Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp.

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We constructed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified two recombinant BACs from the L. monocytogenes pUvBBAC library that each contained the entire virulence gene cluster (vgc) of L. monocytogenes and transferred them to a nonpathogenic Listeria innocua strain. Recombinant L. innocua strains harboring pUvBBAC+vgc1 and pUvBBAC+vgc2 produced the vgc-specific listeriolysin (LLO) and actin assembly protein ActA and represent the first reported cloning of the vgc locus in its entirety. The use of the novel broad-host-range BAC vector pUvBBAC extends the versatility of this technology and provides a powerful platform for detailed functional genomics of gram-positive bacteria as well as its use in explorative functional metagenomics.     

Protective immunity to Listeria monocytogenes infection mediated by recombinant Listeria innocua harboring the VGC locus

In this study we propose a novel bacterial vaccine strategy where non-pathogenic bacteria are complemented with traits desirable for the induction of protective immunity. To illustrate the proof of principle of this novel vaccination strategy, we use the model organism of intracellular immunity Listeria. We introduced a, low copy number BAC-plasmid harbouring the virulence gene cluster (vgc) of L. monocytogenes (Lm) into the non-pathogenic L. innocua (L.inn) strain and examined for its ability to induce protective cellular immunity. The resulting strain (L.inn::vgc) was attenuated for virulence in vivo and showed a strongly reduced host detrimental inflammatory response compared to Lm. Like Lm, L.inn::vgc induced the production of Type I Interferon's and protection was mediated by Listeria-specific CD8(+) T cells. Rational vaccine design whereby avirulent strains are equipped with the capabilities to induce protection but lack detrimental inflammatory effects offer great promise towards future studies using non-pathogenic bacteria as vectors for vaccination.     

Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

A sheep in wolf's clothing: Listeria innocua strains with teichoic acid-associated surface antigens and genes characteristic of Listeria monocytogenes serogroup 4

Listeria monocytogenes serotype 4b has been implicated in numerous food-borne epidemics and in a substantial fraction of sporadic listeriosis. A unique lineage of the nonpathogenic species Listeria innocua was found to express teichoic acid-associated surface antigens that were otherwise expressed only by L. monocytogenes of serotype 4b and the rare serotypes 4d and 4e. These L. innocua strains were also found to harbor sequences homologous to the gene gtcA, which has been shown to be essential for teichoic acid glycosylation in L. monocytogenes serotype 4b. Transposon mutagenesis and genetic studies revealed that the gtcA gene identified in this lineage of L. innocua was functional in serotype 4b-like glycosylation of the teichoic acids of these organisms. The genomic organization of the gtcA region was conserved between this lineage of L. innocua and L. monocytogenes serotype 4b. Our data are in agreement with the hypothesis that, in this lineage of L. innocua, gtcA was acquired by lateral transfer from L. monocytogenes serogroup 4. The high degree of nucleotide sequence conservation in the gtcA sequences suggests that such transfer was relatively recent. Transfer events of this type may alter the surface antigenic properties of L. innocua and may eventually lead to evolution of novel pathogenic lineages through additional acquisition of genes from virulent listeriae.     

Comparison of selective and non-selective enrichment media in the detection of Listeria monocytogenes from meat containing Listeria innocua

AIMS: This study investigated whether the higher incidence of recovery from meat of Listeria innocua compared with L. monocytogenes could be due to the laboratory media used, leading to an artificially lower detection of the pathogenic species, L. monocytogenes. METHODS AND RESULTS: Minced beef was inoculated with L. monocytogenes, L. innocua, or a mixture of these species, and stored at 0 or 10 degrees C under vacuum or aerobic conditions for up to 28 days. Listeria were recovered from the minced beef using selective (University of Vermont Medium, UVM) and non-selective (Buffered Peptone Water, BPW) enrichment broths after 0, 14, and 28 days of storage. In general, there were no significant differences (P < 0.05) between the numbers of L. monocytogenes recovered from minced beef samples after 24 h enrichment in BPW and the numbers recovered using UVM. In addition, the presence of L. innocua in meat samples containing L. monocytogenes did not significantly (P < 0.05) affect the numbers of L. monocytogenes recovered using either enrichment broth. In most cases there were no significant differences (P < 0.05) between the numbers of L. innocua recovered from minced beef samples after 24 h enrichment in BPW compared with numbers recovered using UVM. CONCLUSION: Listeria innocua was found to have no significant competitive advantage over L. monocytogenes in selective or non-selective enrichment media. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that, in some instances, the use of a selective enrichment broth offers no advantage over a non-selective enrichment broth for the recovery of Listeria species from minced beef.     

Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.     

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

Characteristics of maltose transporter activity in an ale and larger strain of the yeast Saccharomyces cerevisiae

R.R. BEUMER, M.C. TE GIFFEL, M.T.C. KOK AND F.M. ROMBOUTS. 1996. All confirmation and identification methods used in this study can be used for the screening of suspected colonies on isolation media for Listeria spp. In traditional enrichment procedures the Microscreen Listeria latex test gives fast results. The DNA probes (Accuprobe and Gene-Trak) are very specific in detecting Listeria monocytogenes . For identification of Listeria spp. both tests (API and Micro-ID) performed equally well. Preference may be given to the API test, since differentiation of L. monocytogenes from L. innocua is based on the absence of arylamidase, through which tests for haemolytic activity and/or CAMP reactions can be omitted. However, the use of Enhanced Haemolysis Agar as isolation medium makes further testing essentially superfluous, since L. monocytogenes strains can be differentiated from L. innocua .     

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.     

Quantification of Listeria spp. contamination on shell and flesh of cooked black tiger prawns (Penaeus monodon)

AIMS: To quantify Listeria levels on the shell and flesh of artificially contaminated cooked prawns after peeling, and determine the efficacy of Listeria innocua as a model for L. monocytogenes in this system. METHODS AND RESULTS: A L. monocytogenes and L. innocua strain were inoculated separately onto cooked black tiger prawns using two protocols (immersion or swabbing with incubation). Prawns were peeled by two methods (gloved hand or scalpel and forceps) and numbers of Listeria on shells, flesh and whole prawn controls were determined. Prawns were exposed to crystal violet dye to assess the penetration of liquids. Regardless of preparation method or bacterial strain there were ca 1log10 CFU more Listeria per shell than per peeled prawn. Dye was able to penetrate to the flesh in all cases. CONCLUSIONS: Shell-on prawns may be only slightly safer than shell-off prawns. Listeria innocua is an acceptable model for L. monocytogenes in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: Reduced risk from L. monocytogenes on prawns can only be assured by adequate hygiene or heating.     

Behaviour of the pathogen surrogates Listeria innocua and Clostridium sporogenes during production of parsley in fields fertilized with contaminated amendments

The survival and transfer of Listeria innocua and Clostridium sporogenes, used as surrogates of the food borne pathogens Listeria monocytogenes and Clostridium botulinum, were quantitatively assessed under field conditions. In the soil, spores of C. sporogenes declined by less than 0.7 log cycles within 16 months and were detected on parsley leaves throughout the experiment. In contrast, L. innocua in the soil declined by 7 log cycles in 90 days and was detected on leaves in low numbers (>0.04 MPN g(-1)) during the first 30 days. Rates of decline in soil were similar in the laboratory at 20 degrees C for two strains of L. innocua and L. monocytogenes ; and in the field for L. innocua over two different years. L. innocua survived better in winter, indicating an important influence of temperature. The major cause of transfer of L. innocua from soil to parsley leaves was splashing due to rain and irrigation. As few as 1 CFU g(-1) Listeria in soil led to contamination of parsley leaves. Internalisation of Listeria through parsley roots was not observed. Under the conditions of soil and climate studied, a delay of 90 days between application of potentially contaminated fertilizer and harvest should be sufficient to eliminate L. monocytogenes.     

Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods

The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O. Forty-six strains were isolated and examined. The lethality of some Listeria isolates for BALB/c mice was also determined. In this study, all isolates identified as L. monocytogenes in the API test gave a positive signal in the PCR. Listeriae identified as L. innocua or L. welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O. All strains identified as L. monocytogenes on the basis of the API test and the PCR produced PI-PLC. However, this activity was not limited to the bacteria of this species. Four out of 17 L. innocua and three out of 10 L. welshimeri isolates were PI-PLC-positive. None of the L. innocua or L. welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice. In contrast, two L. monocytogenes isolates as well as a reference L. monocytogenes strain killed all mice used for the experiment.     

Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

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.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.