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。     

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.     

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.     

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.     

Influence of environmental parameters on phosphatidylcholine phospholipase C production in Listeria monocytogenes: a convenient method to differentiate L. monocytogenes from other Listeria species.

The ability to produce phosphatidylcholine phospholipase C (lecithinase) is associated with virulence in pathogenic species of Listeria. Levels of production vary greatly among members of the genus, and this virulence factor is not readily detectable in many members of the pathogenic species on conventional agar media containing egg yolk, a common substrate for the enzyme. In this study, the influence of a variety of environmental parameters, including temperature, pH, and salt concentration, on the production of lecithinase by a number of strains was evaluated. Lecithinase production by Listeria monocytogenes LO28 in brain heart infusion medium was optimal at 1.75 to 2.0% NaCl; pH 7.0 to 7.3, and 37 to 40 degrees C, and the presence of oxygen had no effect. In a chemically defined medium, the optimal NaCl concentration and temperature were lower at 0.75 to 1.0% NaCl and 33.5 degrees C. As detection of virulence factors is useful to assist in the identification and differentiation of Listeria species, this report shows that lecithinase activity can conveniently be detected within 36 h on a relatively inexpensive medium. Under the conditions described, L. monocytogenes could be distinguished from other members of the genus as a result of distinct lecithin degradation which was not evident in L. innocua, L. seeligeri, L. ivanovii, L. welshimeri, or L. murrayi/grayi.     

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.     

Listeria monocytogenes in pasteurized milk

An haemolytic Listeria monocytogenes strain pathogenic to mice was isolated from 6 out of 28 (21.4%) pasteurized milk samples (3.2% fat milk treated at 78 degrees C for 15 s) marketed by a Madrid processing plant. Listeria grayi was recovered from 25 of the samples (89.2%) and L. innocua from 3 samples (10.7%). One milk sample was contaminated with L. welshimeri. No strains of L. ivanovii, L. seeligeri, L. murrayi, or L. denitrificans were isolated. These results show that pathogenic Listeria strains can be isolated from pasteurized milk and reinforce the hypothesis that this food product may be the source of numerous human listeriosis.     

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.     

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.     

Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis

Seventy-three strains of the seven recognized Listeria species were studied by performing a multilocus enzyme electrophoresis analysis of 18 enzyme loci. The mean number of alleles per locus was 9.5 and all of the loci were polymorphic. A total of 56 electrophoretic types were distinguished. Cluster analysis of a matrix of the genetic distances between paired electrophoretic types revealed that there were six principal clusters at the species level (genetic distances between clusters greater than 0.8). Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Listeria seeligeri, and Listeria ivanovii each corresponded to one of these clusters with no overlap. Our results are in agreement with those of previous DNA hybridization experiments (Rocourt et al., Curr. Microbiol. 7:383-388, 1982). Listeria grayi and Listeria murrayi electrophoretic types formed a unique cluster, thus reinforcing the suggestion of Wilkinson and Jones (J. Gen. Microbiol. 98:399-421, 1977) that these two species should be considered two biovars of a single species.     

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.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

On the specificity of PCR detection of Listeria monocytogenes in food: a comparison of published primers

A total of nine pairs of primers, seven previously published and two newly developed, have been assayed for PCR detection of Listeria monocytogenes in food. They have been tested for specificity on a total of 72 strains including reference and food isolates belonging to L. monocytogenes and other species in the genus. First of all, a polyphasic approach has been carried out in order to establish a reference strain collection. They were biochemically and genetically characterized by API-Lis and randomly amplified polymorphic DNA PCR (RAPD-PCR), respectively. Random amplification of DNA was performed with M13, T7 and T3 universal primers and a data bank was created to compile the RAPD patterns of all the analyzed strains. The UPGMA cluster analysis of RAPD profiles with primer M13 showed eight clusters at 72.3% similarity. Clusters 2 and 7 corresponded to L. monocytogenes. Clusters 1 and 6 grouped L. ivanovii strains. Clusters 3, 4, 5 and 8 corresponded to L. grayi, L. innocua, L. welshimeri and L. seeligeri, respectively. Pattern analysis revealed the existence of miss-identified reference strains which was confirmed by 16S rDNA sequence analysis. RAPD-PCR is a rapid genetic test which helped to confirm strain identity. On the basis of PCR specificity results, primers LM1-LM2 were the best combination for the detection of L. monocytogenes since they only amplified the specific fragment in strains that had been genetically and biochemically assessed as belonging to the species. Specificity of other assayed primers is discussed.