Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (lmaA) in Listeria spp. by the polymerase chain reaction

Johnson, Wendy M.; Tyler, Shaun; Ewan, E P; Ashton, F. E.; Wang, G.; Rozee, K. R.

doi:10.1016/0882-4010(92)90068-y

Cited by 25 publications

(13 citation statements)

References 24 publications

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“…Heterogeneity in the expression of virulence traits in strains belonging to serogroup 4 has repeatedly been reported in the literature (4,38). We also analyzed L. innocua 6a, a serotype in which several strains have previously been reported to harbor the lmaA gene (15). In an L. innocua 6a isolate, we detected a sequence that was 90% homologous to the lmaBA genes, but this strain lacks the genes located upstream of lmaB (36).…”

Section: Discussionmentioning

confidence: 99%

Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes

Schäferkordt

Chakraborty

1997

J Bacteriol

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The lmaA gene of Listeria monocytogenes encodes a protein capable of inducing delayed-type hypersensitivity reactions in L. monocytogenes-immune mice (S. Göhmann, M. Leimeister-Wächter, E. Schiltz, W. Goebel, and T. Chakraborty, M. Microbiol. 4:1091-1099, 1990). Here we show that it is the last gene of the lma operon, which now comprises four genes, lmaDCBA. Maxicell analysis of peptides encoded by the lma operon identified four polypeptides of 16.7, 16.4, 14.9, and 21 kDa which correspond to the gene products encoded by the lmaD, -C, -B, and -A genes, respectively. Northern blot analysis of the lma operon showed that lmaA is expressed by two transcripts: the longer lmaDCBA transcript of 2,100 nucleotides, which was observed at growth temperatures of 37 and 20؇C, and a shorter transcript consisting of lmaBA, which is detected only at low temperatures (20؇C). Two promoters, one preceding the lmaD gene and another located upstream of the lmaB gene, were detected. An extended stem-loop structure resembling box elements found in other gram-positive pathogens was also present in the lmaC-lmaB intergenic region. By immunoblot analysis, we found that although LmaA was produced at both temperatures (20 and 37؇C), it was secreted into culture supernatants only at 20؇C. However, LmaA lacks a bona fide signal peptide sequence and could, like flagellin, be secreted by a type III transport system. DNA hybridization studies indicate that the lma operon is species specific and restricted to pathogenic strains of L. monocytogenes.The gram-positive, facultative intracellular bacterium Listeria monocytogenes is a foodborne pathogen responsible for infections in immunocompromised persons and pregnant women. Listerial infections can result in meningitis, meningoencephalitis, septicemia, and death (10, 12, 16). The consumption of contaminated food has been shown to be the major route of infection. In addition to causing sporadic listeriosis, L. monocytogenes has been implicated in several epidemics in Europe and North America (2, 37). The ability of L. monocytogenes to grow at 4ЊC, a temperature at which many off-theshelf foodstuffs are stored, opens up a unique environment for listerial enrichment (16,43). Listerial products that are expressed at low growth temperatures are therefore a relevant area of study.Virulence factors necessary for entry, intracellular survival, multiplication, and motility have been previously characterized. The genes encoding these factors are organized in two clusters on the listerial chromosome. The main cluster contains the genes coding for hemolysin (hly), phospholipases (plcA, plcB), metalloprotease (mpl), and actin nucleation factor (actA) and includes the virulence regulator protein PrfA (6, 18-21, 27, 28). The internalin genes inlA and inlB, which are required for uptake of the bacteria into the eukaryotic cell, are arranged as an operon close to but separate from the other virulence locus (29). The expression of the inlA and inlB genes is regulated by both PrfA-dependent and -independent mechani...

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Section: Discussionmentioning

confidence: 99%

Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes

Schäferkordt

Chakraborty

1997

J Bacteriol

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“…Multilocus enzyme electrophoresis (4,46), REA (5,43,49), and ribotyping (19,57) have been developed. The use of faster procedures like PCR and immunological or bacteriophage lysis techniques which might allow a more rapid monitoring of all Listeria species is limited because they detect only the genus Listeria or only L. monocytogenes (8,12,16,31,34), thus lacking the ability to simultaneously characterize all the species of Listeria. Moreover, the methods developed so far require pure cultures isolated by traditional methods.…”

Section: Discussionmentioning

confidence: 99%

Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods

Cocolin

Rantsiou

Iacumin

et al. 2002

Appl Environ Microbiol

108

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A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.The genus Listeria comprises a group of ubiquitous, grampositive, nonsporulating bacteria. Previous results of DNA-DNA hybridization experiments unambiguously demonstrated that the species formerly called "Listeria monocytogenes" in fact contained five genomic groups deserving the species rank: and L. seeligeri (48, 49). This subdivision has proven its usefulness for practical and epidemiological purposes, especially when problems caused by food contamination resulted in human listeriosis (29). Only the hemolytic species of Listeria, L. monocytogenes, L. ivanovii, and L. seeligeri, are associated with human pathogenicity. L. monocytogenes is the only species of the genus Listeria that has been involved in known food-borne outbreaks of listeriosis. L. ivanovii has been described to be involved in human pathology only rarely, and L. seeligeri has been reported only once to be the cause of meningitis in a nonimmunocompromised adult (35).Several large outbreaks of listeriosis have been associated with contaminated commercial foodstuffs, such as vegetables, milk, and meat products, on which these bacteria can multiply even at low temperatures (52). Usually, the presence of any Listeria species in food is an indicator of poor hygiene (40).Significant efforts have been dedicated to the development of enrichment media and protocols for L. monocytogenes and Listeria sp. isolation (1,10,11,15,24,47). Ideal enrichment media would facilitate recovery of injured Listeria cells and enrichment of Listeria spp. and L. monocytogenes over competing microflora. In traditional culture-based assays, it becomes very difficult to detect L. monocytogenes at any level when it is greatly outnumbered by other Listeria spp., such as L. innocua, which appears to be present together with L. monocytogenes (10, 51). Consequently, detection of Listeria spp. is oft...

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“…Primers Lis1B, MonoA, Ino2, and Sel1 (6) were used to amplify fragments of the iap gene to confirm the species identification when API and PCR-RFLP data were discordant. Primers hly-a and hly-b (22) were used to amplify the L. monocytogenes hemolysin gene (hlyA). All of these PCR experiments were performed under the following conditions: denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min and a final cycle at 72°C for 7 min.…”

Section: Methodsmentioning

confidence: 99%

Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments

Paillard

Dubois

Duran

et al. 2003

Appl Environ Microbiol

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A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

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Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (lmaA) in Listeria spp. by the polymerase chain reaction

Cited by 25 publications

References 24 publications

Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes

Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes

Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods

Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments

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