Salmonella, the host and its microbiota

The intestine is host to a diverse bacterial community whose structure, at the phylum level, is maintained through unknown mechanisms. Acute inflammation triggered by enteric pathogens, such as Salmonella enterica serotype Typhimurium (S. Typhimurium), is accompanied by changes in the bacterial community structure marked by an outgrowth of the pathogen. Recent studies show that S. Typhimurium can harness benefit from the host response to edge out the beneficial bacterial species that dominate in the healthy gut. The elucidation of how S. Typhimurium alters the bacterial community structure during gastroenteritis is beginning to provide insights into mechanisms that dictate the balance between the host and its microbiota.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Pathogenic effects of Salmonella enterica subspecies enterica serovar Typhimurium on sprouting and growth of maize

The study was undertaken to understand effects and survival of S. enterica subspecies enterica serovar Typhimurium (S. Typhimurium), a zoonotic serovar, on maize seed germination and plant growth. All the four strains of S. enterica subspecies enterica serovar Typhimurium significantly reduced germination of maize seeds in sprouting plates as well as in soil. About > or =2.7x10(3) Salmonella cfu ml(-1) of soaking water, while > or =2.7x10(7) Salmonella cfu g(-1) soil were required to significantly inhibit germination of maize. Similar inhibition of germination could be observed using > or = 16 mg of bacteria free Salmonella cell lysate (CL) protein per g of soil or > or =0.5 mg of CL protein per ml of soaking water in sprouting plates. At the constant dose of 3.6x10(7) to 3.8x10(7) Salmonella cfu or 5 mg cell lysate protein ml(-1) of soaking water, four strains of Salmonella significantly reduced germination, however difference between strains was insignificant. After germination too, maize growth was affected both by Salmonella organism and CL with little strain-to-strain variation. All Salmonella persisted in growing plants from 15 to 35 days of plant age and up to 190 days in soil. Maize plants once grown for a week in sterile soil were resistant to invasion of S. enterica subspecies enterica serovar Typhimurium in their leaves even in doses as high as 7.6x10(9) cfu g(-1) of soil. Salmonella persisted better and longer in plants grown from contaminated seed sown in loam soil, but rarely in plants grew in sandy soil. All maize plants had Salmonella in their stumps even after 35 days of sowing irrespective of kind of soil, primary source of infection (soil or seed) and type of S. enterica subspecies enterica serovar Typhimurium strain. The study revealed that Salmonella is not only zoonotic but a phytopathogen also.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Linkage of avian and reproductive tract tropism with sequence divergence adjacent to the 5S ribosomal subunit rrfH of Salmonella enterica

The 183 bp between the end of the 23S rrlH rRNA gene and the start of the 5S rrfH rRNA gene (ISR-1) and the 197 bp between the end of the rrfH rRNA gene and the start of the transfer RNA aspU (ISR-2) of Salmonella enterica ssp. enterica serotypes Enteritidis, Typhimurium, Pullorum, Heidelberg, Gallinarum, Typhi and Choleraesuis were compared. ISR-1s of D1 serotypes (Pullorum, Gallinarum and Enteritidis), B serotypes (Typhimurium and Heidelberg) and the C2 serotype Newport and the enteric fever pathogens serotype A Paratyphi and serotype D1 Typhi formed three clades, respectively. ISR-2 further differentiated the avian-adapted serotype Gallinarum from avian-adapted Pullorum and Salmonella bongori from S. enterica. The results suggest that serotypes Heidelberg and Choleraesuis share some evolutionary trends with egg-contaminating serotypes. In addition, ISR-1 and ISR-2 sequences that confirm serotype appear to be linked to clinically relevant host associations of the Salmonellae.     

Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.     

Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.     

Hemophagocytic macrophages harbor Salmonella enterica during persistent infection

Salmonella enterica subspecies can establish persistent, systemic infections in mammals, including human typhoid fever. Persistent S. enterica disease is characterized by an initial acute infection that develops into an asymptomatic chronic infection. During both the acute and persistent stages, the bacteria generally reside within professional phagocytes, usually macrophages. It is unclear how salmonellae can survive within macrophages, cells that evolved, in part, to destroy pathogens. Evidence is presented that during the establishment of persistent murine infection, macrophages that contain S. enterica serotype Typhimurium are hemophagocytic. Hemophagocytic macrophages are characterized by the ingestion of non-apoptotic cells of the hematopoietic lineage and are a clinical marker of typhoid fever as well as certain other infectious and genetic diseases. Cell culture assays were developed to evaluate bacterial survival in hemophagocytic macrophages. S. Typhimurium preferentially replicated in macrophages that pre-phagocytosed viable cells, but the bacteria were killed in macrophages that pre-phagocytosed beads or dead cells. These data suggest that during persistent infection hemophagocytic macrophages may provide S. Typhimurium with a survival niche.     

Untangling metabolic and communication networks: interactions of enterics with phytobacteria and their implications in produce safety

Recent outbreaks of vegetable-borne gastrointestinal illnesses across the globe demonstrate that human enteric pathogens can contaminate produce at any stage of production. Interactions of enterics with native plant-associated microbiota influence the microbiological safety of produce by affecting the attachment, persistence and proliferation of human pathogens on plants. Supermarket surveys have revealed that bacteria, but not fungi or mechanical damage, promote the growth of Salmonella enterica on produce. Field and laboratory studies have indicated that some plant pathogenic bacteria and fungi facilitate the entry and internalization of human pathogens in plants. Conversely, some phytobacteria, including those involved in biocontrol of plant diseases, significantly inhibit attachment and plant colonization by non-typhoidal Salmonella and enterovirulent Escherichia coli by producing antibiotics or competing for nutrients in the phyllosphere. In this review, we attempt to elucidate the mechanisms of interactions between human enteric pathogens and plant-associated microbiota, and describe how these interactions affect produce safety.     

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.     

Flagellin in combination with curli fimbriae elicits an immune response in the gastrointestinal epithelial cell line HT-29

Flagellin is the major cytokine-releasing factor when Salmonella enterica serovar Typhimurium (S. Typhimurium) infects intestinal epithelial cells. In this work it is shown that curli, an adhesive proteinaceous surface component of Enterobacteriaceae involved in biofilm formation of S. Typhimurium and Escherichia coli strains can bind flagellin and thus elicit an immune response by the intestinal epithelial cell line HT-29.     

Salmonella biofilm formation on Aspergillus niger involves cellulose--chitin interactions

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose-chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens.     

Regulation of enteric endophytic bacterial colonization by plant defenses

Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethylene-insensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. This ethylene-mediated inhibition of endophytic colonization was reversed by addition of the ethylene action inhibitor, 1-methylcyclopropene. Colonization of Medicago spp. by S. typhimurium also was affected by exogenous ethylene. Mutants lacking flagella or a component of the type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1) colonize the interior of Medicago spp. in higher numbers than the wild type. Arabidopsis defense response-related genotypes indicated that only salicylic acid (SA)-independent defense responses contribute to restricting colonization by Kp342. In contrast, colonization by S. typhimurium is affected by both SA-dependent and -independent responses. S. typhimurium mutants further delineated these responses, suggesting that both flagella and TTSS-SPI1 effectors can be recognized. Flagella act primarily through SA-independent responses (compromising SA accumulation still affected colonization in the absence of flagella). Removal of a TTSS-SPI1 effector resulted in hypercolonization regardless of whether the genotype was affected in either SA-dependent or SA-independent responses. Consistent with these results, S. typhimurium activates the promoter of PR1, a SA-dependent pathogenesis-related gene, while S. typhimurium mutants lacking the TTSS-SPI1 failed to activate this promoter. These observations suggest approaches to reduce contamination of raw produce by human enteric pathogens and to increase the number of growth-promoting bacteria in plants.     

The spectrin cytoskeleton is crucial for adherent and invasive bacterial pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress.     

Effect of the Salmonella pathogenicity island 2 type III secretion system on Salmonella survival in activated chicken macrophage-like HD11 cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.     

Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.     

The two-component sensor kinase KdpD is required for Salmonella typhimurium colonization of Caenorhabditis elegans and survival in macrophages

The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.     

Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.