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PubMedDate not supplied

[Isolation and identification of Listeria monocytogenes and Listeria spp. in dry sausages obtained in markets in the city of La Plata, Argentina].

A total of 60 samples of dry sausages were analyzed (50 of "salami" and 10 of "chorizo" "candelario" type) obtained at random in markets authorized for their commercialization, for the purpose of evidencing the presence of bacteria of the genus Listeria (Listeria monocytogenes and Listeria spp.). The results obtained in salami were the following: 10 (20%) isolates of Listeria spp., were characterized: 1 (2%) strain as L. monocytogenes type 1, 7 (14%) strains as L. innocua, 2 (4%) strains as L. welshimeri. In chorizo candelario type 6 (60%) isolates of Listeria spp., were characterized: 2 (33%) strains as L. monocytogenes type 1 and 4 (66%) strains as L. innocua. The total percentages of isolations were: 26.6% of Listeria spp., 5% of L. monocytogenes type 1, 18.3% of L. innocua and 3.3% of L. welshimeri. In conclusion, we consider that methodologies of control must be developed and implemented in order to guarantee the inocuity of these products.

Animals↗

PubMed2003

Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii.

Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-gamma) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD(50)) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD(50) was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-gamma in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-gamma-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-gamma-inducing activity than LLO. By comparing the IFN-gamma-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-gamma-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-gamma production is responsible for the failure of L. ivanovii to generate effective protective immunity.

Amino Acid Sequence↗

PubMedDate not supplied

Detection and quantification of the iap gene of Listeria monocytogenes and Listeria innocua by a new real-time quantitative PCR assay.

A real-time quantitative polymerase chain reaction (PCR) assay for direct detection and enumeration of Listeria monocytogenes and Listeria innocua was developed and applied to artificially contaminated milk samples. The iap gene present in both species was used as a target for amplification of a 175-bp (L. monocytogenes) and a 309-bp (L. innocua) fragment. To ensure that L. monocytogenes and L. innocua are specifically detectable, tests were carried out using 42 L. monocytogenes strains and 33 L. innocua strains belonging to different serovars. Specificity was also confirmed using 22 bacterial strains not belonging to the genus Listeria, including closely related bacteria. In addition to specificity, the reported assay is characterized by a wide dynamic range of quantification and a high sensitivity, as we could detect as few as six copies of the iap gene per PCR using purified DNA as template. When applied to direct detection and quantification of L. monocytogenes in milk, the more rapid real-time quantitative PCR assay was as sensitive as the traditional plate count method, but real-time quantitative PCR-derived iap gene copy numbers were one to two logs higher than colony-forming units obtained by the plate count method.

Animals↗

PubMed1998

Characterization of plasmids from Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from short-ripened cheeses was analysed. The isolates of L. monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2 kbp), whereas the serogroup 4 isolates did not contain plasmids. One group of L. innocua strains harboured the plasmid pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp). These plasmid groups were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of Listeria isolates. Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp. Plasmid pLI56 did not encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different characteristics to the other three plasmids. It was also the only one cured from the parent strain and the sole plasmid not digested by the restriction enzyme PstI. In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the possibility of a different origin for plasmid pLI56.

Blotting, Southern↗

PubMed1992

Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice.

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.

Animals↗

PubMed1991

Development and characterization of a monoclonal antibody specific for Listeria monocytogenes and Listeria innocua.

BALB/c mice were immunized with crude cell surface proteins of Listeria monocytogenes V7. Approximately 1,680 hybridomas were generated after two fusions, and the monoclone C11E9 was selected and used for further characterization. The monoclonal antibody (MAb) produced by C11E9 was immunoglobulin subclass G2b with kappa light chains. Dot and colony blot results indicated that MAb C11E9 was reactive to all the L. monocytogenes (34 of 34) and Listeria innocua (6 of 6) isolates without any cross-reaction to other organisms tested. Western blot (immunoblot) analysis of crude cell surface proteins in native polyacrylamide gel electrophoresis (PAGE) indicated that MAb C11E9 reacts with a single band in each species, with a molecular mass of 174 kDa for L. monocytogenes and 182 kDa for L. innocua. The MAb reacted with one major protein band in Western blot from acid-urea PAGE for both L. monocytogenes and L. innocua. Isoelectric focusing results indicated two immunoreactive protein bands with pIs of 8.1 and 7.4 for L. monocytogenes. Sodium dodecyl sulfate (SDS)-PAGE and Western blot analysis indicated several proteins with molecular masses of 76, 66, 56, and 52 kDa for L. monocytogenes and 66, 56, and 52 kDa for L. innocua. Reaction of MAb C11E9 to washed live cells indicated the possible binding of antibody to cell surface antigen. These cell surface antigens could be removed by 1 N HCl plus 9 M urea, 2% SDS-0.5% beta-mercaptoethanol, or 4 M guanidine-HCl. The epitope of MAb C11E9 binding site was shown to be protein in nature. Periodic acid-Schiff staining and glycoprotein immunoassay indicated that carbohydrate was absent in the epitope. The cellular locations of the MAb C11E9-reactive antigens were calculated to be 76 and 90% outside and 24 and 10% inside the cell membranes of L. monocytogenes and L. innocua, respectively, for 12- to 14-h cultures.

Animals↗

PubMed1990

Petite colony formation by Listeria monocytogenes and Listeria species grown on esculin-containing agar.

Several strains of Listeria species formed petite-sized colonies from parent stock cultures when grown on agar media containing 0.2-1% (w/v) esculin. This was observed in Listeria monocytogenes (7/22 strains), L. innocua (1/3), L. grayi (1/1), L. seeligeri (1/3), and L. welshimeri (1/1), but not in L. ivanovii (0/1) and L. murrayi (0/1). This phenomenon was only observed on agar media that contained esculin. All petite isolates had biotyping profiles identical to their larger, normal-sized counterpart isolates. Normal and petite-sized isolates from two L. monocytogenes strains, Scott A and V7, were pathogenic to immunosuppressed white mice. On media containing 0.5% (w/v) esculin + ferric iron, Listeria cultures produced colony diameters intermediate in size between those of normal and petite cultures. When pregrown in glucose broth, all petite isolates demonstrated visible beta-glucosidase (esculinase) activity within 5 min, while the normal-sized isolates showed beta-glucosidase activity only after at least 20-70 min. This evidence suggests that cells forming petite colonies are beta-glucosidase constitutive variants within the parent population, while cells that form normal-sized colonies are inducible for beta-glucosidase (esculinase) activity. A possible role for the esculin hydrolysis product, esculetin, in causing petite colony formation is discussed.

Animals↗

PubMed2003

Comparison of the genome sequences of Listeria monocytogenes and Listeria innocua: clues for evolution and pathogenicity.

Listeria monocytogenes, an invasive opportunistic, food-borne pathogen, remains one of the leading causes of mortality from food-borne infections. The recently determined complete genome sequences of L. monocytogenes strain EGDe and of that of the closely related non-pathogenic species Listeria innocua strain CLIP11262 enhance our knowledge of the genetic basis of the virulence of L. monocytogenes and advance our understanding of the evolution of these Listeria species. Both genomes encode a high number of surface, transport and regulatory proteins. Comparison of the genome organisation revealed a perfect synteny between the two Listeria genomes. Comparison with other closely related bacteria also showed a high conservation in genome organisation among the Listeria, Staphylococcus and Bacillus group of low G+C content bacteria. Distinct G+C content of a number of strain-specific genes suggests intensive lateral gene transfer. The identification of a 55-kb locus encoding proteins with high homology to Salmonella enterica serovar Typhimurium vitamin B(12) synthesis proteins as well as those necessary for degradation of ethanolamine and propanediol further indicates acquisition of a complete metabolic pathway by horizontal gene transfer and a probable role of this locus in anaerobic growth in the host.

Anaerobiosis↗

PubMed1995

A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.

The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

Actins↗

PubMed1989

Serovars of Listeria monocytogenes and Listeria innocua from food.

The increase of listeriosis outbreaks in recent years has mainly been due to the consumption of contaminated dairy products. This prompted a revision of prevention and control measures. Cheese and other foods (meat, poultry meat) were investigated. For isolation, samples were prepared and investigated according to the recommendations of the FDA with some modifications including, in particular, sub-enrichment. Suspicious colonies were tested biochemically followed by serological identification with factor sera. Out of 100 chicken (95 broilers, 5 layers) Listeria monocytogenes was isolated from 85 carcases. The contamination rate for minced beef and pork (n = 76) was approximately 30%. That for selected ripened soft cheeses (n = 89) was 9%. The counts of Listeria in red meat were usually very low (approximately 10/g). Serovar 1/2a was predominant (78 out of 102 strains). Further serovars detected were: 1/2b, 1/2c, 3a, 4a, 4b, 4d. For comparison, isolated strains of L. monocytogenes and Listeria innocua were inoculated into chicken embryos. In contrast to the former, L. innocua did not kill the embryos.

Animals↗

PubMed2006

Reliable and rapid identification of Listeria monocytogenes and Listeria species by artificial neural network-based Fourier transform infrared spectroscopy.

Differentiation of the species within the genus Listeria is important for the food industry but only a few reliable methods are available so far. While a number of studies have used Fourier transform infrared (FTIR) spectroscopy to identify bacteria, the extraction of complex pattern information from the infrared spectra remains difficult. Here, we apply artificial neural network technology (ANN), which is an advanced multivariate data-processing method of pattern analysis, to identify Listeria infrared spectra at the species level. A hierarchical classification system based on ANN analysis for Listeria FTIR spectra was created, based on a comprehensive reference spectral database including 243 well-defined reference strains of Listeria monocytogenes, L. innocua, L. ivanovii, L. seeligeri, and L. welshimeri. In parallel, a univariate FTIR identification model was developed. To evaluate the potentials of these models, a set of 277 isolates of diverse geographical origins, but not included in the reference database, were assembled and used as an independent external validation for species discrimination. Univariate FTIR analysis allowed the correct identification of 85.2% of all strains and of 93% of the L. monocytogenes strains. ANN-based analysis enhanced differentiation success to 96% for all Listeria species, including a success rate of 99.2% for correct L. monocytogenes identification. The identity of the 277-strain test set was also determined with the standard phenotypical API Listeria system. This kit was able to identify 88% of the test isolates and 93% of L. monocytogenes strains. These results demonstrate the high reliability and strong potential of ANN-based FTIR spectrum analysis for identification of the five Listeria species under investigation. Starting from a pure culture, this technique allows the cost-efficient and rapid identification of Listeria species within 25 h and is suitable for use in a routine food microbiological laboratory.

Bacteriological Techniques↗

PubMed1996

Identification and purification of novel internalin-related proteins in Listeria monocytogenes and Listeria ivanovii.

Monoclonal antibodies were generated against a 30-kDa protein fraction derived from culture supernatants of a Listeria monocytogenes strain complemented with additional copies of the prfA regulator gene. Several of the antibodies reacted specifically with a hitherto unidentified, secreted 30-kDa polypeptide. By immunoblot analysis, the expression of this 30kDa polypeptide was found to be dependent on the presence of the PrfA regulator protein. Microsequencing of peptides derived from the partially purified 30-kDa protein revealed homologies to the InlA and InlB polypeptides of L. monocytogenes, which are required for the internalization of the bacteria into nonphagocytic cell lines. This prompted us to term the 30-kDa polypeptide internalin-related protein (Irp). Irp-specific monoclonal antibodies cross-reacted with a 24-kDa polypeptide present in culture supernatants of Listeria ivanovii, indicating the existence of an Irp-related protein in this pathogenic Listeria species.

Amino Acid Sequence↗

PubMed2002

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths.

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.

Antibiosis↗

PubMed1992

Minimal water activity levels for growth and survival of Listeria monocytogenes and Listeria innocua.

Following initial range-finding experiments, total count determinations were used to determine minimal water activity (aW) levels for the growth and survival of Listeria monocytogenes (L.m.) and L. innocua (L.i.). Media containing three different humectants; NaCl, sucrose or glycerol were used to determine minimal aW levels for growth in the above media which were 0.92, 0.92 and 0.90 respectively. The growth minima for L.i. were similar, or slightly higher than for L.m. in these media. Survival rates were generally lower in NaCl-adjusted media than in systems adjusted with sucrose or glycerol. Survival of L.m. and L.i. in these experiments was similar.

Culture Media↗

PubMed1997

[Presence of Listeria monocytogenes and Listeria sp. in fresh and cured sausages. Use of different conditions of time and temperature for incubation].

Two different temperatures for enrichment of Listeria monocytogenes and related species have been studied (1) cold enrichment at 4 degrees C (2) enrichment at 30 degrees C (FDA method). Also, two selective media for isolation were tested: Acriflavine-Ceftazidime agar (A.C.) and Palcam agar. We have studied 72 samples of dry-cured sausage (called 'longaniza') at different stages of maturation: fresh, semi-cured and cured samples. The most efficient method was cold enrichment at 4 degrees C during 5 days followed by isolation in Palcam agar, but results were only significant for fresh sausages (P < 0.05).

Animals↗

PubMed1998

Polymorphism of Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes ApaI and SmaI defined five clusters in L. monocytogenes (m1 to m5) and two main clusters in L. innocua (i1 and i2). Cluster i2 was further arranged into three subclusters (i2a, i2b and i2c) based on the different Eco52I (XmaIII) and Crf42I (SacII) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates (m4 and m5) constituted a more homogeneous group than serogroup I isolates (m1, m2 and m3). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes ApaI profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4, the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.

Bacteriophage Typing↗

PubMed2004

Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology.

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).

Bacteriological Techniques↗

PubMed2004

Characterization of Listeria monocytogenes and Listeria innocua from a vegetable processing plant by RAPD and REA.

The incidence of Listeria monocytogenes in a vegetable processing plant was investigated over a 23-month period. Frozen ready-to-eat vegetable samples, well as the plant environment, were sampled. The molecular subtyping techniques, Random Amplified Polymorphic DNA (RAPD) and Restriction Endonuclease Analyses (REA), were performed to help investigate the origin and routes of Listeria dissemination. The low and sporadic incidence of L. monocytogenes made it impossible to establish an epidemiological sequence in the processing plant, though a case of cross-contamination between tomato and ratatouille was detected. Listeria innocua subtyping, however, allowed us to determine the prevalence of several strains in vegetables, and their presence on machinery samples suggested the possibility of cross-contamination during processing. The low incidence of L. monocytogenes indicated that the risk of listeriosis transmission by vegetable consumption is low. On the other hand, the isolation of the same strain of L. innocua in several surveys pointed out the risk of colonisation on surfaces and machinery. The persistence of Listeria spp. is a cause for concern as can lead to future contamination of vegetables processed in the plant and to a possible increased risk for health. Therefore, periodic controls for the presence of Listeria spp. and a further review of the cleaning and disinfection procedures used in frozen vegetable plants are recommended.

Bacterial Typing Techniques↗