Listeria Seeligeri Research Articles

Complementation of Listeria seeligeri with the plcA-prfA genes from L. monocytogenes activates transcription of seeligerolysin and leads to bacterial escape from the phagosome of infected mammalian cells.

Infection experiments have shown that the avirulent species Listeria seeligeri invaded the enterocyte-like cell line Caco-2 with low efficiency but was unable to escape from the phagosome. Introduction of the listeriolysin gene (hly) from L. monocytogenes into L. seeligeri via a recombinant plasmid did not change these characteristics. No measurable transcription of this gene or of the structurally intact chromosomal seeligerolysin gene (lso) was detected. Transformation with a plasmid carrying the bicistronically transcribed plcA-prfA genes from L. monocytogenes resulted in the efficient expression of the plasmid-encoded transcription activator PrfA, a readily detectable synthesis of seeligerolysin and the escape of the bacteria from the phagosome of infected mammalian cells, followed by intracytoplasmic multiplication.

Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands

Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-beta and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-beta, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.

Effect of pH on the Inhibition of Listeria spp. by Vanillin and Vanillic Acid

The antimicrobial effects of vanillin and vanillic acid were verified against several species and strains of Listeria monocytogenes, Listeria innocua, Listeria grayi, and Listeria seeligeri in a laboratory medium adjusted to pH values ranging from 5.0 to 8.0. Medium pH had little influence on the MIC of vanillin as determined by a broth dilution assay, and growth of all test strains was inhibited by concentrations ranging from 23 to 33 mM. In contrast, none of the strains were inhibited by 100mM vanillic acid at pH > 6.0, but complete inhibition was achieved at pH 5.0 with 10 mM. The effect of pH was further characterized by incubation of L. monocytogenes, L. innocua, and L. grayi in media containing 30mM vanillin or 60mM vanillic acid at pH 5.0, 6.0, and 7.0. Bactericidal effects increased with pH in media supplemented with vanillin. An inverse relationship was found for vanillic acid, and the lethality of the compound increased with declining pH. Mixtures of vanillin and vanillic acid exhibited additive inhibitory effects, particularly at lower pH. These natural antimicrobial compounds could prove useful either alone or in mixtures for the control of Listeria spp. in food products.

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.

Antilisterial Properties of Cilantro Essential Oil

The minimum inhibitory concentrations (MIC) of crude essential oil of cilantro (Coriandrum sativum L.) and four fractions recovered by fractional distillation of the crude oil were determined against strains of Listeria monocytogenes, Listeria grayi, Listeria innocua and Listeria seeligeri. Crude oil inhibited all the test strains at concentrations ≤ 0.01% (v/v) and the remaining fractions were effective at concentrations < 0.07% (v/v). One fraction comprising a complex mixture of alcohols, aldehydes, alkanes and terpenes, including high concentrations of (E)-2-decenal, exhibited slightly greater potency than the other fractions. Listericidal activity was demonstrated by a rapid loss in cell viability upon exposure to crude oil dissolved in Tryptic Soy Broth. Cilantro oil is a potent antilisterial plant extract with potential applications as a food preservative or in the formulation of disinfectants for the control of Listeria monocytogenes in the food processing environment.

Species-specific PCR determination of Listeria seeligeri

Listeria seeligeri is a non-pathogenic bacterium coming under the genus Listeria. As this bacterium resembles other Listeria species such as L. monocytogenes and L. ivanovii that are pathogenic to man and animals, it is important that rapid and precise identification techniques be available for L. seeligeri in cases where such determination is desirable. A specific molecular test on the basis of a uniquely present gene region in L. seeligeri will be of particular value under the circumstances. In this report, after comparative screening of genomic DNA from six Listeria species by dot blot hybridization, we isolated one L. seeligeri-specific clone (lse24-315) that contains an insert of 1538 bp. Using primers (lse24-315F and lse24-315R) derived from this clone, we showed that a specific PCR product of 375 bp was generated from genomic DNA of L. seeligeri strains only, but not of other Listeria species or common bacteria. Therefore, the PCR employing primers lse24-315F and lse24-315R provides a rapid, sensitive and specific method for distinguishing L. seeligeri from other Listeria and common bacteria.

Effects of combination treatments of nisin and high-intensity ultrasound with high pressure on the microbial inactivation in liquid whole egg

The consecutive combinations of nisin with high hydrostatic pressure (nisin-HHP) and ultrasound with high hydrostatic pressure (US-HHP) were explored to achieve enhanced microbial inactivation in liquid whole egg processing. The HHP processing conditions were fixed to either 250 MPa for 886 s or 300 MPa for 200 s at the treatment temperature of 5 °C, which have been determined as the optimum HHP processing conditions considering egg protein coagulation and microbial inactivation kinetics. Between the two types of combinations, the nisin-HHP combination showed more promising results. The addition of nisin prior to pressure treatments significantly increased the lethal effects of HHP against Listeria seeligeri up to 5 log cycles. Because the individual effects of each nisin and HHP on the Listeria were almost negligible, the Listeria reductions are considered to be due to the synergistic action of nisin and HHP. However, the combination of nisin-HHP on E. coli showed exactly the same degree of inactivation by HHP alone, which supports protection mechanism of gram negative bacteria against the action of nisin. The US-HHP combination caused no reductions of Listeria and only a slightly increased inactivation of E. coli under the experimental conditions.

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.

Polymerase Chain Reaction Detection of Listeria monocytogenes on Frankfurters Using Oligonucleotide Primers Targeting the Genes Encoding Internalin AB

A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters. Four sets of oligonucleotide primers were evaluated. The set targeting a 902-bp region of the inlAB gene was the most specific. This PCR product was detected in 51 L. monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected in other Listeria spp. (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L. monocytogenes. The detection limit of the PCR assay was 105 CFU per ml of pure cell culture. However, the assay could detect as few as 101 CFU of L. monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30°C. The total assay time including enrichment was approximately 24 h. These results suggest that the PCR assay can be used to rapidly detect L. monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.

Seeligeriolysin O, a cholesterol-dependent cytolysin of Listeria seeligeri, induces gamma interferon from spleen cells of mice.

Seeligeriolysin O (LSO), one of the cholesterol-dependent cytolysins produced by Listeria seeligeri, shows 80% homology to listeriolysin O (LLO) produced by Listeria monocytogenes at the amino acid sequence level. In addition to cytolytic activity, LLO has been shown to exhibit cytokine-inducing activity. In order to determine whether LSO is also capable of exhibiting these two different activities, we constructed a recombinant full-length LSO (rLSO530) and a noncytolytic truncated derivative with a C-terminal deletion (rLSO483) and compared these molecules with recombinant LLO. The cytolytic rLSO530 molecule could induce gamma interferon (IFN-gamma) production in spleen cells when the cytolytic activity was blocked by treatment with cholesterol. The noncytolytic truncated rLSO483 molecule also induced IFN-gamma production. Anti-LLO polyclonal antibody inhibited not only LLO-induced IFN-gamma production but also LSO-induced IFN-gamma production. Both NK cells and CD11b(+) cells were required for LSO-induced IFN-gamma production. Among the various cytokines expressed in CD11b(+) cells, interleukin-12 (IL-12) and IL-18 appeared to be essential. We concluded that LSO exhibits the same biological activity as LLO.

Prevalence and Contamination Levels of Listeria monocytogenes in Smoked Fish and Pâté Sold in Spain

From March to November 2000, 170 samples of smoked fish and 182 samples of pâté for sale in retail outlets and supermarkets in the nine provinces of Castilla and León (Spain) were analyzed for the prevalence of Listeria monocytogenes and other Listeria spp. L. monocytogenes was isolated from 38 (22.3%) of the 170 samples of smoked fish analyzed. Twenty of these positive samples contained L. monocytogenes at >100 CFU/g. Other Listeria spp., such as Listeria innocua (26 isolates), Listeria grayi (9), Listeria welshimeri (3), Listeria seeligeri (3), and Listeria ivanovii (2), were also detected. L. monocytogenes was isolated from 5.4% of the 182 samples of pâté. Only 1 of the 10 positive samples harbored >100 L. monocytogenes CFU/g. Two other species of Listeria were observed in pâté: L. innocua (12 isolates) and L. grayi (2).

Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide

We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively. In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO. To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO. The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding. The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO. These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO.

Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide.

We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively. In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO. To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO. The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding. The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO. These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO.

Listeria in ready-to-eat and unprocessed foods produced in Portugal

Between October 1998 and April 2000, 429 food samples were investigated for the presence ofListeria spp. The foodstuffs included 138 ready-to-eat foods (68 traditional hard and semi-hard Portuguese cheeses, 23 salad vegetables and 47 cooked and/or cured meats) and 291 uncooked foods (14 raw vegetables, 65 raw chicken and 212 raw ewe's, cow's or goat's milk). Listeria spp. were recovered from 63 samples (15%). Listeria monocytogenes was present in 39 (9%) samples, Listeria innocua in 12 (3%), Listeria seeligeri in 23 (5%), Listeria ivanovii in seven (2%) and Listeria grayi in two (0·5%). More than one Listeria species was recovered from 18 samples. A combination of serotyping, phage-typing, cadmium and arsenic sensitivities were used to subtype 36 of the L. monocytogenes isolates: at least nine different strains were recognized. Four food samples yielded two different L. monocytogenes strains.

Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri.

Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

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

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.

Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland

A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes ( n=200), Salmonella ( n=200) and Campylobacter spp.( n=100). L. monocytogenes was not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK.

Listeria Species in Raw and Ready-to-Eat Foods from Restaurants

From September 1999 to March 2000, meat (pork, beef, and chicken), fish (salmon, hake, and sole), vegetable (lettuce and spinach), and Spanish potato omelette samples obtained at restaurants were collected and tested for the occurrence of Listeria spp. Listeria monocytogenes was isolated from 3 (2.9%) out of 103 studied samples. Other species isolated were Listeria grayi (13.6%), Listeria innocua (1.9%), Listeria ivanovii (5.8%), Listeria seeligeri (3.9%), and Listeria welshimeri (1.9%). Listeria was neither isolated from beef nor any type of fish.

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods.

In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.

Incidence and Characterization of Listeria spp. from Foods Available in Korea

A total of 410 domestic Korean food samples were analyzed for the presence of Listeria spp. by the conventional U.S. Department of Agriculture protocol, and presumptive strains were identified by morphological, cultural and biochemical tests according to Bergey's manual and confirmed by API-Listeria kit. Among the total 410 food samples, 46 samples (11.2%) were found to be contaminated with Listeria species. Among the 46 strains of Listeria spp. isolates, 8 strains (17.42%) for Listeria monocytogenes, 3 strains (6.5%) for Listeria seeligeri, 33 strains (71.7%) for Listeria innocua, and 2 strains (4.4%) for Listeria welshimeri were identified, respectively. Also, only beef, chicken, pork, frozen foods, and sausage were contaminated with L. monocytogenes, and the other products were free of L. monocytogenes. Of 46 Listeria spp. isolates, L. innocua (71.7%) was the most predominantly isolated in a variety of foods compared to other Listeria spp. An in vitro virulence assay for Listeria spp. using myeloma and hybridoma cells from murine and human sources was performed. The result showed that only L. monocytogenes killed approximately 95 to 100% hybridoma cells after 6 h and the other Listeria species, such as L. innocua, L. seeligeri, and L. welshimeri strains had about 0 to 10% lethal effect on hybridoma cells. Also, an antibiotic susceptibility test showed that Listeria spp. isolates were very susceptible to the antibiotics tested, except for nalidixic acid. Also, serotyping results showed 75% of L. monocytogenes isolates from beef, chicken, and frozen pizza belonged to serotype 1 and 25% from sauage were type 4.