh) and related proteins (Sleator et al., 2003).
Having survived this initial stage, L. monocytogenes adheres to and is internalized by host cells with the assistance of a family of surface proteins called internalins (Gaillard et al., 1991). The most notable internalins are InlA and InlB. Whereas InlA (an 88 kDa protein encoded by inlA) interacts with E-cadherin to mediate L. monocytogenes entry into epithelial cells, InlB (a 65 kDa protein encoded by inlB) recognizes C1q-R (or Met) to facilitate L. monocytogenes entry into a much broader range of host-cell types, including hepatocytes, fibroblasts and epithelioid cells. Gaining entry to host cells enables L. monocytogenes to evade host immune surveillance functions (Vazquez-Boland et al., 2001).
Following its uptake by host cells, L. monocytogenes is primarily located in single-membraned vacuoles. Two virulence-associated molecules are responsible for lysis of the primary single-membraned vacuoles and subsequent escape by L. monocytogenes: listeriolysin O (LLO) and phosphatidylinositol-phospholipase C (PI-PLC). LLO (a 58 kDa protein encoded by hly) is a pore-forming, thiol-activated toxin that is essential for L. monocytogenes virulence (Portnoy et al., 1992). PI-PLC (a 33 kDa protein encoded by plcA), acting in synergy with phosphatidylcholine-phospholipase C (PC-PLC, a 29 kDa protein encoded by plcB), aids LLO in lysing the primary vacuoles (Vazquez-Boland et al., 2001).
After lysis of the primary single-membraned vacuoles, L. monocytogenes is released to the cytosol, where it undergoes intracellular growth and multiplication. The intracellular mobility and cell-to-cell spread of L. monocytogenes require another surface protein, ActA (a 67 kDa protein encoded by actA), which is cotranscribed with PC-PLC and mediates the formation of polarized actin tails that propel the bacteria toward the cytoplasmic membrane. At the membrane, bacteria become enveloped in filopodium-like structures that are recognized and engulfed by adjacent cells, resulting in the formation of secondary double-membraned vacuoles. A successful lysis of the secondary double-membraned vacuoles signals the beginning of a new infection cycle, which is dependent on PC-PLC upon activation by Mpl (a 60 kDa metalloprotease encoded by mpl) (Vazquez-Boland et al., 2001).
The genes encoding the virulence-associated proteins PI-PLC, LLO, Mpl, ActA and PC-PLC are located in a 9.6 kb virulence gene cluster (Gouin et al., 1994), which is principally regulated by a pleiotropic virulence regulator, PrfA (a 27 kDa protein encoded by prfA). The prfA gene is situated immediately downstream of, and sometimes cotranscribed with, the plcA gene. PrfA activates the transcription of many L. monocytogenes virulence-associated genes. The genes encoding InlA and InlB are positioned elsewhere in the genome. As the inlA and inlB genes possess a transcription binding site similar to that recognized by PrfA, they may also be partially regulated by PrfA. In addition to these virulence-associated genes and proteins, several other genes, such as iap (encoding invasion-associated protein, or Iap), are also involved in L. monocytogenes virulence and pathogenicity (Vazquez-Boland et al., 2001).
Species-specific identification
As L. monocytogenes is morphologically indistinguishable from other Listeria species, and often causes non-specific clinical symptoms, laboratory testing is required to differentiate L. monocytogenes from other Listeria species, and to diagnose listeriosis. The confirmation of Listeria species identity has clinical relevance, that is, the absence of L. monocytogenes in clinical specimens may render antibiotic therapy unnecessary, unless immunocompromised patients are involved. The earlier diagnostic methods for L. monocytogenes are largely phenotype-based, and characterize the gene products of L. monocytogenes through the measurement of biochemical, antigenic and bacteriophage properties. Since these properties may vary with changing external conditions, with growth phase and with spontaneous genetic mutations, the use of phenotypic tests may sometimes lead to equivocal results. Following recent advances in molecular genetic techniques, methods targeting unique genes in Listeria have been designed for the specific differentiation of L. monocytogenes from other Listeria species; these methods are intrinsically more precise and less affected by natural variation than the phenotypic methods.
Biochemical methods
Listeria species are closely related bacteria that share many morphological and biochemical characteristics. Apart from being catalase positive, and indole and oxidase negative, Listeria species can hydrolyse aesculin, but not urea. These common biochemical features have frequently been exploited for the differentiation of Listeria species from other bacteria. On the other hand, Listeria species also possess distinct biochemical properties that can be useful for species-specific identification. For instance, Listeria species show significant variations in their ability to haemolyse horse or sheep red blood cells, and in their ability to produce acid from L-rhamnose, D-xylose and 
-methyl-D-mannoside (Robinson et al., 2000).
Hence, L. ivanovii is differentiated biochemically from L. monocytogenes and other Listeria species by its production of a wide, clear or double zone of haemolysis on sheep or horse blood agar, a positive Christie–Atkins–Munch-Petersen (CAMP) reaction with Rhodococcus equi but not with haemolytic Staphylococcus aureus, and fermentation of D-xylose but not L-rhamnose (Rocourt & Catimel, 1985). In addition, L. ivanovii is distinguished from L. monocytogenes by its strong lecithinase reaction with or without charcoal in the medium, in comparison to L. monocytogenes, which requires charcoal for its lecithinase reaction (Ermolaeva et al., 2003). Similarly, L. innocua is distinguished from L. monocytogenes on the basis of its negative CAMP reaction and its failure to cause ß-haemolysis or to show PI-PLC activity on chromogenic media. L. welshimeri is differentiated from other Listeria species by its negative ß-haemolysis and CAMP reactions, and by its acid production from D-xylose and -methyl-D-mannoside (Robinson et al., 2000).
Nevertheless, the identification of Listeria species by biochemical methods is a laborious process, involving primary isolation with selective and enrichment media, followed by Gram stain and multiple biochemical tests. For the isolation of Listeria that has been injured during food processing, a pre-enrichment in phosphate-buffered broth medium containing inhibitors is also needed. The incorporation of various biochemical procedures into a single testing platform has streamlined the diagnostic process for Listeria species (Rocourt & Catimel, 1985; Bille et al., 1992). However, biochemical testing of Listeria species remains time-consuming (taking up to 6 days to finalize a result) and costly. Furthermore, as biochemical tests measure the phenotypic characteristics of Listeria bacteria, their performance can be influenced by external factors that affect bacterial growth and metabolic mechanisms.
Serological methods
Listeria species possess group-specific surface proteins, such as somatic (O) and flagellar (H) antigens that are useful targets for serological detection with corresponding monoclonal and polyclonal antibodies. While there are 15 Listeria somatic (O) antigen subtypes (I–XV), flagellar (H) antigens comprise four subtypes (A–D) (Seeliger & Höhne, 1979; Seeliger & Jones, 1986), with the serotypes of individual Listeria strains being determined by their unique combinations of O and H antigens (Table 1
). Through examination of group-specific Listeria O and H antigens in slide agglutination, at least 12 serotypes (i.e. 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 7) have been recognized in L. monocytogenes, several (e.g. 1/2a, 1/2b, 3b, 4a, 4b, 4c and 6b) in L. seeligeri, one (i.e. 5) in L. ivanovii, and a few (e.g. 1/2b, 6a and 6b) in L. innocua, L. welshimeri and L. grayi (Seeliger & Jones, 1986; Kathariou, 2002). Since slide agglutination is not easily adapted for high-throughput testing, an ELISA has recently been developed to improve efficiency (Palumbo et al., 2003).
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However, due mainly to the high cost of acquiring subtype-specific antisera, serotyping methods are not routinely performed in clinical laboratories. With both L. monocytogenes and L. seeligeri containing serotypes 1/2a, 1/2b, 3b, 4a, 4b, 4c and 6b, the inability of serotyping methods to correlate serotypes directly with species identities further limits their potential for widespread clinical application. Moreover, as antigen sharing occurs frequently among various L. monocytogenes serotypes, with 1/2a and 3c both containing H antigens A and B; 4a–d, 1/2b and 3b all having H antigens A, B and C; 1/2c and 3a both possessing H antigens B and D; and multiple, common O antigens being present in different serotypes (Table 1
), it can be a challenge to conclusively determine the serotype of some L. monocytogenes strains (Schonberg et al., 1996; Liu et al., 2006a). Like biochemical methods, serotyping methods are also liable to give occasional discrepant results because of their dependence on the phenotypic characteristics of Listeria bacteria. For these reasons, serotyping methods have largely been superseded by molecular procedures that are intrinsically more specific and sensitive for the identification and differentiation of Listeria species.
Molecular methods
Detection by gene probes.
A gene probe is a stretch of specific single-stranded nucleic acid that is enzyme- or radiolabelled and employed for the identification and detection of a (usually membrane-bound) complementary nucleic acid sequence in a target organism. Being a first-generation nucleic acid detection technology, the detection of L. monocytogenes by gene probes is precise and relatively straightforward. Listeria DNA is spotted onto a supporting matrix (e.g. a nitrocellulose filter or nylon membrane), hybridized with an enzyme- or radiolabelled Listeria species-specific gene probe (derived from 16S rRNA, heat-shock protein P60 or other protein-coding gene), and subsequently detected with an appropriate substrate (enzyme label) or by autoradiography (radiolabel) (Klinger et al., 1988; Kohler et al., 1990). As this procedure exploits differences among Listeria species at the genetic level, it is more specific than biochemical and serological methods that are phenotype based. However, since it does not involve nucleic acid amplification, this technique has limited sensitivity, requiring at least 104 copies of target gene per microlitre for reliable detection without signal amplification, although improving to as few as 500 copies of target gene per microlitre with signal amplification; this is inadequate for most clinical samples. Since the introduction of nucleic acid amplification technology, the employment of gene-probe-based detection procedures has steadily declined.
Detection by nucleic acid amplification.
In vitro amplification of nucleic acid is a more recent addition to the genetic detection methods for pathogen identification and diagnosis. Among several elegant approaches to nucleic acid amplification, PCR was the first and remains the most widely applied technique in both research and clinical laboratories. PCR employs two primers (usually 20–30 nucleotides long) that flank the beginning and end of a specific DNA target, a thermostable DNA polymerase that is capable of synthesizing the specific DNA, and double-stranded DNA to function as a template for DNA polymerase. The PCR process begins at a high temperature (e.g. 94 °C) to denature and open the double-stranded DNA template into single-stranded DNA, followed by a relatively low temperature (e.g. 54 °C) to enable annealing between the single-stranded primer and the single-stranded template, and then a temperature of 72 °C to allow DNA polymerase copying (extension) of the template. The whole process is repeated 25–30 times so that a single copy of DNA template can turn into billions of copies within 3–4 h. As PCR has the ability to selectively amplify specific targets present in low concentrations (theoretically down to a single copy of DNA template), it offers exquisite specificity, unsurpassed sensitivity, rapid turnover, and ease of automation for laboratory detection of L. monocytogenes from clinical specimens, in addition to its value for identifying both cultured and non-cultivable organisms. The amplified DNA products can be separated by agarose gel electrophoresis and detected with a DNA stain, or alternatively detected via labelled probes, DNA sequencing, microarray and other related techniques (Wang et al., 1993; Manzano et al., 2000; Volokhov et al., 2002). By exploiting molecular differences within 16S and 23S rRNA genes, intergenic spacer regions, hly, inlA, inlB, iap and other genes (e.g. delayed-type hypersensitivity gene, aminopeptidase gene and putative transcriptional regulator gene lmo0733), L. monocytogenes is rapidly and precisely differentiated from other Listeria species and common bacteria (Table 2) (Aznar & Alarcon, 2002).
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Subtyping
With L. monocytogenes consisting of a diversity of strains, the availability of subtyping procedures to track individual strains involved in listeriosis outbreaks, and to examine the epidemiology and population genetics of L. monocytogenes bacteria, is integral to control and prevention programmes aimed at listeriosis. Similar to species-specific identification, two major subtyping approaches are in common use: phenotypic and genetic (molecular or DNA) subtyping. The phenotypic subtyping approach includes serotyping, phage typing, multilocus enzyme electrophoresis (MLEE) and esterase typing. The genetic subtyping approach encompasses pulsed-field gel electrophoresis (PFGE), ribotyping, PCR-based subtyping techniques [e.g. random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), PCR-restriction fragment length polymorphism (PCR-RFLP) and repetitive element PCR (REP-PCR)] and DNA sequencing-based subtyping techniques [e.g. multilocus sequence typing (MLST)]. While the phenotypic subtyping approach occasionally suffers from low discrimination and reproducibility, the genetic subtyping approach is highly sensitive, discriminatory and reproducible. For improved subtyping discrimination, a combination of two or more subtyping techniques, be they gene or phenotype based, is often used in practice for epidemiologic investigation of L. monocytogenes outbreaks.
Phenotypic subtyping methods
Serotyping.
By separating L. monocytogenes strains into 12 serotypes (i.e. 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 7) on the basis of serological reactions between somatic (O)/flagellar (H) antigens and their corresponding antisera (Table 1) (Seeliger & Höhne, 1979; Seeliger & Jones, 1986), serotyping may potentially be useful for tracking L. monocytogenes strains involved in disease outbreaks. Given that only three serotypes (1/2a, 1/2b and 4b) are commonly associated with human listeriosis, however, the value of serotyping in L. monocytogenes epidemiological investigations is somewhat limited. In addition, the inability of the serotyping procedures to give a fine distinction among serotypes 4a, 4b and 4c has further hampered their potential utility (Liu et al., 2006a). In fact, whereas four serotype 4b strains belonging to lineage I strains as evaluated by prfA virulence gene cluster sequences reacted in PCR with serotypes 4b-, 4d- and 4c-specific ORF2110 virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b strains belonging to lineage III strains were negative by ORF2110 and lmo1134 primers (Ward et al., 2004). Based on their differential reactions in PCR and Southern blot, the four serotype 4b lineage I strains are unquestionably of serotype 4b; however, seven of the nine serotype 4b lineage III strains appear to be of serotype 4c and the other two of serotype 4a (Liu et al., 2006a). Therefore, the serotyping procedure often plays an accessory role in the subtyping and tracking of L. monocytogenes epidemic strains.
Phage typing.
Bacteriophages are viruses that occur naturally in Listeria and other bacteria. Being host specific, bacteriophages have the capacity to lyse closely related Listeria bacteria independently of the bacterial species and serovar identities. Through examination of bacteriophage-induced, host-specific lysis of Listeria bacteria on agar plates, Listeria strains can be separated into distinct phage groups and phagovars, which are useful for tracking the origin and course of listeriosis outbreaks (Audurier et al., 1984). In a phage-typing study involving 16 selected phages, 57 Listeria reference strains and 454 food isolates, Listeria strains were classified into four phage groups, which in turn were divided into 41 distinct phagovars. On the basis of a Listeria strain being lysed by at least one phage at a 100x routine test dilution, an overall typability of 84.5 % was obtained (Loessner & Busse, 1990). By increasing the number of bacteriophages to 21, a typability of 89.5 % was noted after analysis of 1087 Listeria strains (Loessner, 1991). Nonetheless, with close to 10 % of Listeria strains being untypable (especially serovar 3 and L. grayi strains), the usefulness of phage typing as an independent tool for epidemiological investigations is severely constrained.
MLEE.
MLEE is a protein-based, isoenzyme typing method that correlates specific protein band patterns with genotypes. For this method, soluble proteins (or bacterial lysates) from L. monocytogenes strains are separated by starch gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), or isoelectrophoretic focusing under native conditions, followed by visualization with specific enzyme stains. Variations in the electrophoretic mobility of different enzymes (or electrophoretic types, ETs) enable differentiation of L. monocytogenes strains. Since multiple enzymes are present in L. monocytogenes, numerous ETs are often obtained. For instance, assessment of 305 L. monocytogenes strains by MLEE resulted in the detection of 78 ETs (Graves et al., 1994). Based on the similar ETs detected in MLEE, L. monocytogenes serovars 1/2b, 3b and 4b are classified into one distinct division, and serovars 1/2a, 1/2c and 3a in another division (Bibb et al., 1989; Piffaretti et al., 1989). The detection of a large number of electrophoretic types in L. monocytogenes strains by MLEE necessitates careful optimization and standardization of the test procedure so that run-to-run variations are minimized.
Esterase typing.
Esterases are a class of heat-stable enzymes that hydrolyse carboxylic acid esters. Being a variant of MLEE analysis, esterase typing measures the esterase activity from cell extracts of individual L. monocytogenes strains on starch gels following electrophoresis. Upon examination by esterase typing of 219 L. monocytogenes isolates from milk, non-dairy foods, and clinical and veterinary sources, Harvey & Gilmour (1996) detected 59 ETs. Like MLEE, esterase typing produces a high number of ETs that require careful documentation and standardization. Furthermore, as a phenotype-based procedure, the reproducibility of esterase typing is sometimes low.
Genetic subtyping methods
PFGE.
PFGE is a highly reproducible, discriminatory and effective molecular typing method that is based on restriction fragment length polymorphisms (RFLPs) of bacterial DNA. In RFLP analysis, bacterial genomic DNA is digested with restriction enzymes to yield hundreds of fragments, which are then separated by conventional agarose gel electrophoresis to form distinctive banding patterns for individual strains. Given its complex band patterns, however, the interpretation of RFLP results is notably tedious and technically demanding. PFGE uses selected restriction enzymes to yield between 8 and 25 large DNA bands of 40–600 kb in size, and alternating currents to cause DNA fragments to move back and forth, resulting in a higher level of fragment resolution. For this method, L. monocytogenes bacteria are first placed in agarose plugs, where they are lysed, and the DNA is then digested with selected restriction enzymes. The plugs containing the digested DNA are transferred into an agarose gel and electrophoresed for 30–50 h with alternating currents. On the basis of distinct DNA band patterns, PFGE classifies L. monocytogenes into subtypes (or pulsotypes), providing sensitive subtype discrimination that is considered the reference standard (Brosch et al., 1994, 1996; Graves et al., 1994). Indeed, after a comparative examination of 35 L. monocytogenes strains by serotyping, esterase typing, ribotyping, RAPD and PFGE, PFGE along with ribotyping produced the most discriminatory outcomes for L. monocytogenes (Kerouanton et al., 1998). However, due to its time-consuming nature (taking 30 h or longer to perform) and its requirement for special equipment, PFGE is not widely used outside reference laboratories.
Ribotyping.
Ribotyping is a derivative of RFLP analysis that uses a ribosomal DNA (rDNA) probe to detect the restriction fragment patterns of chromosomal DNA digested with appropriate restriction enzymes, resulting in much simpler and more consistent band patterns. For this method, Listeria DNA is initially digested with restriction enzymes (e.g. EcoRI, PvuII and XhoI) into many pieces (>300–500) of small-sized fragments (1–30 kb). The resultant DNA fragments are then separated by agarose gel electrophoresis, transferred to a membrane (via Southern blot), and detected with a probe derived from the Escherichia coli gene that encodes rRNA (rDNA). Thus, only DNA fragments that contain rRNA genes are recognized (Graves et al., 1994). In a study involving 1346 L. monocytogenes strains, Bruce et al. (1995) showed that 50 band patterns can be detected after digestion of L. monocytogenes DNA with EcoRI and detection with the E. coli rrnB rRNA operon.
On the whole, ribotyping is a robust, reproducible typing technique that has a similar discriminatory power to, but produces fewer bands than, PFGE. As ribotyping does not demand special equipment, it has become a practical tool that has been frequently employed for the tracking and subtyping of L. monocytogenes bacteria. Indeed, with an automated ribotyping test becoming available commercially (RiboPrinter Microbial Characterization System, Qualicon, Wilmington, DE), it is envisaged that ribotyping will play an increasingly dominant role in the epidemiologic investigation of disease due to L. monocytogenes.
PCR-based subtyping techniques
RAPD and arbitrarily primed PCR (AP-PCR).
Both RAPD and AP-PCR use low-stringency PCR amplification with a single primer of an arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments. For the method, a single short random primer (usually 10 bases long for AP-PCR, and 10–15 bases long for RAPD) is used in PCR at a relatively low temperature (around 36 °C) to generate amplified products from L. monocytogenes DNA that form distinct band patterns after agarose gel electrophoresis. The possible mechanism behind RAPD and AP-PCR is that by reducing the stringency of the primer-annealing temperature, a random primer that shows no complete homology to a genome may have a perfect match of two to three nucleotides between the 3' end of the primer and the template strand to allow annealing and the priming of complementary strand synthesis by DNA polymerase, given that a putative three-nucleotide sequence can in principle be found once in each 64 nucleotide sequence (43 permutations). When two such annealing and priming events occur within a certain distance of each other and in proper orientation, the sequence between the matching sites can be amplified effectively. RAPD (or AP-PCR) is more economical and faster than other typing methods, and is particularly suitable for testing fewer than 50 strains. However, the discriminatory ability of RAPD and AP-PCR is sometimes inconsistent (Farber & Addison, 1994; O'Donoghue et al., 1995). In a recent study, RAPD gave less robust results than PCR ribotyping for subtyping L. monocytogenes isolates involved in invasive and non-invasive listeriosis outbreaks (Franciosa et al., 2001).
AFLP.
AFLP is a modification of RFLP through the addition of adaptors to restriction enzyme-digested DNA, followed by PCR amplification and electrophoretic separation of PCR products. In this procedure, L. monocytogenes DNA is digested with two restriction enzymes, one with an average cutting frequency (e.g. EcoRI) and one with a higher cutting frequency (e.g. MseI or TaqI). The restriction fragments are ligated with double-stranded oligonucleotide adapters (i.e. linkers and indexers), and then amplified by PCR with adapter-specific primers. The resultant PCR products are separated by PAGE to generate highly informative, polymorphic patterns of 40–200 bands for individual L. monocytogenes strains. Apart from differentiating among the L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species, AFLP is also useful for separating L. monocytogenes strains into different genotypes. Overall, AFLP is highly discriminatory, sensitive and reproducible, thus representing a valuable tool in the characterization of L. monocytogenes strains, and also in the identification of Listeria species (Ripabelli et al., 2000; Guerra et al., 2002; Keto-Timonen et al., 2003). An obvious shortcoming of AFLP is its requirement for the ligation of linkers and indexers to enzyme-digested DNA from individual strains, which apart from being another time-consuming step, adds an extra dimension of uncertainty to the testing procedure.
PCR-RFLP.
In contrast to AFLP, in which L. monocytogenes DNA is digested with restriction enzymes, linked with adaptors to facilitate PCR amplification, PCR-RFLP undertakes PCR amplification of one or more L. monocytogenes housekeeping or virulence-associated genes (e.g. hly, actA and inlA), followed by digestion with selected restriction enzymes (e.g. HhaI, SacI or HinfI) and separation by agarose gel electrophoresis. Subsequent examination of the distinct band patterns permits differentiation of L. monocytogenes subtypes (Wiedmann et al., 1997). As it involves nucleic acid amplification, PCR-RFLP requires only a small amount of starting DNA. It has the added advantage over AFLP of obviating the need to ligate linkers and indexers before PCR amplification. Used in combination with other subtyping procedures, PCR-RFLP provides a sensitive, discriminatory and reproducible method for tracking and epidemiological investigation of L. monocytogenes bacteria.
REP-PCR.
Like other prokaryotic organisms, L. monocytogenes possesses a genome that contains randomly dispersed, repetitive sequence elements, such as repetitive extragenic palindromes (REPs) of 35–40 bp with an inverted repeat, and intergenic repeat units or enterobacterial repetitive intergenic consensus sequences (ERICs) of 124–147 bp with a highly conserved central inverted repeat. The REP and ERIC sequences represent useful primer binding sites for PCR amplification of the L. monocytogenes genome to achieve species and strain discrimination. Using REP-PCR, L. monocytogenes strains have been divided into four clusters that match the origin of isolation, each consisting of multiple subtypes (Jersek et al., 1999). As it produces a similar level of discrimination to PFGE and ribotyping techniques, REP-PCR offers a valuable alternative for the rapid subtyping of L. monocytogenes strains.
DNA sequencing-based subtyping techniques.
DNA sequencing of one or more selected genes is increasingly used for genetic subtyping of L. monocytogenes. MLST focuses on multiple genes or gene fragments (e.g. housekeeping or virulence-associated genes) to determine the subtypes and genetic relatedness of L. monocytogenes isolates. The availability of DNA sequencing data also aids the reconstruction of ancestral and evolutionary relationships among L. monocytogenes isolates. Compared to other typing methods, such as PFGE and ribotyping, MLST is less ambiguous and easier to interpret (Ward et al., 2004). With the cost of DNA sequencing decreasing rapidly, MLST is poised to play a more important role in L. monocytogenes subtyping and phylogenetic studies.
Although each of the subtyping procedures above represents on its own an elegant approach to the tracking of L. monocytogenes strains, the combined use of two or more procedures is generally more discriminatory and powerful than each applied alone. In terms of technical simplicity and test reliability, ribotyping and PCR-RFLP stand out clearly. Indeed, by using ribotyping together with PCR-RFLP and other subtyping procedures, L. monocytogenes strains have been grouped into three genetic lineages (or divisions), with lineage I consisting of serotypes 1/2b, 3b, 4b, 4d and 4e; lineage II of serotypes 1/2a, 1/2c, 3a and 3c; and lineage III of serotypes 4a and 4c (Rasmussen et al., 1995; Wiedmann et al., 1997, 2002; Jeffers et al., 2001; Nadon et al., 2001; Gray et al., 2004; Meinersmann et al., 2004). Interestingly, L. monocytogenes isolates from sporadic and endemic human listeriosis mostly belong to lineages I and II, whereas those from animal and environmental specimens are of lineage III. This information has been invaluable for tracking and population genetic studies of L. monocytogenes strains involved in disease outbreaks.
While a general consensus on the compositions and divisions of lineages I and II exists, there is some uncertainty concerning the make-up and taxonomic status of lineage III. Within lineage III, three subsets (i.e. G8.1, H7.1; G5.8, H7.1; and E/G5.8, H7.1) are delineated through Southern blot analysis of EcoRI-digested L. monocytogenes DNA with a probe from the rrnB rRNA operon of E. coli (Bruce et al., 1995). In turn, the subset G8.1, H7.1 is subdivided into seven ribotypes (dd0648, dd11903, dd8842, dd0652, dd11696, dd11698 and dd12388) by ribotyping hly, inlA and actA polymorphisms, with ribotype dd0648 being represented by ATCC 19114 (Wiedmann et al., 1997). On the other hand, while the subset G5.8, H7.1 comprises a single ribotype, dd3823, the subset E/G5.8, H7.1 includes two ribotypes, dd6821 and dd6824. Since the ribotype dd0648 strain ATCC 19114 in the subset G8.1, H7.1 demonstrates only a 72 % DNA–DNA homology with the L. monocytogenes type strain (barely above the 70 % DNA–DNA relatedness required for the phylogenetic definition of a species) and a 54 % homology with the L. innocua type strain, it has been postulated that ATCC 19114 is a new subspecies (Wiedmann et al., 1997). In addition, as strains in the subsets G5.8, H7.1 and E/G5.8, H7.1 possessing inlA and the G5.8 fragment are rhamnose negative, in contrast to the L. monocytogenes type strain, which is rhamnose positive, the existence of either two new subspecies or a new species has been also hypothesized (Wiedmann et al., 1996, 1997).
After the recent examination of seven lineage III strains from subsets G8.1, H7.1, G5.8, H7.1 and E/G5.8, H7.1, as well as ribotypes DUP1061 and DUP10142, by using PCR, Southern blot and DNA sequencing techniques, the following have become apparent: (a) ribotype dd0648 strains (ATCC 19114 and FSL-J1-031) in the subset G8.1, H7.1, and ribotype dd3823 strain (FSL-X1-008) in the subset G5.8, H7.1, as well as ribotype DUP1061 strain (FSL-J1-168), are clearly of L. monocytogenes serotypes 4a and 4c; (b) a close genetic relationship exists among three serotype 4c strains of ribotype dd0648 (FSL-J1-031) in the subset G8.1, H7.1, ribotype dd3823 (FSL-X1-008) in the subset G5.8, H7.1 and ribotype DUP1061 (FSL-J1-168) in comparison with the serotype 4a strain from ribotype dd0648 (ATCC 19114) in the subset G8.1, H7.1; (c) being rhamnose negative and possessing inlA, lmo2672 and ORF2819, strains of ribotypes dd6821 (FSL-X1-009) and dd6824 (FSL-X1-010) in the subset E/G5.8, H7.1 are likely of serotype 7, which may represent a distinct genotype (or subspecies) within the species of L. monocytogenes; and (d) ribotype DUP10142 strain (FSL-J1-158), being related to ribotypes dd3823 (subset G5.8, H7.1), dd6821 and dd6824 (subset E/G5.8, H7.1) through a shared reaction with lmo2672 primers, may constitute part of the genotype that encompasses ribotypes dd6821 and dd6824. Alternatively, because of its negative reaction with ORF2819 primers, ribotype DUP10142 strain (FSL-J1-158) may form another genotype (or subspecies) distinct from the one that covers ribotypes dd6821 and dd6824, which does not fit into the current serotype scheme for L. monocytogenes (Liu et al., 2006b).
In addition, the transcriptional regulator lmo2672 gene (Liu et al., 2004a) has also proven valuable as a target for the specific identification of uncommon L. monocytogenes lineage III strains [e.g. ribotypes dd6821 (FSL-X1-009), dd6824 (FSL-X1-010) and DUP10142 (FSL-J1-158)], as these strains are undetected by the species-specific lmo0733 primers (Liu et al., 2003a). Therefore, a combination of lmo0733 and lmo2672 primers in a multiplex PCR format will facilitate the detection of all L. monocytogenes strains, including the rare lineage III isolates, in a rapid, sensitive and specific manner (Liu et al., 2006b).
Virulence determination
Despite being pathogenic at the species level, L. monocytogenes is in fact made up of a spectrum of strains or genotypes with varying pathogenic potential. While many L. monocytogenes strains are highly pathogenic and sometimes deadly, others are relatively avirulent and cause little harm in the host. The availability of laboratory methods to accurately assess the pathogenic potential of L. monocytogenes strains is therefore vitally important to the effective control and prevention of listeriosis. Over the years, a variety of methods have been developed to gauge the virulence of L. monocytogenes strains. These include the mouse virulence assay, in vitro cell assays, and the detection of virulence-associated proteins and genes. These methods have not only contributed to the improved understanding of L. monocytogenes virulence and pathogenicity, but also helped devise appropriate control measures against listeriosis.
Mouse virulence assay The mouse virulence assay was one of the first methods described for L. monocytogenes virulence assessment. Being capable of providing an in vivo measurement of all virulent determinants, the mouse virulence assay is regarded as the gold standard for any newly developed tests for L. monocytogenes virulence (Nishibori et al., 1995; Pine et al., 1991; Roche et al., 2001; Liu et al., 2003a). In general, the mouse virulence assay is conducted by inoculating groups of mice with various doses of L. monocytogenes bacteria via the oral, nasal, intraperitoneal, intravenous or subcutaneous routes. The virulence of a given L. monocytogenes strain is determined by the mouse mortality resulting from infection, after estimation of c.f.u. by plate counts, and is commonly expressed as median lethal dose (LD50) (Reed & Muench, 1938; Welkos & O'Brien, 1994). Alternatively, the virulence of a given L. monocytogenes strain can be determined by the number of L. monocytogenes bacteria that reach the spleen following experimental infection.
Although an essential step in LD50 calculation, the estimation of c.f.u. by plate counts is a task requiring considerable attention and consistency, as it has a narrow margin of error. The observation that L. monocytogenes strains with varying levels of virulence often display vastly different growth rates on many selective media further exacerbates the problem (Gracieux et al., 2003). Even on a non-selective medium (e.g. BHI agar), L. monocytogenes strains causing greater mouse mortality tend to yield higher numbers of c.f.u. than those causing less or no mouse mortality (Liu, 2004). Thus, the number of c.f.u. for a given L. monocytogenes strain may vary from run to run and from laboratory to laboratory, resulting in different LD50 values for an identical test strain.
Recently, relative virulence (%) has been described as an alternative to LD50 measurement for the practical and direct interpretation of the mouse virulence assay for L. monocytogenes (Liu, 2004). The relative virulence (%) is obtained by dividing the number of dead mice by the total number of mice tested for a particular strain, using a known virulent strain (e.g. L. monocytogenes EGD) as reference. Being independent of c.f.u. estimation, the relative virulence (%) requires fewer dosage groups, and appears to give a more accurate assessment of L. monocytogenes virulence. That is, while the LD50 values provide a somewhat imprecise measure of L. monocytogenes virulence, the relative virulence (%) is much more direct and precise (Table 3). Nonetheless, given the high cost associated with animal experimentation, the mouse virulence assay is not routinely used for determining L. monocytogenes virulence.
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Detection of virulence-associated proteins and genes Early attempts to determine L. monocytogenes virulence through the detection of virulence-associated proteins and genes were largely unsuccessful, since many of the target proteins and genes are present in both virulent and avirulent strains. While in vitro demonstration of LLO, PC-PLC and PI-PLC activities often provides general guidance on the pathogenic potential of L. monocytogenes strains, its reliability as a virulence indicator is by no means satisfactory. Indeed, the association between haemolytic, PC-PLC and PI-PLC activities and in vitro virulence has not been convincingly established (Roche et al., 2001; Olier et al., 2002). Similarly, PCR detection of L. monocytogenes virulence-associated genes, such as inlA, inlB, actA, hly, plcA and plcB, and genetic lineage analysis has not resulted in a clear correlation between these genes and the underlying virulence of L. monocytogenes (Piffaretti et al., 1989; Nishibori et al., 1995; Rasmussen et al., 1995; Wiedmann et al., 1997; Jaradat et al., 2002). Although some naturally virulence-attenuated L. monocytogenes strains (particularly those isolated from human carrier cases) often contain mutations in their prfA, hly, actA and inlA genes, resulting in the expression of truncated or non-functional PrfA, LLO, ActA and InlA proteins (Olier et al., 2002; Roberts et al., 2005; Roche et al., 2005), targeting these gene mutations as a means of determining L. monocytogenes virulence does not constitute a sound option in practical terms. On the one hand, a screening assay for genetic alterations in multiple L. monocytogenes genes can be cumbersome and costly. On the other hand, many L. monocytogenes isolates will not be covered by the assay due to their lack of changes in the genes targeted. Therefore, an optimal strategy for L. monocytogenes virulence testing remains the detection of virulence-specific gene(s) that are present only in virulent strains, but absent in avirulent strains.
The recent completion of the whole genome sequences of several L. monocytogenes and L. innocua strains (Glaser et al., 2001; Nelson et al., 2004) has facilitated the identification of novel virulence-specific genes with potential for improved determination of L. monocytogenes virulence and pathogenicity (Liu et al., 2003a; Liu, 2004). Using PCR primers derived from the genes encoding putative transcriptional regulators (i.e. lmo0833, lmo1116, lmo1134 and lmo2672), putative internalins (i.e. lmo2821 and lmo2470) and unknown proteins (i.e. lmo0834 and lmo1188), L. monocytogenes virulent strains could be readily differentiated from avirulent strains. In particular, PCR targeting the putative internalin gene lmo2821 offers a rapid, sensitive and precise means of distinguishing virulent from avirulent L. monocytogenes strains (Table 3).
The virulence of L. monocytogenes as determined by PCR has subsequently been confirmed by a mouse model. Examination of the representative L. monocytogenes strains by a mouse virulence assay indicates that being the only serotype not recognized by lmo2821 primers, serotype 4a strains (e.g. HCC23, HCC25 and ATCC 19114) are unable to produce mouse mortality, and are thus truly non-pathogenic, while other serotype strains are more or less virulent (using relative virulence as a criterion) (Table 3) (Liu et al., 2003a; Liu, 2004). For instance, L. monocytogenes avirulent strains with a relative virulence of 0 % are negative by PCR, whereas L. monocytogenes virulent strains with a relative virulence of 30–100 % are positive by PCR targeting lmo2821. Recently, lmo2821 has been confirmed to be a novel internalin gene (inlJ) directly involved in L. monocytogenes virulence (Sabet et al., 2005). Being present in L. monocytogenes strains/serotypes that are capable of causing human listerial outbreaks and mouse mortality, but absent in avirulent, non-pathogenic strains/serotypes (Doumith et al., 2004b; Liu, 2004), lmo2821 (i.e. inlJ) represents the target of choice for laboratory differentiation of virulent from avirulent L. monocytogenes strains.
Future perspectives
Being an opportunistic intracellular pathogen capable of surviving various food manufacturing processes, L. monocytogenes has become recognized as a major cause of human foodborne infection during recent decades. As a consequence, a concerted research effort has been directed toward an improved understanding of the L. monocytogenes bacterium and its pathogenic mechanisms. This in turn has facilitated the development of laboratory procedures for enhanced identification, subtyping and virulence determination of L. monocytogenes, and contributed to the implementation of appropriate and effective control and prevention strategies against listeriosis.
As one of the earlier techniques developed, serotyping is a phenotype-based diagnostic technique that has made the early success in the detection and subtyping of L. monocytogenes possible. However, since serotyping measures the phenotypic characteristics of L. monocytogenes, it may sometimes give variable results (Schonberg et al., 1996; Liu et al., 2006a). Attempts have been made to design and apply PCR-based procedures for serotyping purposes, but these methods fall short of achieving serotype-specific determination of L. monocytogenes (Borucki & Call, 2003; Doumith et al., 2004a). Therefore, an improved understanding of the molecular mechanisms underlying the regulation of L. monocytogenes somatic (O) and flagellar (H) proteins is essential. Toward this goal, a novel L. monocytogenes serotype 4b-specific gene cassette (gltA–gltB) has been characterized (Lei et al., 2001; Kathariou, 2002). Further research on the genes encoding other serotype-specific antigens will help illuminate the mechanisms behind the production and regulation of L. monocytogenes serotype proteins, and lead to the development of novel molecular tests for reliable and precise determination of L. monocytogenes serotypes. In addition, considering that three strains (FSL-X1-009, FSL-X1-010 and FSL-J1-158) from ribotypes dd6821, dd6824 and DUP10142, respectively, appear phenotypically and genetically distinct from other members within the species of L. monocytogenes, but are probably related to serotype 7, further study is required to verify the taxonomic status of these strains and serotype 7 strains (e.g. the possibility of their being either one or two separate novel subspecies).
The application of various genetic subtyping procedures has resulted in the classification of L. monocytogenes strains into three lineages. While L. monocytogenes isolates from sporadic and endemic human listeriosis are often in lineage I (comprising serotypes 1/2b, 3b, 4b, 4d and 4e) and lineage II (containing serotypes 1/2a, 1/2c, 3a and 3c), those from animal and environmental specimens are of lineage III (consisting of serotypes 4c and 4a). However, in spite of being in the same genetic lineage III, serotypes 4c and 4a demonstrate marked differences in the mouse virulence assay. That is, while serotype 4c strains (e.g. ATCC 19116 and 874) display a relative virulence equal to that of the control virulent strain EGD (i.e. 100 %), serotype 4a strains (e.g. ATCC 19114 and HCC25) show a relative virulence of 0 % (Table 3) (Liu, 2004). The fact that a negligible number of serotype 4c isolates originate from human listeriosis cases suggests the inability of serotype 4c strains to establish in human hosts. As L. monocytogenes serotype 4c strains lack many virulence-specific putative transcriptional regulator genes in comparison with strains of other serotypes (with the exception of serotype 4a) (Liu et al., 2003a), it is possible that some key invasion-associated proteins are not produced by serotype 4c strains as a result. It appears that these proteins may be required for L. monocytogenes passage through the intestine and subsequent phases before the establishment of infection. Further experimentation is warranted to pinpoint the events at which L. monocytogenes serotype 4c strains fail to complete the infection cycle. This may involve characterization of the virulence-specific, putative transcriptional regulator genes and their protein products, as well as the identification of the genes controlled by these transcriptional regulators.
The internalin gene lmo2821 (i.e. inlJ) appears to be an excellent target for determining L. monocytogenes virulence, as strains capable of causing mouse mortality invariably harbour this gene (Table 3) (Liu et al., 2003a; Liu, 2004). With InlJ playing a direct role in L. monocytogenes virulence (Sabet et al., 2005), it is important to further examine the regulation of lmo2821 (inlJ) through identification of its possible regulator(s) and related proteins. In addition, while past research has largely been directed to elucidating the molecular mechanisms of L. monocytogenes virulence and pathogenicity, relatively little has been undertaken with regard to the inability of L. monocytogenes avirulent strains to cause disease. Given that both virulent and avirulent L. monocytogenes strains are equally resistant to acid (pH 3.0 or lower) (Liu et al., 2005a), it is likely that avirulent strains are capable of surviving the acidic stomach environment once ingested by hosts. Therefore, the inability of L. monocytogenes avirulent strains to result in listeriosis is most likely due to their failure to go through other key stages of infection, such as internalization, escape from vacuoles, intracellular growth and cell-to-cell spread. Extending our research to this area may help provide a more comprehensive picture of L. monocytogenes virulence.
ACKNOWLEDGEMENTS
This study was made possible with a grant from US Department of Agriculture Agricultural Research Service (Agreement No. 58-6202-5-083). The author is indebted to Drs M. L. Lawrence, A. J. Ainsworth and F. W. Austin for their interest in and support of the Listeria research project.
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