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Detection of listeriolysin, the thiol-dependent hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seeligeri.

The listeriolysin gene from a weakly hemolytic but virulent strain of Listeria monocytogenes serotype 1/2a was cloned in Escherichia coli K-12. Recombinants were identified on the basis of their cross-reactivities to hyperimmune antisera raised against streptolysin O and listeriolysin. Low levels of hemolytic activity were detected in crude lysates of strains harboring the listeriolysin gene. In DNA hybridization studies with five DNA probes that encoded the listeriolysin gene and surrounding sequences, highly homologous listeriolysin genes were found to be present in the species L. monocytogenes, Listeria ivanovii, and Listeria seeligeri. Immunoblotting performed with affinity-purified antibody to listeriolysin allowed the detection of this protein in supernatants of all three species. This study demonstrates for the first time that listeriolysin is produced by L. seeligeri and documents the genetic homology between the various listeriolysins produced by Listeria spp. Sequences unique to the species L. monocytogenes were found to be located downstream of the listeriolysin gene. Furthermore, the restriction fragment length polymorphisms detected with probes flanking the hlyA gene may be useful epidemiological markers in identifying and distinguishing virulent Listeria strains from each other.

Bacterial Toxins↗

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri.

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.

Animals↗

The use of listeriolysin O in an ELISA, a skin test and a lymphocyte blastogenesis assay on sheep experimentally infected with Listeria monocytogenes, Listeria ivanovii or Listeria innocua.

Purified listeriolysin O (LLO) was evaluated as a specific antigen to detect both humoral and cell mediated immune responses of sheep infected with Listeria monocytogenes. Six sheep (two in each group) were orally inoculated with 10(10) organisms of L. monocytogenes, L. ivanovii, or L. innocua. Only the L. monocytogenes inoculated sheep had an elevated temperature (> 42 degrees C) and after 15 days had anti-LLO antibodies as assessed by an ELISA. In a blastogenesis assay, only peripheral blood mononuclear cells (PBMC) from L. monocytogenes-infected sheep responded to LLO, while PBMC from all the sheep responded somewhat to heat-killed L. monocytogenes bacteria. In a skin test, only L. monocytogenes-infected sheep exhibited a positive reaction to injected LLO, while all the Listeria-infected sheep reacted to heat-killed bacteria. On day 120 postinfection, all of the sheep were orally inoculated with L. monocytogenes. Only the four that had not been previously given L. monocytogenes exhibited an elevated temperature (> 42 degrees C). 80 days later, sera from all of the animals were positive for anti-LLO antibodies. Thus, prior exposure to L. ivanovii or L. innocua does not protect against a L. monocytogenes challenge. These results suggest LLO is an excellent antigen for use in detecting Listeria infection in sheep. However, whether LLO will be useful in differentiating chronically infected animals from animals that have recovered, has yet to be investigated.

Animals↗

Differentiation of Listeria monocytogenes, Listeria innocua, Listeria ivanovii, and Listeria seeligeri by pulsed-field gel electrophoresis.

Clamped homogeneous electric field analysis of Listeria DNA with ApaI, AscI, SmaI, or NotI revealed species- and serotype-specific differences in genomic fingerprints. Clamped homogeneous electric field analysis may prove useful, therefore, in epidemiologic studies. Also, the summation of individually sized AscI fragments of genomic DNA from L. monocytogenes serotype 4b 101M and Scott A indicated genome lengths of 2,925 and 3,046 kb, respectively.

DNA Fingerprinting↗

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.

Base Sequence↗

Elevated intraocular pressure, pigment dispersion and dark hypopyon in endogenous endophthalmitis from Listeria monocytogenes.

Listeria monocytogenes endophthalmitis occurred in an immunologically competent patient with no identifiable extraocular septic focus. The patient presented with a dark hypopyon and markedly elevated intraocular pressure, and the diagnosis was established by culture and histopathologic examination of ocular fluid. Four of the fourteen reported cases of Listeria monocytogenes endophthalmitis also presented with a dark hypopyon, and all cases had markedly elevated intraocular pressure. The presence of a dark hypopyon and elevated intraocular pressure may indicate endogenous intraocular infection with Listeria monocytogenes, even in an apparently healthy host.

Aged↗

[Early onset neonatal septicemia caused by Listeria monocytogenes].

Listeria monocytogenes can cause sepsis and meningitis during the neonatal period. Six cases of early onset neonatal sepsis caused by Listeria monocytogenes are reported here. These cases were diagnosed in a private hospital at Santiago, Chile from December 1984 throughout November 1986. The incidence rate was 1.4 x 1,000 liveborns. Clinical findings included prematurity (6), meconium stained amniotic fluid (6), hepatomegaly (6), splenomegaly (6), maculopapular exanthem (4), anal prolapse (3) and meningitis (1). Additionally 5 patients developed respiratory distress and 4 required ventilatory support. Overall mortality was 50% (3/6). All deaths were related to respiratory failure and occurred during the first week of disease. All patients received ampicillin and amikacin early in the course of their infection. Listeriosis of the newborn infant might be preventable by prompt recognition and treatment of maternal infections. Since Listeria infection in pregnancy is usually mild and symptoms and signs are nonspecific, prevention may be difficult. Pregnant women with fever of no clear origin or with an influenza like syndrome should be screened for listeriosis with cultures from blood, vagina and cervix samples.

Humans↗

Perinatal infection with Listeria monocytogenes.

Listeria monocytogenes has been increasingly recognized as a cause of intrauterine sepsis with associated perinatal wastage. The condition is mostly acquired through dietary intake and appropriate advice should be given to all pregnant women. The most common presentations in pregnancy include premature labour, an influenza-like illness and reduced fetal movements. In this report, we present a series of 24 cases of perinatal listeria infection presenting to either our obstetric or neonatal units and confirmed by the microbiology department of the hospital. In particular, we wish to highlight 3 cases in which antenatal diagnosis and aggressive therapy was associated with a successful outcome. Amongst the remaining 21 cases in which an antenatal diagnosis was not made, there were 5 perinatal deaths and 1 mid-trimester loss at 18 weeks. Clinicians must maintain a high index of suspicion for listeria, particularly in gravid patients who present with fever in the setting of a persistent 'flu-like' illness and premature labour. Once suspected, appropriate specimens for listeria culture should include blood, cervical swabs and midstream urine. Empirical antibiotic therapy with amoxicillin should be instituted while waiting for culture results in patients with possible Listeria monocytogenes sepsis.

Adult↗

[Neonatal systemic infection caused by Listeria monocytogenes].

Listeria monocytogenes is a gram positive cocco-bacillus which causes perinatal infections and also attacks immunocompromised hosts. Little is known about it in our medium. As part of a prospective study on neonatal systemic infections, its participation at the National Institute of Perinatology was researched. During a period of 18 months, 9,283 live newborns were observed, 141 of them were diagnosed with neonatal septicemia. During this period seven neonates had systemic infections due to Listeria monocytogenes: three had septicemia (two of these with meningitis) and all seven cases had pneumonia. The gestational age of the neonates was 26.1 to 41 weeks (X + DS = 35 + 4.3), with a weight of 830 g to 2,975 g (X + DS 1,958 + 773), four out of seven weighed less than 2,000 grams. The most frequent clinical manifestation was respiratory related causing a need for a differential diagnosis with hyaline membrane disease, transitory tachypnea and meconium swallowing at birth. All of the strains isolated were found to be susceptible to ampicillin, penicillin, gentamicin and amikacin; requiring high CMI levels of cephalosporins.

Humans↗

Influence of ibuprofen on the infection with Listeria monocytogenes.

Listeria monocytogenes is a bacterial infection, which is facultatively localized in monocytes and macrophages. The influence of ibuprofen (CAS 15687-27-1), a nonsteroidal anti-inflammatory drug (NSAID), on this bacterial infection in balb/c mice was investigated. One day prior to sublethal infection, balb/c mice were treated intravenously with various therapeutic concentrations of ibuprofen alone or ibuprofen in combination with a suboptimal dosage of murine recombinant interferon gamma, a lymphokine produced by T-helper cells. Three days post-infection, parasite burdens of the mainly infected organs, spleen and liver, were determined by the colony-forming unit assay. It was shown that the prophylactic treatment with ibuprofen in a concentration of 4 mg/kg body weight resulted in a more than 10-fold reduction of viable Listeria monocytogenes in the spleen, whereas in liver 12 mg/kg Ibuprofen was necessary for a comparable kill of viable bacteria. A higher concentration of ibuprofen did not resulted in a higher antibacterial efficacy. In order to clarify the mechanism of ibuprofen action, molecular-biological experiments were performed to measure the messenger RNA (mRNA) induced by ibuprofen. It is presented here that therapeutic concentrations of ibuprofen induced significant higher amounts of mRNA for interleukin-1 in human monocytes compared to untreated cells. These findings support the hypothesis that ibuprofen influences the complex immune system to overcome a bacterial infection.

Animals↗

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat.

An immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca. This technique involved enrichment of the suspect sample at 30 degrees C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1 x 10(3) cfu ml-1 was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua. The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.

Animals↗

Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes.

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.

DNA Primers↗

Differential regulation of Ia expression and antigen presentation by listeriolysin-producing versus non-producing strains of Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterial pathogen. A single gene product, listeriolysin (LLO), is critical for the induction of protective immunity. We now show that listeria that produce functional LLO augment Ia expression by macrophages and are better presented to a Th1, CD4+ anti-listeria T cell line. We used two genetically engineered strains of listeria which differed only in their ability (Ly+) or inability (Ly-) to produce functional LLO. Ia-negative murine macrophages ingested either Ly+ or Ly-, and then were stimulated by interferon-gamma (IFN-gamma). Increasing numbers of live Ly+, but not Ly-, augmented IFN-gamma-induced Ia expression. Ly+ by itself did not induce Ia expression. Heat-killed Ly+ and Ly- did not augment IFN-gamma-induced Ia expression. The abundance of Ia on the macrophage cell surface is one major determinant of antigen presentation to CD4+ T cells. Consistent with their ability to augment la expression, Ly+ were better presented than Ly- to a CD4+, Th1, anti-listeria T cell line. When macrophages and T cells were from different inbred mouse strains, antigen presentation required identity at the class II region of the MHC gene complex. This indicated that antigen presentation occurred via Ia molecules. The increased ability of macrophages to present Ly+ is a product of the macrophage-listeria interaction, not a property of the T cell tine 86. If Ia-negative macrophages ingested Listeria and were then stimulated by IFN-gamma, Ly+ was presented more efficiently than Ly-. On the other hand, if Ia-positive macrophages ingested Listeria, then Ly+ and Ly- were presented equally well to T cells. Altogether our data is consistent with the hypothesis that macrophages interact differently with Ly+, and that this contributes to the ability of only live Ly+ to induce protective immunity.

Animals↗

Oral pretreatment of mice with CpG DNA reduces susceptibility to oral or intraperitoneal challenge with virulent Listeria monocytogenes.

Listeria monocytogenes is an enteroinvasive intracellular bacterial pathogen that infects humans and other animals, including mice, sometimes resulting in severe systemic infections. Previous studies showed that intraperitoneal (i.p.) pretreatment of susceptible BALB/c mice with immune-stimulatory CpG DNA 48 to 96 h prior to i.p. challenge with virulent L. monocytogenes reduces bacterial numbers in livers by greater than 100-fold, correlating with recovery from infection. Here we show that oral pretreatment of BALB/c mice with CpG DNA results in decreased susceptibility to either oral or i.p. challenge with L. monocytogenes. A single dose of 200 microg of CpG DNA administered to BALB/c mice orally by gavage 48 h or 7 days before oral challenge with virulent L. monocytogenes reduces bacterial numbers approximately 10- to 100-fold in livers and spleens. Lymphotoxin alpha knockout mice lacking Peyer's patches (PPs) and pretreated orally with CpG DNA 48 h prior to oral challenge with L. monocytogenes also have reduced susceptibility to infection, suggesting that PPs are required neither for oral infection nor for CpG-induced resistance against oral infection with L. monocytogenes. Surprisingly, 48-h oral pretreatment of BALB/c mice with 100 to 200 microg of CpG DNA results in approximately 100-fold-decreased bacterial numbers in livers following i.p. challenge with L. monocytogenes, suggesting, along with other data in this report, that orally delivered CpG DNA induces systemic resistance to infection. These results indicate that oral administration of CpG DNA induces systemic innate immune defenses against either oral or systemic infection with virulent L. monocytogenes.

Adjuvants, Immunologic↗

Bacteriocin inhibition of two glucose transport systems in Listeria monocytogenes.

Listeria monocytogenes transports glucose by proton motive force-mediated and phosphoenolpyruvate-dependent phosphotransferase systems (PEP-dependent PTS). Inhibition of both systems by nisin, pediocin JD and leuconosin S is reported here for four strains of L. monocytogenes. Intracellular and extracellular adenosine triphosphate (ATP) and extracellular inorganic phosphate were measured in energized L. monocytogenes Scott A cells to determine whether inhibition of the PEP-dependent PTS might occur as a result of bacteriocin-induced leakage of intracellular components. Addition of nisin resulted in a decrease in intracellular ATP with an increase in extracellular ATP. Leuconosin S and pediocin JD induced a depletion of intracellular ATP. ATP efflux was low for the leuconosin S-treated cells and barely detectable for pediocin JD-treated cells. Addition of nisin, leuconosin S and pediocin JD induced efflux of inorganic phosphate. It appears that bacteriocin-mediated inhibition of the glucose PEP-dependent PTS occurs as a result of hydrolysis or efflux of ATP, PEP and other essential molecules from L. monocytogenes cells.

Adenosine Triphosphate↗

Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes.

Listeria monocytogenes was used as a model intracellular parasite to study stages in the entry, growth, movement, and spread of bacteria in a macrophage cell line. The first step in infection is phagocytosis of the Listeria, followed by the dissolution of the membrane surrounding the phagosome presumably mediated by hemolysin secreted by Listeria as nonhemolytic mutants remain in intact vacuoles. Within 2 h after infection, each now cytoplasmic Listeria becomes encapsulated by actin filaments, identified as such by decoration of the actin filaments with subfragment 1 of myosin. These filaments are very short. The Listeria grow and divide and the actin filaments rearrange to form a long tail (often 5 microns in length) extending from only one end of the bacterium, a "comet's tail," in which the actin filaments appear randomly oriented. The Listeria "comet" moves to the cell surface with its tail oriented towards the cell center and becomes incorporated into a cell extension with the Listeria at the tip of the process and its tail trailing into the cytoplasm behind it. This extension contacts a neighboring macrophage that phagocytoses the extension of the first macrophage. Thus, within the cytoplasm of the second macrophage is a Listeria with its actin tail surrounded by a membrane that in turn is surrounded by the phagosome membrane of the new host. Both these membranes are then solubilized by the Listeria and the cycle is repeated. Thus, once inside a host cell, the infecting Listeria and their progeny can spread from cell to cell by remaining intracellular and thus bypass the humoral immune system of the organism. To establish if actin filaments are essential for the spread of Listeria from cell to cell, we treated infected macrophages with cytochalasin D. The Listeria not only failed to spread, but most were found deep within the cytoplasm, rather than near the periphery of the cell. Thin sections revealed that the net of actin filaments is not formed nor is a "comet" tail produced.

Actin Cytoskeleton↗

A predictive model to determine the effects of pH, milkfat, and temperature on thermal inactivation of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of significance because of its comparatively high heat resistance, zero tolerance in ready-to-eat foods, and growth at refrigeration temperatures. A 3 x 3 x 3 factorial study was done to determine the effects of milkfat (0%, 2.5%, 5.0%), pH (5.0, 6.0, 7.0), and processing temperature (55 degrees C, 60 degrees C, 65 degrees C) on the thermal resistance of L. monocytogenes in a formulated and homogenized milk system. Data were fit to a modified Gompertz equation where parameter estimates characterized three regions of a survival curve: the shoulder, maximum slope, and tail. Statistical analysis was done for each of the 27 individual treatment sets to visualize individual effects on parameter estimates and to evaluate how well the Gompertz equation represented the data. A regression model for the Gompertz equation was generated to predict the logarithmic surviving fraction of L. monocytogenes based on all 27 treatments and their single and interactive effects. The shoulder region of the survival curve was affected by pH; however, the maximum slope was affected by temperature, milkfat, and the interaction of temperature x milkfat. Validation of the model suggests that the predictions are best suited for processing above 62 degrees C. Trends over time for a 4-log reduction in cells (4D values) were evaluated using results from the 27 individual treatment sets, the regression model for the Gompertz equation, and a linear equation. At lower temperatures, 4D values by the three methods varied by twofold. At higher temperatures, all methods gave similar 4D values, suggesting that death became more linear. Based on this study all three factors affect heat resistance for specific regions of a survival curve, and a predictive model was developed that can be used as a preliminary estimate for L. monocytogenes inactivation.

Animals↗

Early effects of lead on bone marrow cell responsiveness in mice challenged with Listeria monocytogenes.

Listeria monocytogenes challenge of lead-treated mice results in increased mortality. Since macrophage development constitutes the initial phase of the immune response to L. monocytogenes, bone marrow and spleens from Pb-treated mice that were infected with L. monocytogenes were analyzed for their ability to form colonies when exposed to the macrophage growth factor CSF-1. Serum colony-stimulating activity also was evaluated. Data obtained indicate the Pb exposure results in decreased responsiveness of bone marrow and spleen cells to CSF-1 while colony-stimulating activity in serum rises. This lack of bone marrow-derived macrophage development may contribute to the increased mortality observed with L. monocytogenes challenged. Pb-treated mice.

Animals↗