Identification Of Listeria Research Articles

In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes

Simultaneous in situ analysis of the structure and function of bacterial cells present within complex communities is a key for improving our understanding of microbial ecology. A protocol for the in situ identification of Listeria spp. using fluorescently tagged, rRNA-targeted oligonucleotide probes was developed. Ethanol fixation and enzymatic pretreatment with lysozyme and proteinase K were used to optimize whole cell hybridization of exponential phase and stationary phase Listeria spp. cells. In parallel, transcript probes carrying multiple digoxigenin molecules were combined with anti-digoxigenin Fab antibody fragments labeled with horseradish peroxidase to detect, via the catalytic deposition of fluorescein-tyramide, the iap-mRNA in single Listeria monocytogenes cells. The iap gene encodes the associated virulence factor p60. Application of the new signal amplification technique resulted in strong signals comparable in intensity to those obtained with fluorescently labeled rRNA-targeted oligonucleotide probes.

In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes.

Simultaneous in situ analysis of the structure and function of bacterial cells present within complex communities is a key for improving our understanding of microbial ecology. A protocol for the in situ identification of Listeria spp. using fluorescently tagged, rRNA-targeted oligonucleotide probes was developed. Ethanol fixation and enzymatic pretreatment with lysozyme and proteinase K were used to optimize whole cell hybridization of exponential phase and stationary phase Listeria spp. cells. In parallel, transcript probes carrying multiple digoxigenin molecules were combined with anti-digoxigenin Fab antibody fragments labeled with horseradish peroxidase to detect, via the catalytic deposition of fluorescein-tyramide, the iap-mRNA in single Listeria monocytogenes cells. The iap gene encodes the associated virulence factor p60. Application of the new signal amplification technique resulted in strong signals comparable in intensity to those obtained with fluorescently labeled rRNA-targeted oligonucleotide probes.

Evaluation of the Serobact ™ and Microbact ™ systems for the detection and identification of Listeria spp.

The Serobact ™ Listeria latex agglutination reagent and Microbact ™ 12L system were evaluated, respectively, for the detection and identification of Listeria in foods. The Serobact ™ reagent proved effective, compared with conventional cultural methods, in the detection of low levels (1–10 cells/25 g) of both L. monocytogenes and L. innocua artificially inoculated into a range of foods, following enrichment. Extended incubation of either the primary or secondary enrichment increased the number of samples positive for Listeria by both Serobact ™ and conventional culture, but also increased the number of false positive reactions. The Serobact ™ reagent also proved effective in screening enrichment cultures of potentially contaminated retail food samples. Using the USDA enrichment procedure, the Serobact ™ reagent yielded 100% sensitivity and 93.9% specificity, when compared to conventional culture. Identification of 81 Listeria isolates using the Microbact ™ 12L system was compared with conventional biochemical and physiological tests. While identification after 4 h was poor, due to negative or incomplete fermentation of carbohydrates and lack of haemolysis, identification of all but two of 81 isolates was achieved after 24 h.

Simple color tests based on an alanyl peptidase reaction which differentiate Listeria monocytogenes from other Listeria species.

The hydrolysis of DL-alanine-beta-naphthylamide and D-alanine-p-nitroanilide for identification of Listeria spp. has been studied with 227 cultures. All species of Listeria, except L. monocytogenes, hydrolyzed these substrates. The reactions were detected by simple chromogenic reactions and could substitute for the CAMP test.

The identification of Listeria species

The purpose of this study was to compare methods for the identification of Listeria species. Three hundred and fifty cultures representing the six species of Listeria were tested using conventional sugar fermentation and haemolytic reactions, as well as the hydrolysis of the dl-alanine β-naphthylamide (DLABN), and the API Listeria identification test kit. Using conventional tests, 99% of cultures were correctly identified: four L. monocytogenes were misidentified as L. innocua. The DLABN hydrolysis test distinguished L. monocytogenes from the remainder of the genus for 98% of the cultures: 6 out of 14 L. ivanovii isolates gave atypical results. There was correct identification for 97% of the cultures using the API Listeria test kit and no misidentifications were obtained: nine cultures (six L. monocytogenes and three L. innocua) gave equivocal profiles which were not ascribed to any species.

Detection and identification of Listeria monocytogenes from milk and cheese by a single-step PCR.

Primers of iap gene were used as a target to develop a PCR technique for detecting Listeria monocytogenes in milk and cheese. The PCR technique gives good results in the detection of Listeria monocytogenes either in artificially or naturally contaminated foodstuffs and has a high sensitivity and specificity. Application of this rapid diagnostic tool could provide further information about the spread of L. monocytogenes in milk and cheese.

Confirmation and identification of Listeria spp.

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.

A simple colony-blot method for identification of Listeria in food samples.

A method for identification of Listeria in food samples was developed. It consisted of cultivating of suspected specimen on standard agar medium, direct absorption of grown colonies onto nitrocellulose membrane and processing of the latter with rabbit serum raised against purified cell wall protein Lm79/39 of L. monocytogenes. Analysis using anti-rabbit peroxidase conjugate and 4-chloro-1-naphthol and H2O2 solutions allowed direct detection of Listeria colonies which remained readily available for subsequent isolation.

A Multiplex PCR Method for the Identification of Listeria spp. and Listeria monocytogenes in Dairy Samples

A multiplex polymerase chain reaction (PCR) method for the identification of Listeria spp. and Listeria monocytogenes has been developed. For the identification of Listeria spp. two specific primers were derived from the 16S rRNA sequence. For the identification of Listeria monocytogenes specific primers were derived from the gene sequence of the invasion-associated protein. The two amplified DNA fragments, respectively 1003 bp and 593 bp, were separated by agarose electrophoresis. The results obtained by the multiplex PCR method were in agreement with those obtained by the classical bacteriological method for the 64 dairy samples with presumptive positives.

Comparison of Nucleic Acid Hybridization Assays and Biochemical Characterization Tests for the Confirmation of Listeria monocytogenes

A comparison was made of various biochemical and nucleic acid hybridization assays used for the confirmation of Listeria monocytogenes. Identification and confirmation tests included traditional carbohydrate utilization tests, several types of hemolysis assays, an immunocompromised mouse-pathogenicity test, the API® LISTERIA identification system, the Biolog GP MicroPlate™, a prototype Biolog Listeria MicroPlate™, and two nucleic acid hybridization assays (a slot-blot assay and the commercially available AccuProbe™L. monocytogenes culture confirmation test). A panel of 44 Listeria isolates, including representatives from all six existing Listeria species, was tested. A considerable number of the L. monocytogenes isolates tested gave ambiguous reactions on the CAMP-hemolysis assay. In many instances, the Biolog GP MicroPlate™ was unable to differentiate L. monocytogenes from other Listeria species. Reactions on the prototype Listeria MicroPlate™ mirrored those of traditional biochemical tests, but additional analyses were necessary to differentiate between L. monocytogenes and Listeria innocua. The API® LISTERIA identification system could readily differentiate between the latter two Listeria species without the need for additional tests. Atypical L. monocytogenes isolates (nonhemolytic and nonpathogenic or hemolytic and nonpathogenic) were correctly identified by the API® LISTERIA system. A perfect correlation was observed between results from the slot-blot format hybridization assay and the AccuProbe™ test. The probes used in the two assays hybridized with all L. monocytogenes isolates, regardless of hemolytic reaction or mouse pathogenicity. The commercially available assays examined in this study were all easy to use and capable of providing results within 24 hours.

Comparison of MICRO-ID Listeria Method with Conventional Biochemical Methods for Identification of Listeria Isolated From Food and Environmental Samples: Collaborative Study

Fourteen laboratories participated in a collaborative study to evaluate the ability of the MICRO-ID Listeria identification method to correctly identify Listeria isolated from food and environmental sources. Each collaborator received 60 isolates consisting of 51 Listeria and 9 non-Listeria cultures. All isolates were identified by conventional biochemical analyses in the principal laboratory. Cultures were checked for purity by Gram staining and examined for oxidase and catalase activities. Only Gram positive, oxidase negative, catalase positive cultures were tested with the method. Colonies from trypticase soy agar with 0.6% yeast extract were suspended in 4.6 mL physiological saline to a MacFarland No. 1 turbidity standard and used to inoculate the test strip. In addition, the hemolytic reaction of each isolate was determined by using the Christie-Atkins-Munch-Peterson (CAMP) test and by stabbing sheep blood agar. Identification of Listeria is based on the octal code obtained from the strip and the hemolytic reaction of the isolate. The MICRO-ID Listeria method agreed with conventinal biochemical identification for 98.0% of L. monocytogenes, 77.1% of L. seeligeri, 90.0% of L. ivanovii, 96.4% of L. grayi/L. murrayi, 73.9% of L. welshimeri, and 100% of L. innocua isolates. A large percentage of errors in identification of the L. seeligeri and L. ivanovii cultures was caused by inaccurate reading of the CAMP and hemolysis tests rather than errors in the test strip. The method was adopted first action by AOAC International.

The homologous and heterologous regions within the iap gene allow genus- and species-specific identification of Listeria spp. by polymerase chain reaction.

The iap gene of Listeria species encodes protein p60. The comparison of iap-related genes from different Listeria species indicated common and variable regions within these genes which appeared to be specific for each Listeria species. On the basis of the iap gene sequences, pairs of polymerase chain reaction (PCR) primers which allowed the unambiguous identification of all members of the genus Listeria, of groups of related Listeria species, and of L. monocytogenes, exclusively, were selected. The PCR primers specific for L. monocytogenes yielded PCR products which represented essentially the repeat region of the iap gene. The size of these PCR products allowed an estimate of the number of the TN repeat units within the repeat region of the p60 protein of an L. monocytogenes strain. The data indicated that the number of repeat units differed among L. monocytogenes isolates.

Catalase-Negative Listeria monocytogenes Causing Meningitis in an Adult Clinical and Laboratory Features

A 63-year-old previously healthy woman presented with acute meningitis. Cultures of the cerebrospinal fluid yielded a serotype 1/2a isolate of Listeria monocytogenes that was biochemically typical in all respects, other than the reproducible lack of catalase production. During therapy, the patient developed oculomotor dysfunction that was attributed to an abscess in the internal capsule. This case report documents the existence of catalase-negative L. monocytogenes indicating that catalase production should not be a strict criterion for identification of Listeria. Furthermore, this clinical experience extends in vitro and experimental animal studies indicating that catalase production is not a necessary virulence factor for invasion by Listeria.

Overlay technique for direct detection and identification of haemolytic Listeria on selective plating medium

An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.

Evaluation of the API-ZYM system for identification of Listeria

The API-ZYM system (Analytab Products) was evaluated for potential use for confirming the identity of Listeria isolates. A total of 68 Listeria strains from various sources were assayed using standard procedures. All isolates were positive for esterase (C4), esterase lipase (C8), phosphohydrolase (one exception), β-glucosidase, acid phosphatase and lipase (one exception). Negative responses were observed consistently for valine aminopeptidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-fucosidase and cystine aminopeptidase (one exception). Variable responses were noted for alkaline phosphatase, leucine aminopeptidase, chymotrypsin, α-glulucosidase and N-acetyl glucosaminidase. Comparison of these results with those of other Gram positive species indicated that the API-ZYM system could be used for rapid confirmation of Listeria to the subgenus level.

Environmental Survey for Listeria Species in California Milk Processing Plants

This survey was undertaken to determine the prevalence of Listeria monocytogenes and Listeria spp. in environmental samples obtained from milk processing plants located throughout California. Identification of Listeria spp. other than L. monocytogenes was not made. Milk processing plants were categorized as to type of product produced and subdivided into areas based on manufacturing activity occurring in that area. A total of 597 environmental samples from 156 plants were analyzed in the six month period of January through July 1987. Listeria spp. were isolated from 75 (12.6%) of the samples. Thirty-eight of the Listeria spp. (50.7%) were identified as L. monocytogenes. Forty-six plants (29.5%) yielded positive results for Listeria spp., of which, 31 plants (19.9%) were positive for L. monocytogenes. Listeria was recovered more frequently from fluid milk product plants and frozen milk product plants. Likewise, Listeria was isolated most frequently from the packaging-filling room location within a plant and least frequently from the raw milk receiving room. Although Listeria spp. was isolated most frequently from the conveyor site, the drain site within a plant location gave a Listeria isolation rate similar to the total number of Listeria positive sites within that plant location.

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).