Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR.

The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.     

Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.     

李斯特菌毒力因子及其进化

李斯特菌属包含6个种,毒力各有差异。在细菌耐受外界环境、黏附侵袭及细胞内感染过程中,毒力因子各司其职又相互协作。毒力基因常聚集为毒力岛,其中PrfA依赖型毒力基因簇(LIPI-1)与内化素岛(LIPI-2)是致病种最重要的两个毒力岛。李斯特菌各个种可能来源于同一个携带有完整毒力岛的祖先,在长期进化过程中,通过基因水平转移或重组、整合等事件,演化为目前流行的6个种。噬菌体、转座子、质粒等可能扮演着毒力进化执行者的角色。一些天然非典型菌株是目前研究的热点,如含有LIPI-1的无害李斯特菌和缺失LIPI-1的塞氏李斯特菌,其演化进程可能尚未达到或已超越目前流行的状态,为李斯特菌毒力进化的研究提供了重要线索。     

Structural and functional properties of the p60 proteins from different Listeria species.

The major extracellular protein p60 of Listeria monocytogenes seems to be required for this microorganism's adherence to and invasion of 3T6 mouse fibroblasts but not for adherence to human epithelial Caco-2 cells. Western blot analysis with polyclonal antibodies against p60 of L. monocytogenes indicated the presence of cross-reacting proteins in the culture supernatants of all Listeria species. Protein p60 of L. monocytogenes could restore adhesion of the L. monocytogenes mutant RIII (impaired in the synthesis of p60) to mouse fibroblasts more efficiently than that of Listeria grayi. The amino acid sequences of the p60-related proteins of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi indicated highly conserved regions of about 120 amino acids at both the N-terminal and the C-terminal ends. The middle portions of these proteins, consisting of about 240 amino acids, varied considerably. These parts include the repeat domain consisting of repetitions of Thr (T) and Asn (N) which was present only, albeit in different arrangements, in the p60 proteins of L. monocytogenes and L. innocua. The p60-related proteins of L. grayi, L. ivanovii, L. seeligeri, and L. welshimeri each contained an insertion of 54 amino acids which was absent in the p60 proteins of L. monocytogenes and L. innocua.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Isolation and PCR amplification of a species-specific oxidoreductase-coding gene region in Listeria grayi

Listeria grayi is a nonpathogenic Gram-positive bacterium that demonstrates considerable similarities to other members in the genus Listeria, including the foodborne human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. A rapid diagnostic test to identify and diagnose listeriosis would be valuable, especially in cases where the presence of L. grayi may complicate diagnosis. This test would be based on a unique gene present in L. grayi. In this study, after comparative screening of a recombinant L. grayi DNA library by dot blot hybridization, an L. grayi specific clone (lgr20-246) with an insert of 722 bp was isolated. By applying PCR primers derived from a distinct region of the clone not shared by other bacteria, a specific band of 420 bp was amplified from the genomic DNA of L. grayi only and not of other Listeria species or common bacteria. These results suggest that the PCR assay employing primers lgr20-246F and lgr20-246R provides an independent and precise means of distinguishing L. grayi from other Listeria species and common bacteria. Therefore, it would be another useful technique for laboratory differentiation of Listeria bacteria.     

Monoclonal antibodies which identify a genus-specific Listeria antigen.

Fifteen murine monoclonal antibodies (MAbs) which react specifically with a protein antigen found in all species of Listeria were developed and characterized. These MAbs were tested extensively by both enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses for cross-reaction with non-Listeria organisms, such as Staphylococcus, Streptococcus, Citrobacter, Pseudomonas, and Salmonella species, and were found to be nonreactive. The genus-specific antigen was identified as a heat-stable protein with a molecular weight in the range of 30,000 to 38,000 (under both reducing and nonreducing conditions), depending on the species of Listeria tested. In Listeria monocytogenes, L. innocua, L. ivanovii, and L. seeligeri the antigen has a molecular weight of approximately 30,000 to 34,000. In L. grayi and L. murrayi it has a molecular weight of approximately 35,000 to 38,000. In addition, several of the MAbs recognize lower-molecular-weight protein bands. There appear to be at least two groups of Listeria-specific MAbs based upon isotype and results of enzyme-linked immunosorbent assay and Western blot analyses. These MAbs have proven to be useful in the development of a diagnostic assay for Listeria species in food products.     

Monoclonal antibodies which identify a genus-specific Listeria antigen

Fifteen murine monoclonal antibodies (MAbs) which react specifically with a protein antigen found in all species of Listeria were developed and characterized. These MAbs were tested extensively by both enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses for cross-reaction with non-Listeria organisms, such as Staphylococcus, Streptococcus, Citrobacter, Pseudomonas, and Salmonella species, and were found to be nonreactive. The genus-specific antigen was identified as a heat-stable protein with a molecular weight in the range of 30,000 to 38,000 (under both reducing and nonreducing conditions), depending on the species of Listeria tested. In Listeria monocytogenes, L. innocua, L. ivanovii, and L. seeligeri the antigen has a molecular weight of approximately 30,000 to 34,000. In L. grayi and L. murrayi it has a molecular weight of approximately 35,000 to 38,000. In addition, several of the MAbs recognize lower-molecular-weight protein bands. There appear to be at least two groups of Listeria-specific MAbs based upon isotype and results of enzyme-linked immunosorbent assay and Western blot analyses. These MAbs have proven to be useful in the development of a diagnostic assay for Listeria species in food products.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Taxonomic studies on Brochothrix, Erysipelothrix, Listeria and atypical lactobacilli

One hundred and eighty-seven strains of listeriae, atypical lactobacilli, Brochothrix, Erysipelothrix and related Gram-positive bacteria were tested for 140 characters based on morphology, physiology and biochemistry. Computer analyses of the data resulted in the recovery of 20 phenons. Representative strains were examined for cytochrome content, cell wall, fatty acid and isoprenoid quinone composition and the G + C content of the DNA. The results indicate that the species Listeria monocytogenes, L. innocua and L. ivanovii are phenotypically very similar. L. grayi and L. murrayi are more distinct from these but are so similar to each other that L. murrayi should be regarded as a subspecies of L. grayi. Some of the atypical lactobacilli studied are members of the genus Brochothrix; the others probably represent four distinct species in a new genus.     

Identification of hypervariable and conservative fragments of Listeria genome

The computer analysis revealed hypervariable and highly conservative fractions in the genes of Gram-positive bacteria of the Listeria genus. As a result of analysis of gene iap coding protein p60, PCR based test systems for detection of 6 Listeria species, L. monocytogenes, L. seeligeri, L. ivanovii, L. innocua, L. grayi and L. welshimeri have been developed. Species-specific and conservative gene fragments coding Listeria pathogenicity factors, listeriolysin and cytolysin, were detected. The sets of primers for detection and gene typing of L. monocytogenes, L. seeligeri and L. ivanovii containing cytolysin have been made. The gene typing of Listeria may be carried out in one reaction with the use of multiplex PCR: amplified fragments for different Listeria species differ in the length of the amplified product. The developed sets of primers have a 95-100% degree of homology and may be recommended for the detection and gene typing of Listeria.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Serological studies on Listeria grayi and Listeria murrayi

A cross-agglutination study between somatic antigens from reference strains of Listeria grayi and Listeria murrayi with rabbit antisera was done. L. murrayi antisera reacted, at low titres, with L. grayi but L. grayi antisera did not react with L. murrayi antigen. These results, together with agglutinin-absorption tests, led to the conclusion that the serologic relationship between L. grayi and L. murrayi is not as close as is thought. The two species seem to differ in at least one somatic factor, that might be designated O-XVI for L. grayi and O-XVII for L. murrayi. The serologic relationship of L. grayi and L. murrayi with other serovars of Listeria is discussed. The agglutination titre of 180 healthy ruminants against O-antigens of L. grayi and L. murrayi was also investigated; almost all the sera reacted with the antigens of these species, with similar titres (that reached 640) to those detected against O-antigens of serogroups 1/2 and 4.     

Serological studies on Listeria grayi and Listeria murrayi

A cross-agglutination study between somatic antigens from reference strains of Listeria grayi and Listeria murrayi with rabbit antisera was done. L. murrayi antisera reacted, at low titres, with L. grayi but L. grayi antisera did not react with L. murrayi antigen. These results, together with agglutinin-absorption tests, led to the conclusion that the serologic relationship between L. grayi and L. murrayi is not as close as is thought. The two species seem to differ in at least one somatic factor, that might be designated O-XVI for L. grayi and O-XVII for L. murrayi. The serologic relationship of L. grayi and L. murrayi with other serovars of Listeria is discussed. The agglutination titre of 180 healthy ruminants against O-antigens of L. grayi and L. murrayi was also investigated; almost all the sera reacted with the antigens of these species, with similar titres (that reached 640) to those detected against O-antigens of serogroups 1/2 and 4.     

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Specific gene probe for detection of biotyped and serotyped Listeria strains

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Isolation of micro-organisms of the species Listeria from raw milk intended for human consumption

Refrigerated mixtures of raw milk provided by a dairy which was supplied by farms from west and central Spain were tested for the presence of Listeria microorganisms. A total of 95 samples were taken at regular intervals over a 16-month period. Listeria grayi was isolated from 89.5% of the samples, Listeria monocytogenes s. str. from 45.3%, Listeria innocua from 15.8%, Listeria welshimeri from 3.1%, and Listeria seeligeri from 1.05%. Listeria ivanovii, Listeria murrayi, and Listeria denitrificans were not isolated.