Molecular characteristics and virulence potential of Listeria monocytogenes isolates from Chinese food systems.

In this study, we examined Listeria monocytogenes isolates from Chinese food sources in an attempt to gain further insights on the molecular characteristics and virulence potential of this important foodborne pathogen. Of the 88 L. monocytogenes food isolates recovered, 42 (47.7%) were of serovars 1/2a or 3a; 23 (26.1%) of serovars 1/2b or 3b; 15 (17.0%) of 1/2c or 3c; 6 (6.8%) of serovars 4b, 4d or 4e; and 2 (2.2%) of serovars 4a or 4c. In contrast to inlAB locus conserved in all serovars, internalin cluster between ascB and dapE varies with different serovars, with inlC2DE, inlGC2DE and inlGHE predominantly in serovars 1/2b or 4b, serovar 1/2a and serovar 1/2c. While inlF existed in all the inlGHE- and inlGC2DE-containing isolates but 17.4% of those having inlC2DE, lmo2026 existed in all the inlGHE-containing isolates but 20.0% of those bearing inlGC2DE, suggesting that inlF might have co-evolved with inlGC2DE and inlGHE while lmo2026 with inlGHE only. With the exception of serovar 4a isolate, most serovar isolates demonstrated remarkable ability to form plaques on L929 cells and produced significant mouse mortality irrespective of the internalin gene organization and whether an intact actA gene is present or not. These results indicate that majority of these food isolates may have the potential to cause human diseases if ingested via contaminated foods. Given that serovar 4b accounts for nearly half of human clinical listeriosis cases documented, the relative low proportion of serovar 4b food isolates suggests that this serovar is probably more tolerant of the adverse conditions in the host's stomach and/or more efficient in entering host cells than serovars 1/2a, 1/2b and 1/2c.

Listeria monocytogenes serovar 4a is a possible evolutionary intermediate between L. monocytogenes serovars 1/2a and 4b and L. innocua.

The genus Listeria consists of six closely related species and forms three phylogenetic groups: L. monocytogenes- L. innocua, L. ivanovii-L. seeligeri-L. welshimeri, and L. grayi. In this report, we attempted to examine the evolutionary relationship in the L. monocytogenes-L. innocua group by probing the nucleotide sequences of 23S rRNA and 16S rRNA, and the gene clusters lmo0029-lmo0042, ascBdapE, rplS-infC, and prs-ldh in L. monocytogenes serovars 1/2a, 4a, and 4b, and L. innocua. Additionally, we assessed the status of L. monocytogenes-specific inlA and inlB genes and 10 L. innocua-specific genes in these species/serovars, together with phenotypic characterization by using in vivo and in vitro procedures. The results indicate that L. monocytogenes serovar 4a strains are genetically similar to L. innocua in the lmo0035-lmo0042, ascB-dapE, and rplS-infC regions and also possess L. innocua-specific genes lin0372 and lin1073. Furthermore, both L. monocytogenes serovar 4a and L. innocua exhibit impaired intercellular spread ability and negligible pathogenicity in mouse model. On the other hand, despite resembling L. monocytogenes serovars 1/2a and 4b in having a nearly identical virulence gene cluster, and inlA and inlB genes, these serovar 4a strains differ from serovars 1/2a and 4b by harboring notably altered actA and plcB genes, displaying strong phospholipase activity and subdued in vivo and in vitro virulence. Thus, by possessing many genes common to L. monocytogenes serovars 1/2a and 4b, and sharing many similar gene deletions with L. innocua, L. monocytogenes serovar 4a represents a possible evolutionary intermediate between L. monocytogenes serovars 1/2a and 4b and L. innocua.

Preparation of Listeria monocytogenes specimens for molecular detection and identification.

Listeria monocytogenes is a common foodborne pathogen that has the capacity to cause severe clinical illness in vulnerable human population groups. The availability of rapid and specific laboratory tests to identify this bacterium is essential for preventing an otherwise easily treated malaise from developing into a life-threatening disease. To this end, a variety of rapid, sensitive and precise nucleic acid-based assays have been developed, contributing to the improved diagnosis of listeriosis. Nonetheless, since many molecular assays rely on enzymatic reaction for template amplification, which is liable to interference from inhibitory substances present in clinical, food and environmental specimens, they often require purified nucleic acids as starting material for test consistency. As a consequence, considerable efforts have been directed toward the development of innovative and efficient sample handling procedures that reduce and eliminate inhibitory elements present in the specimens. By reviewing the recent progresses in the sample preparation methods that have been described for enhanced molecular detection and identification of L. monocytogenes, including rapid procedures for cultured isolates, more elaborate techniques for processing clinical, food and environmental samples, and specific considerations in preparing samples for quantitative PCR analysis, this article highlights further research requirement in the specimen processing protocols that form the basis for continued improvement in the overall performance of molecular assays for listeriosis.

A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes.

Listeria monocytogenes internalin gene inlJ has been described previously for differentiation of virulent from avirulent strains. However, a recent report indicated that there exist some unusual lineage IIIB strains (e.g., serotype 7 strain R2-142) that possess no inlJ gene but have the capacity to cause mouse mortality via intraperitoneal inoculation. Therefore, a multiplex PCR incorporating inlA, inlC and inlJ gene primers was developed in this study for rapid speciation and virulence determination of L. monocytogenes. Although inlB gene was also assessed for species-specific recognition, it was not included in the multiplex PCR due to the negative reaction observed between the inlB primers and serotypes 4a-e strains. The species identity of the 36 L. monocytogenes strains under investigation was verified through the amplification of an 800 bp fragment with the inlA primers and the virulence of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ primers, respectively. Whereas L. monocytogenes pathogenic strains with capacity to cause mortality (showing relative virulence of 30-100%) in A/J mice via the intraperitoneal route were invariably detected by the inlC and/or inlJ primers, naturally non-pathogenic strains (showing relative virulence of 0%) were negative with these primers. While 8 of the 10 L. ivanovii strains reacted with the inlC primers, they could be effectively excluded as non-L. monocytogenes through their negative reactions with the inlA primers in the multiplex PCR. Thus, the use of the multiplex PCR targeting inlA, inlC and inlJ genes facilitates simultaneous confirmation of L. monocytogenes species identity and virulence.

Toward an improved laboratory definition of Listeria monocytogenes virulence.

Listeria monocytogenes is an opportunistic foodborne pathogen that encompasses a diversity of strains with varied virulence. The ability to rapidly determine the pathogenic potential of L. monocytogenes strains is integral to the control and prevention campaign against listeriosis. Early methods for assessing L. monocytogenes virulence include in vivo bioassays and in vitro cell assays. While in vivo bioassays provide a measurement of all virulence determinants of L. monocytogenes, they are not applied routinely due to their reliance on experimental animals whose costs have become increasingly prohibitive. As a low cost alternative, in vitro cell assays are useful for estimating the virulence of L. monocytogenes strains. However, these assays are often slow, and at times variable. Prior attempts to ascertain L. monocytogenes virulence by targeting virulence-associated proteins and genes have been largely unsuccessful, since many of the assay targets are present in both virulent and avirulent strains. Recent identification of novel virulence-specific genes (particularly internalin gene inlJ) has opened a new avenue for rapid, sensitive, and precise differentiation of virulent L. monocytogenes strains from avirulent strains. The application of DNA sequencing technique also offers an additional tool for assessing L. monocytogenes virulence potential. By providing an update on the laboratory methods that have been reported for the determination of L. monocytogenes pathogenicity, this review discusses future research needs that may help achieve an improved laboratory definition of L. monocytogenes virulence.

Identification, subtyping and virulence determination of Listeria monocytogenes, an important foodborne pathogen.

Listeria monocytogenes is an opportunistic intracellular pathogen that has become an important cause of human foodborne infections worldwide. Given its close relationship to other Listeria species and its tendency to produce non-specific clinical symptoms, the availability of rapid, sensitive and specific diagnostic tests for the differentiation of L. monocytogenes from other Listeria species is helpful for selecting appropriate treatment regimens. In addition, with L. monocytogenes comprising a diversity of strains of varying pathogenicity, the ability to precisely track the strains involved in listeriosis outbreaks and speedily determine their pathogenic potential is critical for the control and prevention of further occurrences of this deadly disease. Extensive research in recent decades has revealed significant insights regarding the molecular mechanisms of L. monocytogenes infection. This in turn has facilitated the development of laboratory procedures for enhanced detection and identification of L. monocytogenes, and has also contributed to the implementation of improved control and prevention strategies against listeriosis. The purpose of this review is to summarize recent progress in the species-specific identification, subtyping and virulence determination of L. monocytogenes strains, and to discuss future research needs pertaining to these important areas of listeriosis.

PCR amplification of a species-specific putative transcriptional regulator gene reveals the identity of Enterococcus faecalis.

Enterococcus faecalis, an opportunistic bacterial pathogen, has become prominent in recent decades due to its ability to develop resistance to various antibiotics. The ability to rapidly identify E. faecalis, followed by prompt treatment, is therefore vital for the control of this pathogen. In this report, upon comparative BLAST search of the E. faecalis genome against other bacterial DNA sequences at GenBank, an E. faecalis-specific gene Ef0027, that codes for a putative phosphosugar binding transcriptional regulator, was identified. Using PCR primers derived from this gene (i.e., Ef0027F/R), a specific DNA fragment of 518 bp was detected in E. faecalis strains only, and it was not found in other Enterococcus species and common bacteria. These results suggest that PCR amplification of the putative transcriptional regulator gene Ef0027 provides a useful means of identifying E. faecalis from other bacteria.

Rapid identification of Streptococcus pyogenes with PCR primers from a putative transcriptional regulator gene.

Streptococcus pyogenes (GAS) is a common bacterial pathogen that has emerged as an increasingly important health concern in many parts of the world. Although GAS may appear harmless in healthy individuals, the ability of this bacterium to take advantage of a weakened or compromised host defense system is extraordinary. Following the recent publication of the genome sequences of several S. pyogenes strains, we undertook an investigation of a specialized gene group in GAS that encodes transcriptional regulators. By screening S. pyogenes transcriptional regulator genes from the complete genome of M1 strain SF370 against other DNA sequences at GenBank by BLAST searches, we identified a gene (i.e., Spy1258) that is uniquely present in the bacterium. Application of PCR primers (spy1258F and spy1258R) derived from this gene facilitated amplification of a 407-bp DNA fragment from S. pyogenes only, but not from other species of the genus Streptococcus and common bacteria. Apart from offering an additional target for specific confirmation of GAS, further analysis of the putative transcriptional regulator gene Spy1258 and its related protein product may lead to new insights into the molecular mechanisms of S. pyogenes maintenance and pathogenicity.

Comparative assessment of acid, alkali and salt tolerance in Listeria monocytogenes virulent and avirulent strains.

Listeria monocytogenes is an opportunistic bacterial pathogen of man and animals that has the capacity to survive under extreme environmental conditions. While our knowledge on L. monocytogenes and its ability to sustain within wide pH and temperature ranges and salt concentrations has been largely built on the virulent strains of this species, relatively little is known about avirulent strains in this regard. In this study, we extend our analysis on avirulent L. monocytogenes strains. By subjecting three virulent (EGD, 874 and ATCC 19196) and three avirulent (ATCC 19114, HCC23 and HCC25) strains to various pH and salt concentrations, it was found that L. monocytogenes recovered well after treatment with 100 mM Tris at pH 12.0, and to a lesser extent at pH 3.0. Interestingly, avirulent L. monocytogenes strains showed a somewhat higher tolerance to alkali than virulent strains. This unique feature of avirulent L. monocytogenes strains may potentially be exploited for the development of a rapid technique for differentiation between avirulent and virulent strains. Furthermore, all L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M, or 40% w/v) for a long period of time (20 h and possibly longer). Together, these results highlight that acid, alkali, and/or salt treatments commonly used in food product processing may not be sufficient to eliminate L. monocytogenes, and therefore stringent quality control measures at the beginning and end of the food manufacturing process is essential to ensure that such food products are free of listerial contamination.

Species-specific PCR determination of Listeria seeligeri.

Listeria seeligeri is a non-pathogenic bacterium coming under the genus Listeria. As this bacterium resembles other Listeria species such as L. monocytogenes and L. ivanovii that are pathogenic to man and animals, it is important that rapid and precise identification techniques be available for L. seeligeri in cases where such determination is desirable. A specific molecular test on the basis of a uniquely present gene region in L. seeligeri will be of particular value under the circumstances. In this report, after comparative screening of genomic DNA from six Listeria species by dot blot hybridization, we isolated one L. seeligeri-specific clone (lse24-315) that contains an insert of 1538 bp. Using primers (lse24-315F and lse24-315R) derived from this clone, we showed that a specific PCR product of 375 bp was generated from genomic DNA of L. seeligeri strains only, but not of other Listeria species or common bacteria. Therefore, the PCR employing primers lse24-315F and lse24-315R provides a rapid, sensitive and specific method for distinguishing L. seeligeri from other Listeria and common bacteria.

Listeria monocytogenes: comparative interpretation of mouse virulence assay.

Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity. The availability of laboratory procedures to ascertain the pathogenic potential of L. monocytogenes bacteria would greatly enhance the control and prevention of listerial infections. As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L. monocytogenes virulence. The pathogenic potential of a given L. monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD(50)). In this report, we describe an alternative method [i.e., relative virulence (%)] that does not involve CFU estimation, and is comparable to LD(50) for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L. monocytogenes EGD) as reference. Besides providing a more direct interpretation in comparison with LD(50) values for mouse virulence assay, this method requires fewer dosage groups per L. monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories.

Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene.

Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L. monocytogenes. The presence of L. innocua may cause difficulty or confusion in the laboratory identification of L. monocytogenes or other Listeria spp. In this report, through examining the recently published genome sequence of L. innocua strain CLIP 11262 (serovar 6a), we identified a L. innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR). The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L. innocua strains only. We expect that this assay will be useful in confirming identification of L. innocua or in studies where rapid detection of L. innocua is necessary.

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes.

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes.

Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches. After comparison of transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P. multocida. By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P. multocida only, but not from other bacteria. These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P. multocida from other bacteria. Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P. multocida virulence and pathogenicity.

Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes.

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.

PCR detection of a putative N-acetylmuramidase gene from Listeria ivanovii facilitates its rapid identification.

Listeria ivanovii is a Gram-positive bacterial pathogen that is capable of causing abortions and stillbirths in farm animals, particularly sheep and cattle. In terms of morphological, biochemical and molecular characteristics, L. ivanovii resembles other Listeria species such as L. monocytogenes, a pathogen of both man and animals. In this study, through comparative analysis of genomic DNA from the six Listeria species, a L. ivanovii specific clone (liv22-228) containing a 946 bp insert was isolated. This clone contained the 5' ends of two divergently transcribed L. ivanovii genes and an intergenic spacer region, similar in organization to homologous regions from the L. innocua and L. monocytogenes genomes. Regions of low homology in the clone were identified by comparing to the L. innocua and L. monocytogenes genomes, and oligonucleotide primers (liv22-228F and liv22-228R) were designed. These primers amplified a 463 bp band from genomic DNA of L. ivanovii strains only, but not from other Listeria species or common bacteria. Thus, PCR employing L. ivanovii specific primers (liv22-228F and liv22-228R) provides a useful and straightforward method for rapid and precise determination of L. ivanovii.

Molecular approaches to the identification of pathogenic and nonpathogenic listeriae.

The genus Listeria consists of a closely related group of Gram-positive bacteria that commonly occur in the environment and demonstrate varied pathogenic potential. Of the 10 species identified to date, L. monocytogenes is a facultative intracellular pathogen of both humans and animals, L. ivanovii mainly infects ungulates (eg., sheep and cattle), while other species (L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, L. rocourtiae, L. fleischmannii and L. weihenstephanensis) are essentially saprophytes. Within the species of L. monocytogenes, several serovars (e.g., 4b, 1/2a, 1/2b and 1/2c) are highly pathogenic and account for a majority of clinical isolations. Due to their close morphological, biological, biochemical and genetic similarities, laboratory identification of pathogenic and nonpathogenic Listeria organisms is technically challenging. With the development and application of various molecular approaches, accurate and rapid discrimination of pathogenic and nonpathogenic Listeria organisms, as well as pathogenic and nonpathogenic L. monocytogenes strains, has become possible.

Characteristics of cell-mediated, anti-listerial immunity induced by a naturally avirulent Listeria monocytogenes serotype 4a strain HCC23.

The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did not. Compared with EGD, HCC23 induced a relatively low level of gamma interferon (IFN-gamma) in mice, and ATCC 15313 stimulated no detectable IFN-gamma. The percentages of gated CD4 T cells from mice immunized with EGD and HCC23 showed a notable drop (to 30%) at 21 days post exposure in comparison with that (about 50%) from ATCC 15313-injected or untreated mice; and the percentage of gated NK cells from EGD-immunized group was markedly higher than those from other treatment groups. Mice immunized with HCC23 and EGD developed an equally strong protective immunity against listeriosis that was effective in both short and long terms, but those injected with ATCC 15313 or saline succumbed to listeriosis within 6 days of challenge.

Listeria-based anti-infective vaccine strategies.

Listeria monocytogenes is a Gram-positive intracellular bacterium that has accounted for a significant proportion of human foodborne infections in recent decades. Although infection with sublethal level of L. monocytogenes generates enduring immunity, it is impractical to apply intact virulent strains as vaccine. Through use of killed, attenuated, naturally avirulent, subcellular and DNA vaccine preparations, significant protection has been achieved in experimental animals against listeriosis. Being a robust bacterium capable of eliciting all aspects of cell-mediated immunity, L. monocytogenes has the potential to become an ideal vector for vaccine delivery against other infective agents. Furthermore, with a high safety threshold, L. monocytogenes has increasingly been recognized as a vaccine vector of choice for a range of bacterial, viral and parasitic pathogens. Not surprisingly, a large number of patents have been filed in recent years that exploit attenuated or naturally avirulent L. monocytogenes strains or its listeriolysin (LLO) protein to enhance specific T cell response in the vaccine recipients or to direct vaccine molecule to the cytoplasm of host cells. Following a brief review on the key features of L. monocytogenes infection and host immune responses to listeriosis, this article summarizes the current state and recent progresses in the Listeria-based vaccine strategies that have been developed against bacterial, viral and parasitic diseases, together with a discussion on the future development trends in the utilization of L. monocytogenes as vaccine vector.

Listeria monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.