Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.     

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.     

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC₅₀ values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO dimethyl sulfoxide - HPC hepatocyte primary culture - IC₅₀ 50% inhibitory concentration - LDH lactate dehydrogenase     

Cloning and expression of the Listeria monocytogenes haemolysin in Escherichia coli

Abstract Listeriolysin, an SH-activated haemolysin probably involved in Listeria pathogenicity, has been cloned into the cosmid vector pHC79 and was expressed in Escherichia coli HB101 cells. Chromosomal DNA of Listeria monocytogenes serovar 1/2 was partially digested with Mbo I and ligated to the Bam HI cleaved cosmid. From 2000 recombinant clones examined, 12 (0.6%) produced haemolysin in solid and liquid media. All of them contained chromosome fragments of Listeria of about 40 kb. The cloning of the listeriolysin determinant will lead to a better understanding of the basis of Listeria pathogenicity.     

Interaction of Listeria monocytogenes autolysin amidase with glycosaminoglycans promotes listerial adhesion to mouse hepatocytes

Adherence to the cell surface is a key event during infection of pathogenic microorganisms. We have previously reported that autolysin amidase (Ami) of Listeria monocytogenes promotes an efficient listerial adherence to mouse hepatocytes and requires for listerial pathogenicity. Cell wall anchoring (CWA) domain of Ami has been shown to bind lipoteichoic acid on listerial cell wall but the binding of Ami to host cell surface molecules remains to be determined. In this study, we present evidence here that Ami promotes efficient adherence of L. monocytogenes to mouse hepatocytes mediated by glycosaminoglycans (GAGs). The adhesion of L. monocytogenes wild type but not Ami-deficient mutant to the hepatocytes was dramatically attenuated by 4-nitrophenyl-β-D-xylopyranoside, a specific inhibitor of GAG association to cell surface. Full-length and truncated Ami were used to investigate the binding of Ami to GAGs and we found that four-repeated CWA of Ami is sufficient to bind GAGs on the host cell surface. Competitive assay and surface plasmon resonance demonstrated that Ami interacts with sulfated GAGs but not non-sulfated GAGs. The results suggest that Ami acts as an adhesin of L. monocytogenes to hepatocytes by interaction via its four-repeated CWA domain and sulfated GAGs.     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.     

Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes

Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed.     

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

天然心房肽对实验性心功能与心肌酶的影响

以天然心房肽灌流豚鼠离体工作心脏,药后１分钟心率开始减慢,左室压及其微分开始升高;５分钟后逐渐恢复,至１０分钟恢复正常。体外培养大鼠心肌细胞,培养液中乳酸脱氢酶含量在药后２小时减少至４８．５±６．８ｕ,为药前的４２．７％（Ｐ＜０．０１）,药后８小时仍明显低于药前水平。为心房肽的新药开发提示了新思路。     

Bacillus cereus-Induced Fluid Accumulation in Rabbit Ileal Loops

The usefulness of the ligated rabbit ileal loop as an experimental model of Bacillus cereus food poisoning was investigated. Positive responses, as measured by fluid accumulation in the loop, were obtained from 19 of 22 strains of B. cereus. Four of six strains of B. thuringiensis also elicited fluid accumulation, but eight strains of other Bacillus spp. failed to evoke a response. The growth medium employed markedly affected the ability of a given strain of B. cereus to provoke a response. Brain heart infusion broth (BHI) (Difco) proved to be best for this purpose. Loop fluid-inducing activity was produced by exponentially growing cells and was present in cell-free culture filtrates and associated with washed vegetative cells. Intraluminal growth of B. cereus did not elicit fluid accumulation. Cultures grown at temperatures in the range of 18 C to 43 C were loop active. When BHI cultures of selected loop positive strains were injected intraluminally into the normal ileum of rabbits, they failed to elicit diarrhea.     

A role for proline synthesis and transport in Listeria monocytogenes barotolerance

Aims: To assess the contribution of proline biosynthesis to listerial barotolerance. Methods and Results: Using a Listeria monocytogenes proBA deletion mutant, incapable of synthesizing proline, together with a proline‐overproducing strain, the contribution of proline synthesis to listerial barotolerance was determined. The ΔproBA strain does not survive as well as the wild type when subjected to treatment of 500 MPa in rich media and 400 MPa in minimal media (c. 1 log lower survival in both conditions). Betaine and carnitine decrease the ability of the wild type to survive at low pressures (300 MPa), but confer normal or slightly increased levels of protection at higher pressures (350 and 400 MPa). Conclusions: A functional proline synthesis system is required for optimal survival of Listeria following treatment at high‐pressure (HP) levels (500 MPa in brain heart infusion and 400 MPa in defined medium), particularly where other compatible solutes are absent or limiting. Significance and Impact of the Study: Given the potential of HP processing as an effective food processing/safety strategy, understanding how pathogens such as Listeria have evolved to cope with such stresses is an important food safety consideration. In this context, the work presented here may help to develop safer and more effective processing regimens.     

Specific detection and quantitative enumeration of Listeria spp. using fluorescent in situ hybridization in combination with filter cultivation (FISHFC)

Aims:  To design a rapid specific method for enumeration of viable Listeria spp. using the fluorescence in situ hybridization with filter cultivation (FISHFC) method.
Methods and Results:  The probe, Lis-1400, was designed from the 23S rRNA region of the Listeria genome, and labelled with 5'-carboxy-tetramethyl-rhodamine- N - hydroxy-succinimide-ester. Fluorescence was observed for all Listeria species but not for any organisms from the other genera, suggesting Lis-1400 is highly specific for Listeria spp. For purposes of filter cultivation prior to hybridization, hydrophilic polypropylene membrane filters gave better contrast between fluorescing colonies and background fluorescence. This was because of a high S/N ratio (fluorescence intensity of each microcolony/fluorescence intensity of background noise) after FISH treatment. Results were achievable in 14 h using Lis-1400-aided FISHFC as compared with 4–7 days required for confirmation of Listeria spp. by conventional plate count methods. Moreover, viable Listeria counts in selected food samples showed no significant differences between Lis-1400-aided FISHFC and conventional methods.
Conclusions:  The Lis-1400-aided FISHFC method is more efficient than conventional methods for enumeration of viable Listeria spp. in food samples.
Significance and Impact of the Study:  For enumeration of Listeria spp., Lis-1400-aided FISHFC method is equally accurate yet faster than conventional plate count methods, and can be valuable in the control of listeriosis.     

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.     

Toxic exoproducts of citrobacter freundii

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.     

Influence of immunomodulation on the development of Listeria monocytogenes infection in aged guinea pigs

We investigated the impact of immunomodulation on the development of listeriosis within an aged population of guinea pigs after an intragastric challenge with Listeria monocytogenes. Supplementation with vitamin E for 35 days significantly increased the level of cytotoxic T cells (CD8(+)), while treatment with cyclosporin A resulted in a 25% decrease of CD8(+) T cells. In the animals receiving the low dose (10(2) CFU) of L. monocytogenes, 50% of the control-group animals became infected. Only 22% of animals receiving the orthomolecular dose of vitamin E became infected, whereas animals that were immunosuppressed had an infection rate of 89%. In the immunosuppressed group three animals (16%) developed listerial infection with a quantifiable bacterial level of 0.3-3 log CFU g(-1) of organ in the spleen and liver. In the high-dose study, the population of L. monocytogenes was consistently 1 log CFU g(-1) lower in the spleen or liver of the vitamin E-supplemented group, compared with the control and cyclosporin A-treated animals. At day 4, a significant increase in the levels of CD8(+) during listerial infection occurred in vitamin E-supplemented animals, suggesting an increased ability to produce CD8(+) T cells. The results suggest that immunomodulation of the host can influence listerial infection within an aged population of guinea pigs.     

Protective effect of S-adenosylmethionine against galactosamine-induced injury of rat hepatocytes in primary culture

The protective effect of S-adenosylmethionine (SAMe) on D-galactosamine (GalN)-induced damage to rat hepatocytes was tested in primary cultures. SAMe at concentrations of 50 and 1000 mg/l significantly reduced lactate dehydrogenase release from cells injured by 40 mM GalN after 24 h of incubation. There were no significant changes in urea production after 24 h among tested groups, including control hepatocytes. Exposure of hepatocytes to GalN leads to 3.5-fold decrease in urea synthesis after 48 h in comparison with control cell cultures. Addition of the highest dose of SAMe (1000 mg/l) into the culture media attenuated this decrease by 180 %. None of the tested doses of SAMe (5, 25, 50 and 1000 mg/l) affected considerably the reduced activity of mitochondrial dehydrogenases. The content of reduced and oxidized glutathione in GalN-exposed cells was diminished to 1.5 % and 16 %, respectively, of the control values after 24 h. Using only the highest concentration SAMe increased significantly these contents. SAMe had no effect on dramatically decreased albumin synthesis. These findings indicate beneficial effect of SAMe, especially of the highest concentration, on GalN-induced toxicity to rat hepatocytes in primary culture. This action of SAMe seems to be associated with reduction of plasma membrane damage and increased synthesis of glutathione.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species

Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.