Production of Autoinducer 2 in Salmonella enterica Serovar Thompson Contributes to Its Fitness in Chickens but Not on Cilantro Leaf Surfaces

Food-borne illness caused by Salmonella enterica has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of S. enterica serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of S. enterica serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of luxS. The population size of the S. enterica serovar Thompson parental strain was significantly higher than that of its LuxS⁻ mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS⁻ strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of S. enterica serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS⁻ mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS⁻ mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to S. enterica in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of S. enterica in these environments.     

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

An Altered Immune Response,but Not Individual Cationic Antimicrobial Peptides,Is Associated with the Oral Attenuation of Ara4N-Deficient Salmonella enterica Serovar Typhimurium in Mice

Salmonella enterica serovar Typhimurium (S. Typhimurium) uses two-component regulatory systems (TCRS) to respond to stimuli in the local microenvironment. Upon infection, the Salmonella TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals in the intestinal lumen and within host cells. TCRS-mediated gene expression results in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide resistance. The PmrA-regulated pmrHFIJKLM operon mediates 4-amino-4-deoxy-L-arabinose (Ara4N) production and attachment to the lipid A of LPS. A ΔpmrF S. Typhimurium strain cannot produce Ara4N, exhibits increased sensitivity to cationic antimicrobial peptide (CAMP)-mediated killing, and attenuated virulence in mice upon oral infection. CAMPs are predicted to play a role in elimination of Salmonella, and may activate PhoPQ and PmrAB in vivo, which could increase bacterial resistance to host defenses. Competition experiments between wild type (WT) and ΔpmrF mutant strains of S. Typhimurium indicated that selection against this mutant first occurs within the intestinal lumen early during infection. However, CRAMP and active cryptdins alone are not responsible for elimination of Ara4N-deficient bacteria in vivo. Investigation into the early immune response to ΔpmrF showed that it differed slightly from the early immune response to WT S. Typhimurium. Further investigation into the early immune response to infection of Peyer’s patches suggests a role for IL-13 in the attenution of the ΔpmrF mutant strain. Thus, prominent CAMPs present in the mouse intestine are not responsible for the selection against the ΔpmrF strain in this location, but limited alterations in innate immune induction were observed that affect bacterial survival and virulence.     

CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg²⁺ transporter and at least three other inducible open reading frames belonged to the Mg²⁺ regulon, repression of the Mg stimulon by Mg²⁺ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni²⁺, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni²⁺-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

rpoS-Regulated Core Genes Involved in the Competitive Fitness of Salmonella enterica Serovar Kentucky in the Intestines of Chickens

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky''s prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella''s adaptation to its animal host and especially for S. Kentucky''s emergence as the dominant serovar in poultry.     

Vaccination of Chickens with SPI1-lon and SPI1-lon-fliC Mutant of Salmonella enterica Serovar Enteritidis

The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.     

Low-Shear Modeled Microgravity Alters the Salmonella enterica Serovar Typhimurium Stress Response in an RpoS-Independent Manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.     

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.     

Induction and Resuscitation of Viable but Nonculturable Salmonella enterica Serovar Typhimurium DT104†

Salmonella enterica serovar Typhimurium DT104 11601was tested for its ability to maintain viability in minimal, chemically defined solutions. Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms (~235 days) at 5°C. Organisms maintained at a higher temperature (21°C) showed continuous, equivalent CFU per milliliter (~10⁶) up to 400 days after inoculation. Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity. Temperature upshift (56°C ± 0.5, 15 s) of the nonculturable organisms (5°C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation. Interestingly, S. enterica serovar Typhimurium DT104 from the microcosms at either 5°C (1 to 200 days) or 21°C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA. Furthermore, cells from 21°C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells. It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans. The fact that S. enterica serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that S. enterica serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions.     

Interaction of Saccharomyces boulardii with Salmonella enterica Serovar Typhimurium Protects Mice and Modifies T84 Cell Response to the Infection

Background

Salmonella pathogenesis engages host cells in two-way biochemical interactions: phagocytosis of bacteria by recruitment of cellular small GTP-binding proteins induced by the bacteria, and by triggering a pro-inflammatory response through activation of MAPKs and nuclear translocation of NF-κB. Worldwide interest in the use of functional foods containing probiotic bacteria for health promotion and disease prevention has increased significantly. Saccharomyces boulardii is a non-pathogenic yeast used as a probiotic in infectious diarrhea.

Methodology/Principal Findings

In this study, we reported that S. boulardii (Sb) protected mice from Salmonella enterica serovar Typhimurium (ST)-induced death and prevented bacterial translocation to the liver. At a molecular level, using T84 human colorectal cancer cells, we demonstrate that incubation with Sb before infection totally abolished Salmonella invasion. This correlates with a decrease of activation of Rac1. Sb preserved T84 barrier function and decreased ST-induced IL-8 synthesis. This anti-inflammatory effect was correlated with an inhibitory effect of Sb on ST-induced activation of the MAPKs ERK1/2, p38 and JNK as well as on activation of NF-κB. Electron and confocal microscopy experiments showed an adhesion of bacteria to yeast cells, which could represent one of the mechanisms by which Sb exerts its protective effects.

Conclusions

Sb shows modulating effects on permeability, inflammation, and signal transduction pathway in T84 cells infected by ST and an in vivo protective effect against ST infection. The present results also demonstrate that Sb modifies invasive properties of Salmonella.     

Effect of Environmental Temperatures on Proteome Composition of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium (STM) is a major cause of gastroenteritis and transmitted by consumption of contaminated food. STM is associated to food originating from animals (pork, chicken, eggs) or plants (vegetables, fruits, nuts, and herbs). Infection of warm-blooded mammalian hosts by STM and the underlying complex regulatory network of virulence gene expression depend on various environmental conditions encountered in hosts. However, less is known about the proteome and possible regulatory networks for gene expression of STM outside the preferred host. Nutritional limitations and changes in temperature are the most obvious stresses outside the native host. Thus, we analyzed the proteome profile of STM grown in rich medium (LB medium) or minimal medium (PCN medium) at temperatures ranging from 8 °C to 37 °C. LB medium mimics the nutritional rich environment inside the host, whereas minimal PCN medium represents nutritional limitations outside the host, found during growth of fresh produce (field conditions). Further, the range of temperatures analyzed reflects conditions within natural hosts (37 °C), room temperature (20 °C), during growth under agricultural conditions (16 °C and 12 °C), and during food storage (8 °C). Implications of altered nutrient availability and growth temperature on STM proteomes were analyzed by HPLC/MS-MS and label-free quantification. Our study provides first insights into the complex adaptation of STM to various environmental temperatures, which allows STM not only to infect mammalian hosts but also to enter new infection routes that have been poorly studied so far. With the present dataset, global virulence factors, their impact on infection routes, and potential anti-infective strategies can now be investigated in detail. Especially, we were able to demonstrate functional flagella at 12 °C growth temperature for STM with an altered motility behavior.     

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.