The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens

The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells.     

Factors affecting the adsorption of buchnericin LB,a bacteriocin produced by Lactobacillus [correction of Lactocobacillus] buchneri

Buchnericin-LB adsorbs to gram-positive but not to gram-negative bacteria. The tested gram-positive bacteria were species of Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus, Listeria, Bacillus, Staphylococcus; gram-negative bacteria belonged to the genera Salmonella, Escherichia, Yersinia and Pseudomonas. Buchnericin-LB adsorption depended on pH but not on time and temperature. Also some anions of salts and lipoteichoic acid reduced or inhibited its adsorption. Treatment of cells and cell walls of sensitive bacteria with detergents, organic solvents or enzymes did not affect subsequent binding of buchnericin-LB. Treatment with buchnericin-LB caused sensitive cells to lose high amounts of intracellular K+ ions and UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-galactopyranoside. Buchnericin-LB (640-2560 AU/ml) decreased the colony forming units (99%) and absorbance values of Listeria monocytogenes and Bacillus cereus. These results indicate that the mode of action of buchnericin-LB is bactericidal and its lethal effect is very rapid.     

The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells

Yersinia virulence is dependent on the expression of plasmid-encoded secreted proteins called Yops. After bacterial adherence to receptors on the mammalian cell membrane, several Yops are transported by a type III secretion pathway into the host cell cytoplasm. Two Yops, YopH and YopE, prevent macrophages from phagocytosing Yersinia by disrupting the host cell cytoskeleton and signal transduction pathways. In contrast to this active inhibition of phagocytosis by Yersinia , other pathogens such as Salmonella , Shigella , Listeria and Edwardsiella actively promote their entry into mammalian cells by binding to specific host surface receptors and exploiting existing cell cytoskeletal and signalling pathways. We have tested whether Yersinia Yops can prevent the uptake of these diverse invasive pathogens. We first infected epithelial cells with Yersinia to permit delivery of Yops and subsequently with an invasive pathogen. We then measured the level of bacterial invasion. Preinfection with Yersinia inhibited invasion of Edwardsiella , Shigella and Listeria , but not Salmonella . Furthermore, we found that either YopE or YopH prevented Listeria invasion, whereas only YopE prevented Edwardsiella and Shigella invasion. We correlated the inhibitory effect of the Yops with the inhibitory action of the cell-signalling inhibitors Wortmannin, LY294002 and NDGA, and concluded that the four invasive pathogenic species enter epithelial cells using at least three distinct host cell pathways. We also speculate that YopE affects the rho pathway.     

The role of clathrin-dependent endocytosis in bacterial internalization

Internalization of bacteria into mammalian host cells has been studied extensively in the past two decades. These studies have highlighted the amazingly diverse strategies used by bacterial pathogens to induce their entry in non-phagocytic cells. The roles of actin and of the whole cytoskeletal machinery have been investigated in great detail for several invasive organisms, such as Salmonella, Shigella, Yersinia and Listeria. Recent results using Listeria highlight a role for the endocytosis machinery in bacterial entry, suggesting that clathrin-dependent endocytic mechanisms are also involved in internalization of large particles. This contrasts with the generally accepted dogma but agrees with previous studies of bacterial and viral infections and also of phagocytosis.     

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion

Abstract Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.     

Occurrence of sublethal injury after pulsed electric fields depending on the micro-organism, the treatment medium ph and the intensity of the treatment investigated

AIMS: The objective was to investigate the occurrence of sublethal injury after pulsed electric field (PEF) depending on the treatment time, the electric field strength and the pH of the treatment media in two Gram-positive (Bacillus subtilis ssp. niger, Listeria monocytogenes) and six Gram-negative (Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella serotype Senftenberg 775W, Salmonella serotype Typhimurium, Yersinia enterocolitica) bacterial strains. METHODS AND RESULTS: A characteristic behaviour was observed for the Gram-positive and Gram-negative bacteria studied. Whereas Gram-positive bacteria showed a higher PEF resistance at pH 7.0, the Gram-negative were more resistant at pH 4.0. In these conditions, in which bacteria showed their maximum resistance, a large proportion of sublethally injured cells were detected. In most cases, the longer the treatment time and the higher the electric field applied, the greater the proportion of sublethally injured cells that were detected. No sublethal injury was detected when Gram-positive bacteria were treated at pH 4.0 and Gram-negative at pH 7.0. CONCLUSIONS: Sublethal injury was detected after PEF so, bacterial inactivation by PEF is not an 'all or nothing' event. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could be useful for improving food preservation by PEF.     

Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae.

Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway. The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion. Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant. Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA. In addition, yopE-containing S. typhimurium expressed a YopE-mediated cytotoxicity on cultured HeLa cells. YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity. Both translocation and cytotoxicity were prevented by the addition of gentamicin. Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed. These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria. We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.     

Defense response mechanisms of ginseng callus cultures induced by Yersinia pseudotuberculosis, a human pathogen

The effect of Yersinia pseudotuberculosis, the bacterial pathogen affecting humans and animals, on growth of ginseng (Panax ginseng C.A. Mey) cell cultures was studied. The bacteria strongly induced the expression of phenylalanine ammonia-lyase and β-1,3-glucanase, the proteins encoded by the defense-related genes of ginseng and inhibited the normal ginseng callus growth but did not affect the resistant cell cultures. The thermostable and thermolabile protein toxins of these bacteria are lethal to mice when induced parentherally, and they also induced the expression of the defense-related genes in ginseng callus cultures. At the same time, the ginseng cells completely suppressed the bacterial cell growth. These data suggest that the ginseng cells recognized the yersinia and developed the immune response to this pathogen. The interaction between the ginseng cells and Y. pseudotuberculosis is similar to the hypersensitive response of plants to plant pathogens.     

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes

CH. NIEDERHAUSER, CH. HÖFELEIN, M. ALLMANN, P. BURKHALTER, J. LÜTHY AND U. CANDRIAN. 1994. The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.     

Rapid clearance of a recombinant Salmonella vaccine carrier prevents enhanced antigen-specific CD8 T-cell responses after oral boost immunizations

The type III secretion system of Salmonella enterica serovar Typhimurium can be used to target heterologous antigens directly into the cytosol of antigen-presenting cells. Our laboratory has previously reported that the single oral immunization of mice with a recombinant Salmonella strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal wild-type Listeria challenge infection. In the present study, we investigated whether these antigen-specific cytotoxic T lymphocytes induced by the prime immunization contribute to a more rapid clearance of the vaccine carrier after subsequent boost immunizations and whether oral boost immunizations lead to an augmented p60-specific CD8 T-cell response. We found that the ability of recombinant Salmonella strains to colonize the intestine, mesenteric lymph nodes, and spleen was markedly impaired after boost immunizations but that this effect was independent of existing CD8 T cells reactive with p60(217-225). A significant elevation of antigen-specific CD8 T cells could not be detected by enzyme-linked immunospot assay after the second or the third oral immunization, possibly due to the rapid clearance of the bacterial vaccine carrier from lymphatic organs.     

Growth of food-borne pathogenic bacteria in oil-in-water emulsions: I—Methods for investigating the form of growth

Methods are presented for investigating the site and form of growth of bacteria in model oil-in-water emulsions and in dairy cream. Following growth of the bacteria, the continuous aqueous phase is gelled using agarose and the oil phase removed using a mixture of chloroform and methanol. Using this method, the authors have found that Listeria monocytogenes, Salmonella typhimurium and Yersinia enterocolitica grow in the form of colonies in concentrated oil-in-water emulsions. Colonies of L. monocytogenes and Y. enterocolitica also form in artificially-inoculated fresh and tinned dairy cream. If information about the precise site of growth is not required, the authors have discovered that intact colonies can be liberated from the model emulsions by dissolving away the oil phase with chloroform: methanol.     

Metabolism of innate immune cells in bacterial infections

Metabolic activity of innate immune cells infected by various doses of Gram-negative (Yersinia pseudotuberculosis, Salmonella enteritidis) and Gram positive (Staphylococcus aureus, Listeria monocytogenes) bacteria has been investigated. Using various animal models we found that in during the initial period (up to 2 days) the changes in cellular responses depend on the type of the pathogen. In response to infection caused by Gram-negative bacteria predominant of neutrophil accumulation in the foci of inflammation was observed, while Gram-positive bacteria induced preferential accumulation of macrophages. The study of metabolism of these cells showed that the response of terminally differentiated primed phagocytes to pathogen appearance was higher than in cells circulating in blood. In addition to the priming state the phagocyte reactivity is influenced by the bacterial load. At a low phagocyte/microbe ratio the cells reaction is almost undetectable, while an excess of microorganisms causes (despite of the increase of the phagocytic parameters) the hyperactivation of cell metabolism and production of maximal amounts of bactericide agents, which exhibit a damaging effect on the cell itself.     

Pre-existing anti-Salmonella vector immunity prevents the development of protective antigen-specific CD8 T-cell frequencies against murine listeriosis

Our laboratory has focused its research on the use of the type III secretion system of Salmonella enterica serovar Typhimurium to translocate heterologous antigens directly into the cytosol of antigen-presenting cells. We have previously reported that the single oral immunization of mice with a recombinant Salmonella aroA/sptP mutant strain expressing the translocated Yersinia outer protein E fused to the immunodominant antigen p60 from Listeria monocytogenes in a type III-mediated fashion results in the efficient induction of p60-specific CD8 T cells and confers protection against a lethal Listeria challenge infection. In the present study, we determined whether pre-existing anti-Salmonella vector immunity influences the induction of p60-specific CD8 T cells and modulates protective immunity against listeriosis after oral vaccination with recombinant Salmonella. After single oral immunization, the Salmonella aroA/sptP double mutant strain was found to colonize spleens of mice for 21days. In contrast, the period of colonization was significantly shortened to 6days due to anti-Salmonella vector immunity after second oral immunization. The latter scenario led to the induction of low-level frequencies of antigen-specific CD8 T cells. Compared to the significantly higher numbers of p60-specific T lymphocytes elicited after single oral immunization, the low amount of Listeria-specific CD8 T cells did not confer protection against listeriosis.     

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA(s)-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.     

Survival of pathogenic bacteria during mesophilic anaerobic digestion of animal waste

The survival of pathogenic bacteria was investigated during the operation of a full-scale anaerobic digester which was fed daily and operated at 28°C. The digester had a mean hydraulic retention time of 24 d. The viable numbers of Escherichia coli, Salmonella typhimurium, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter jejuni were reduced during mesophilic anaerobic digestion. Echerichia coli had the smallest mean viable numbers at each stage of the digestion process. Its mean T₉₀ value was 76.9 d. Yersinia enterocolitica was the least resistant to the anaerobic digester environment; its mean T₉₀ value was 18.2 d. Campylobacter jejuni was the most resistant bacterium; its mean T₉₀ value was 438.6 d. Regression analysis showed that there were no direct relationships between the slurry input and performance of the digester and the decline of pathogen numbers during the 140 d experimental period.     

Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication

Strains of Escherichia, Salmonella, Shigella and Yersinia actively enter eukaryotic cells. Several techniques were used to compare and contrast the invasion mechanisms of Salmonella cholerae-suis, Yersinia enterocolitica and Shigella flexneri. Three animal cell lines (CHO, HEp-2 and MDCK) were examined for susceptibility to bacterial entry by these strains. Levels of intracellular bacteria varied widely between cell lines, but CHO cells were the most susceptible to bacterial invasion, HEp-2 invasion levels were intermediary, whereas polarized MDCK cells were invaded to a lesser extent. This illustrates that tissue culture models can be optimized to study bacterial invasion and intracellular replication. We used these tissue culture models to examine the interactions between host cells and these invasive bacteria. The use of lysosomotropic agents (methylamine and ammonium chloride), cationic ionophores (monensin) and acidification-defective CHO cell lines demonstrated that endosome acidification is not required for bacterial invasion or intracellular replication. Drugs which inhibited microfilament formation (cytochalasins B and D) prevented internalization of S. cholerae-suis, Y. enterocolitica and S. flexneri, indicating that invasion is a microfilament-dependent event. The microtubule inhibitors, colchicine, vincristine and vinblastine, did not affect bacterial internalization.     

Intestinal carriage of campylobacters, salmonellas, yersinias and listerias in pigeons in the city of Barcelona

L. CASANOVAS, M. DE SIMÓN, M.D. FERRER, J. ARQUÉS AND G. MONZÓN. 1995. The faecal bacterial flora of pigeons, which may be the source of infectious diseases in man, was studied in the city of Barcelona. Four hundred cloacal specimens were examined for Campylobacter jejuni, Camp. coli, Salmonella spp., Yersinia spp. and Listeria spp., over a 12 month period.
Campylobacter jejuni was the most frequently isolated micro-organism, found in 105 pigeons (26.2%), with a greater incidence in the districts of the city with a high density of pigeons and without seasonal variation. Salmonella spp. were isolated from six specimens (1.5%) and Yersinia intermedia was isolated from only one pigeon.     

Yersinia outer protein P inhibits CD8 T cell priming in the mouse infection model

Pathogenic yersiniae translocate a mixture of effector proteins called Yersinia outer proteins (Yops) into the cytosol of eukaryotic cells by their type III secretion system. YopP is one of the best characterized of these effector proteins and known to inhibit the proinflammatory response of the host by interfering with NF-kappaB signal transduction and inducing apoptosis of macrophages. The effects of YopP on the immune response were studied by a Yersinia Ag-independent approach using bacteria that translocate the well-characterized model Ag listeriolysin O of Listeria monocytogenes via their type III secretion system. In this study we demonstrate a novel function for YopP in vivo. It is shown for the first time that YopP not only counteracts the innate immune defense but also inhibits the adaptive immune system by suppressing the development of an effective CD8 T cell response in a mouse model. A possible mechanism for this could be the inhibition of Ag presentation by dendritic cells (DC). In vitro this is shown to be due to the rapid induction of programmed DC death and to inhibition of DC maturation. Using this approach we could further show that the listeriolysin O-specific CD8 T cells generated in vivo by the yopP mutant are functional and are able to protect mice against a lethal challenge with wild type Listeria.     

Enhancement of mice susceptibility to infection with Listeria monocytogenes by the treatment of morphine

The effect of morphine on the susceptibility of BALB/c mice to diarrheagenic Escherichia coli, Shigella flexneri, Listeria monocytogenes, Salmonella Enteritidis, Yersinia enterocolitica, was examined via the intraperitoneal inoculation. Morphine treatment increased the susceptibility to S. Enteritidis and L. monocytogenes, resulting in bacteremia and central nervous system (CNS) invasion (for L. monocytogenes), while the infection with other bacteria did not show the systemic dissemination in the morphinetreated mice. Notably, L. monocytogenes infection caused 100% mortality with a mean survival time (MST) of 1.3 days in morphine-treated mice, but untreated mice did not die. The present data suggested that individuals using heroin or treated with morphine derivatives might be at high risk for listeriosis, especially those who are immunocompromised. Recent increasing consumption of morphine may propose the necessity for further epidemiological surveillance on infectious diseases.