Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

李斯特菌毒力因子及其进化

李斯特菌属包含6个种,毒力各有差异。在细菌耐受外界环境、黏附侵袭及细胞内感染过程中,毒力因子各司其职又相互协作。毒力基因常聚集为毒力岛,其中PrfA依赖型毒力基因簇(LIPI-1)与内化素岛(LIPI-2)是致病种最重要的两个毒力岛。李斯特菌各个种可能来源于同一个携带有完整毒力岛的祖先,在长期进化过程中,通过基因水平转移或重组、整合等事件,演化为目前流行的6个种。噬菌体、转座子、质粒等可能扮演着毒力进化执行者的角色。一些天然非典型菌株是目前研究的热点,如含有LIPI-1的无害李斯特菌和缺失LIPI-1的塞氏李斯特菌,其演化进程可能尚未达到或已超越目前流行的状态,为李斯特菌毒力进化的研究提供了重要线索。     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Comparison of the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity of Listeria spp.

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.     

Listeria monocytogenes in spontaneous abortions in humans and its detection by multiplex PCR

AIM: To assess the extent of Listeria monocytogenes in causation of human spontaneous abortions by isolation methods and PCR analysis for the presence of virulence-associated genes. METHODS AND RESULTS: A total of 305 samples comprising blood, urine, placental bits, faecal and vaginal swabs were collected from 61 patients with spontaneous abortions. Listeria spp. were isolated from 10 samples collected from nine (14.8%) patients. Confirmation of these isolates was based on biochemical tests, haemolysis on blood agar, CAMP test, phosphatidylinositol-specific phospholipase C (PI-PLC) assay followed by in vivo pathogenicity tests and multiplex PCR to detect virulence-associated genes (prfA, plcA, hlyA, actA and iap). Three isolates were confirmed as L. monocytogenes. Of these, two isolates turned out to be pathogenic and found to posses all five genes. However, the remaining two haemolytic L. monocytogenes isolates lacking the plcA gene and activity in the PI-PLC assay were found to be nonpathogenic by in vivo tests. CONCLUSIONS: The occurrence of pathogenic L. monocytogenes in cases of spontaneous abortions was 3.3%. It seems that the plcA gene and its expression have an important role as essential virulence determinants in pathogenic Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of pathogenic L. monocytogenes isolates from cases of spontaneous abortion indicates the significance of listeric infection in pregnant women.     

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36 kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol-specific phospholipases C (PI-PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI-PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI-PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI-PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI-PLC of L. monocytogenes might be involved in virulence.     

Influence of environmental parameters on phosphatidylcholine phospholipase C production in Listeria monocytogenes: a convenient method to differentiate L. monocytogenes from other Listeria species.

The ability to produce phosphatidylcholine phospholipase C (lecithinase) is associated with virulence in pathogenic species of Listeria. Levels of production vary greatly among members of the genus, and this virulence factor is not readily detectable in many members of the pathogenic species on conventional agar media containing egg yolk, a common substrate for the enzyme. In this study, the influence of a variety of environmental parameters, including temperature, pH, and salt concentration, on the production of lecithinase by a number of strains was evaluated. Lecithinase production by Listeria monocytogenes LO28 in brain heart infusion medium was optimal at 1.75 to 2.0% NaCl; pH 7.0 to 7.3, and 37 to 40 degrees C, and the presence of oxygen had no effect. In a chemically defined medium, the optimal NaCl concentration and temperature were lower at 0.75 to 1.0% NaCl and 33.5 degrees C. As detection of virulence factors is useful to assist in the identification and differentiation of Listeria species, this report shows that lecithinase activity can conveniently be detected within 36 h on a relatively inexpensive medium. Under the conditions described, L. monocytogenes could be distinguished from other members of the genus as a result of distinct lecithin degradation which was not evident in L. innocua, L. seeligeri, L. ivanovii, L. welshimeri, or L. murrayi/grayi.     

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.     

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 of Listeria sp. in droppings from urban rooks (Corvus frugilegus)

Droppings from 112 urban rooks ( Corvus frugilegus ) were cultured for the presence of Listeria sp. Overall, 46% of rooks sampled harboured one or more Listeria species. Of all birds examined, 33%, 24% and 8%, respectively, were infected with Listeria monocytogenes, Listeria innocua and Listeria seeligeri. Differentiation of L. monocytogenes and L. seeligeri carried out by several phenotypic typing methods proved the diversity of strains and the major role of rooks which widely contribute to spreading this bacteria in our environment. The results also suggest that the ability to recover specific Listeria strains from the same sample is at least partially dependent on the methodology. These findings reinforce the need for strain-specific typing of multiple L. monocytogenes isolates from the same sample.     

Evolutionary history of the genus Listeria and its virulence genes

The genus Listeria contains the two pathogenic species Listeria monocytogenes and Listeria ivanovii and the four apparently apathogenic species Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi. Pathogenicity of the former two species is enabled by an approximately 9 kb virulence gene cluster which is also present in a modified form in L. seeligeri. For all Listeria species, the sequence of the virulence gene cluster locus and its flanking regions was either determined in this study or assembled from public databases. Furthermore, some virulence-associated internalin loci were compared among the six species. Phylogenetic analyses were performed on a data set containing the sequences of prs, ldh, vclA, and vclB (all directly flanking the virulence gene cluster), as well as the iap gene and the 16S and 23S-rRNA coding genes which are located at different sites in the listerial chromosomes. L. grayi represents the deepest branch within the genus. The remaining five species form two groupings which have a high bootstrap support and which are consistently found by using different treeing methods. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch. Based on this perception, we tried to reconstruct the evolution of the virulence gene cluster. Since no traces of lateral gene transfer events could be detected the most parsimonious scenario is that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri and that the pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. This hypothesis is also supported by the location of the putative deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri.     

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.     

Listeria monocytogenes in pasteurized milk

An haemolytic Listeria monocytogenes strain pathogenic to mice was isolated from 6 out of 28 (21.4%) pasteurized milk samples (3.2% fat milk treated at 78 degrees C for 15 s) marketed by a Madrid processing plant. Listeria grayi was recovered from 25 of the samples (89.2%) and L. innocua from 3 samples (10.7%). One milk sample was contaminated with L. welshimeri. No strains of L. ivanovii, L. seeligeri, L. murrayi, or L. denitrificans were isolated. These results show that pathogenic Listeria strains can be isolated from pasteurized milk and reinforce the hypothesis that this food product may be the source of numerous human listeriosis.     

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.     

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments

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.     

Bacteriocin-like substance (BLS) production in Aeromonas hydrophila water isolates

30 Aeromonas hydrophila water isolates were tested for bacteriocin-like substance (BLS) production using a target panel of closely related microorganisms and other Gram-positive and Gram-negative bacteria, including food-borne pathogens. A. hydrophila showed antibacterial activity against one or more indicator microorganisms, but the activity emerged only with non-phylogenetically related genera or species. In particular all A. hydrophila showed antibacterial activity against one or more of the tested Staphylococcus strains, five against Listeria spp. (Listeria seeligeri, Listeria welshimeri and Listeria ivanovii), and eight presented a weak antagonistic activity towards Streptococcus agalactiae and Lactobacillus spp. Inhibitory activity was not observed against the other Gram-positive (Listeria monocytogenes, Listeria innocua and Enterococcus spp.) and Gram-negative tested strains, including Aeromonas sobria, Aeromonas caviae and the same A. hydrophila, when used as indicator. Anti-staphylococcal activity was observed with a gradual increase of the inhibition zone during incubation and seemed to be influenced by A. hydrophila hemolytic expression. Extrachromosomal analysis showed the presence, in 70% of the strains, of one to five plasmids with molecular masses ranging from 2.1 to 41.5 MDa, but it was not possible to relate this result with BLS production.     

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.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Production of thiol-dependent haemolysins by Listeria monocytogenes and related species

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml-1) and the type strain CIP 82110T (10 HU ml-1). The haemolytic activity reached a maximum level by 8-10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (Mr about 60,000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml-1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9-30 HU ml-1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of Mr about 60,000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.