Spread Of Listeria Research Articles

Adhesion and kinetics of biofilm formation and related gene expression of Listeria monocytogenes in response to nutritional stress

Listeria monocytogenes is a gram-positive pathogen, that usually adheres to stainless steel (SS), and other abiotic surfaces in food processing that undergo repeated cleaning and cause the spread of Listeria. Through the enumeration of biofilm cells, extracellular polymeric substance (EPS) component and the scanning electron microscopy (SEM) analysis of biofilms, it was found that the ratio of cells and extracellular matrix is affected by nutrition status. Regardless of the temperature, all strains exhibited a higher adhesion ability when exposed to 10-fold diluted TSB-YE (DTSB-YE, nutrition deficiency). Three hour initial adhesion was significantly positively correlated with biofilm formation (p＜0.01). DTSB-YE enhances initial attachment and subsequently promotes biofilm formation. The SEM analysis also showed that in DTSB-YE the adhesion and covered area of the attached cells were higher than those in TSB-YE (rich media). The amount of both extracellular polysaccharides and proteins was significantly higher when incubated in DTSB-YE than TSB-YE. The highest biofilm formation of Lm83 was observed in DTSBYE independent of temperature. The effects of nutrition deficiency on the expression of critical biofilm-associated genes of Lm 83 planktonic and biofilm cells were measured. The gene expression levels of inlA and sigB in biofilm cells in TSB-YE and DTSB-YE were approximately 95.7% and 88.0% and 42.2% and 45.7% lower than those in planktonic cells, respectively. However, the expression of inlA in DTSB-YE was significantly higher (p＜0.05) than that in TSB-YE for the same cell state. Interestingly, the gene expression of motB was considerably higher in DTSB-YE than in TSBYE, regardless of the state. These results indicate that better cell motility in nutrient deficiencies might facilitate the cell aggression to promote biofilm formation.

Molecular Mechanisms of Intercellular Dissemination of Bacterial Pathogens.

Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexerni, and Rickettsia spp. use an actin-based motility process to spread in mammalian cell monolayers. Cell-to-cell spread is mediated by protrusive structures that contain bacteria encased in the host cell plasma membrane. These protrusions, which form in infected host cells, are internalized by neighboring cells. In this review, we summarize key findings on cell-to-cell spread, focusing on recent work on mechanisms of protrusion formation and internalization. We also discuss the dynamic behavior of bacterial populations during spread, and highlight recent findings showing that intercellular spread by an extracellular bacterial pathogen.

Host endoplasmic reticulum COPII proteins control cell-to-cell spread of the bacterial pathogen Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that uses actin-dependent motility to spread between human cells. Cell-to-cell spread involves the formation by motile bacteria of plasma membrane-derived structures termed 'protrusions'. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co-precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild-type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host-mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13 or Sar1 or brefeldin A treatment also perturbed the structure of cell-cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.

Listeria monocytogenesantagonizes the human GTPase Cdc42 to promote bacterial spread

The bacterial pathogen Listeria monocytogenes uses actin-based motility to spread from infected human cells to surrounding healthy cells. Cell-cell spread involves the formation of thin extensions of the host plasma membrane ('protrusions') containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco-2 BBE1 induced a decrease in the level of Cdc42-GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen-induced downregulation of host Cdc42.

Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria

The bacterial pathogen Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42. These three human proteins form a complex that generates tension at apical cell junctions. Listeria relieves this tension and facilitates protrusion formation by secreting a protein called InlC. InlC interacts with a Src Homology 3 (SH3) domain in Tuba, thereby displacing N-WASP from this domain. Interaction of InlC with Tuba is needed for efficient Listeria spread in cultured human cells and infected animals. Recent structural data has elucidated the mechanistic details of InlC/Tuba interaction, revealing that InlC and N-WASP compete for partly overlapping binding surfaces in the Tuba SH3 domain. InlC binds this domain with higher affinity than N-WASP, explaining how InlC is able to disrupt Tuba/N-WASP complexes.

Generation of airborne Listeria innocua from model floor drains.

Listeria monocytogenes can colonize floor drains in poultry processing and further processing facilities, remaining present even after cleaning and disinfection. Therefore, during wash down, workers exercise caution to avoid spraying hoses directly into drains in an effort to prevent the escape and transfer of drain microflora to food contact surfaces. The objective of this study was to examine the extent to which an inadvertent water spray into a colonized floor drain can cause the spread of airborne Listeria. Listeria innocua was used to inoculate a polyvinyl chloride model floor drain, resulting in approximately 10(8) cells per ml of phosphate-buffered saline and 10(4) attached cells per square centimeter of inner surface. Each model drain was subjected to a 2-s spray of tap water at 68.9 kPa from a distance of 1 m. Drains were sprayed while filled and again after emptying. Airborne cells were collected by using sedimentation plates containing Listeria selective agar which were placed on the floor and walls of a contained room at incremental horizontal and vertical distances of 0.6, 1.2, 2.4, or 4.0 m from the drain. Sedimentation plates were exposed for 10 min. A mechanical sampler was used to also collect air by impaction on the surface of Listeria selective agar to determine the number of cells per liter of air. The experiment was conducted in triplicate rooms for each of four replications. L. innocua was detected on sedimentation plates on the floor as far as 4.0 m from the drain and on walls as high as 2.4 m above the floor and 4 m from the drain. A 2-s spray with a water hose into a contaminated drain can cause airborne spread of Listeria, resulting in the potential for cross-contamination of food contact surfaces, equipment, and exposed product.

Spread of Listeria in fauna of the central region of Russia

The results of studying the spread of Listeria in nature, among environmental objects, wild ungulates, and hydrobionts are given. Both pathogenic and nonpathogenic Listeria species circulate in the territories of the Tver and Kaluga oblasts.

The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria

Introductory ParagraphSeveral pathogenic bacteria, including Listeria monocytogenes, use an F-actin motility process to spread between mammalian cells1. Actin ‘comet tails’ propel Listeria through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which Listeria overcomes cortical tension to generate protrusions is unknown. Here, we identify bacterial and host proteins that directly regulate protrusions. We show that efficient spreading between polarized epithelial cells requires the secreted Listeria virulence protein InlC. We next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP2. InlC promotes protrusion formation by inhibiting Tuba and N-WASP, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells3. We demonstrate that, by inhibiting Tuba and N-WASP, InlC makes taut apical junctions become slack. Experiments with myosin II inhibitors indicate that InlC-mediated perturbation of junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, our results suggest that InlC promotes bacterial dissemination by relieving cortical tension, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions.

Listeria monocytogenes translocates throughout the digestive tract in asymptomatic sheep

Ruminants are fed forage which is often contaminated with Listeria, and frequently shed Listeria monocytogenes with their faeces. The present study was designed to localize the sites of infection in the digestive tract concomitant with Listeria faecal excretion in a sheep model. Ten Listeria-free sheep were inoculated per os with a dose of 10(10) c.f.u. of a pathogenic L. monocytogenes strain. Listeria received by two of the ten animals were radiolabelled with (111)indium oxine. The dissemination of the Listeria was assessed by in vivo imaging, by culture of bacteria in the faeces, organs and digesta samples taken at slaughter on days 1, 2, 6, 10 and 14 post-inoculation, and by measuring gamma radioactivity of samples on day 6. It was shown that Listeria spread through the entire volume of the forestomachs within 4 h, and through the whole gastrointestinal tract (GIT) within 24 h. Faecal shedding of Listeria lasted 10 days. Rumen, duodenum, jejunum, ileum, caecum and colon walls and digesta, mesenteric lymph nodes, liver and spleen were temporarily infected. However, Listeria persisted for at least 14 days in rumen digesta and retropharyngeal lymph nodes, and at a relatively high level (1 x 10(4) c.f.u. g(-1)) in palatine tonsils. These findings suggest that L. monocytogenes can translocate from all parts of the GIT, with the rumen digesta, but not the gallbladder, serving as a reservoir. The results indicate that brief and low-level faecal excretion of L. monocytogenes is concomitant with a transitory asymptomatic infection in sheep.

Reduction of Yersinia enterocolitica and Listeria spp. on pig carcasses by enclosure of the rectum during slaughter

• By sealing off the rectum with a plastic bag immediately after it had been freed, the spread of Y. enterocolitica O:3/biovar 4 to pig carcasses could be considerably reduced. The organism was recovered from only 0.8% of carcasses when the plastic bag technique was employed. Y. enterocolitica O:3/biovar 4 was recovered from 10% of pig carcasses when eviscerating procedures did not include the use of the plastic bag technique. There was thus an obvious risk of the bacteria further contaminating meat cuts and other meat products. The plastic bag technique was effective both in connection with manual excision of the rectum/low throughput (90 per h), and mechanical freeing of the rectum/high slaughter rate (240 per h). • L. monocytogenes was not detected in any of the samples taken from 120 pig carcasses in Norway or from 120 pig carcasses in Sweden. The plastic bag technique was used on half of these pigs. L. innocua was tested for in 120 pigs slaughtered in Sweden. The bacterium was recovered from 33% of the carcasses eviscerated without using a plastic bag, and from 10% of the carcasses in which this technique was employed. The results suggested that there were other, non-faecal, sources of contamination. Other measures in addition to the plastic bag technique are therefore required to limit the spread of Listeria spp. • By incorporating the plastic bag technique into the slaughtering procedures, the meat industry would contribute to preventing the dissemination of Y. enterocolitica and other pathogens which spread via the faces.

Listeria in effluents from the food-processing industry

There is general agreement that listeriosis has a significant impact on Man as well as on animals. Listeria monocytogenes has been isolated from the faeces of healthy human and animal carriers and from various environmental sources. L. monocytogenes is the pathogenic species most responsible for abortion, septicaemia and meningitis in animals and Man. Listeria ivanovii is a primary cause of abortion in animals. Owing to a number of epidemics and single cases caused by food contaminated with L. monocytogenes, listeriosis has received more attention in the past ten years than ever before. Entry of the organism into food-processing plants is primarily caused by animals which excrete Listeria in their faeces. Other sources of entry are raw foods of animal origin and personnel in food establishment. Proliferation of Listeria is promoted by high humidity and nutrient waste in certain food production plants. Removal of Listeria is almost impossible by routine disinfection. Listeria-contaminated sites pose a serious risk of recontamination of food-processing equipment and processed foods. Moreover, such sites represent an inexhaustible source of entry for Listeria in plant effluents. There is no denying that effluents from food-processing plants increase the spread of Listeria in the environment. However, considering the existence of other sources of entry, such as human and animal husbandry wastes, and that circulation and recontamination within the environment itself are also possible, this may not be a particularly important risk.

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.