Salmonella enterica Serovar Enteritidis Antimicrobial Peptide Resistance Genes Aid in Defense against Chicken Innate Immunity,Fecal Shedding,and Egg Deposition

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major etiologic agent of nontyphoid salmonellosis in the United States. S. Enteritidis persistently and silently colonizes the intestinal and reproductive tract of laying hens, resulting in contaminated poultry products. The consumption of contaminated poultry products has been identified as a significant risk factor for human salmonellosis. To understand the mechanisms S. Enteritidis utilizes to colonize and persist in laying hens, we used selective capture of transcribed sequences to identify genes overexpressed in the HD11 chicken macrophage cell line and in primary chicken oviduct epithelial cells. From the 15 genes found to be overexpressed in both cell types, we characterized the antimicrobial peptide resistance (AMPR) genes, virK and ybjX, in vitro and in vivo. In vitro, AMPR genes were required for natural morphology, motility, secretion, defense against detergents such as EDTA and bile salts, and resistance to antimicrobial peptides polymyxin B and avian β-defensins. From this, we inferred the AMPR genes play a role in outer membrane stability and/or modulation. In the intestinal tract, AMPR genes were involved in early intestinal colonization and fecal shedding. In the reproductive tract, virK was required in early colonization whereas a deletion of ybjX caused prolonged ovary colonization and egg deposition. Data from the present study indicate that AMPR genes are differentially utilized in various host environments, which may ultimately assist S. Enteritidis in persistent and silent colonization of chickens.     

Prevalence of Salmonella enterica in Poultry and Eggs in Uruguay during an Epidemic Due to Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis (S. Enteritidis) is frequently associated with food-borne disease worldwide. Poultry-derived products are a major source. An epidemic of human infection with S. Enteritidis occurred in Uruguay, and to evaluate the extent of poultry contamination, we conducted a nationwide survey over 2 years that included the analysis of sera from 5,751 birds and 12,400 eggs. Serological evidence of infection with Salmonella group O:9 was found in 24.4% of the birds. All positive sera were retested with a gm flagellum-based enzyme-linked immunosorbent assay, and based on these results, the national prevalence of S. Enteritidis infection was estimated to be 6.3%. Salmonellae were recovered from 58 of 620 pools made up of 20 eggs each, demonstrating a prevalence of at least 1 in every 214 eggs. Surprisingly, the majority of the isolates were not S. Enteritidis. Thirty-nine isolates were typed as S. Derby, 9 as S. Gallinarum, 8 as S. Enteritidis, and 2 as S. Panama. Despite the highest prevalence in eggs, S. Derby was not isolated from humans in the period of analysis, suggesting a low capacity to infect humans. Microarray-based comparative genomic hybridization analysis of S. Derby and S. Enteritidis revealed more than 350 genetic differences. S. Derby lacked pathogenicity islands 13 and 14, the fimbrial lpf operon, and other regions encoding metabolic functions. Several of these regions are present not only in serovar Enteritidis but also in all sequenced strains of S. Typhimurium, suggesting that these regions might be related to the capacity of Salmonella to cause food-borne disease.Salmonella enterica is a major cause of food-borne disease worldwide (14, 18, 46). Poultry-derived products, particularly chicken eggs, are considered a major source of human infection with Salmonella (2, 20, 38). Chickens can be infected with many different serovars of Salmonella. Of these, S. enterica serovars Pullorum and Gallinarum (S. Pullorum and S. Gallinarum, respectively) are host specific and represent a major concern for the poultry industry but have no impact on public health. Other S. enterica serovars frequently isolated from chickens, such as Typhimurium, Enteritidis, and Heidelberg, can infect a wider range of hosts and frequently reach the human food chain, causing food-borne disease.A peculiar epidemiological feature of human salmonellosis is that epidemics are commonly associated with a particular prevalent serovar of S. enterica that shows temporal and geographical variation. Until the 1980s, S. enterica serovar Typhimurium (S. Typhimurium) was the serovar most commonly isolated from humans worldwide, but by the late 1980s, S. enterica serovar Enteritidis (S. Enteritidis) emerged as the most common cause of salmonellosis in Europe, and during the 1990s, it became the most prevalent serovar in many countries worldwide (9, 22, 33, 40, 43). The reasons for this worldwide serovar shift are still not understood, and several hypotheses have been proposed, including the existence of a rodent reservoir for S. Enteritidis or the epidemiological change induced by vaccination of poultry against the closely related bacterium S. Gallinarum (47).In Uruguay, S. Typhimurium was the most frequently isolated serovar until 1994, and S. Enteritidis was only sporadically isolated (3, 24, 37). In 1995, a first outbreak of S. Enteritidis occurred, starting an epidemic that lasted almost 10 years. This outbreak was traced back to sandwiches prepared with contaminated mayonnaise that were distributed nationwide by a local catering service. According to data provided by the national public health authorities, the outbreak affected an estimated 600 individuals countrywide. From then on, several other outbreaks of various sizes occurred and S. Enteritidis was identified as the cause in 89% of Salmonella food poisoning episodes. In most of these cases (80%, according to official records), eggs or chicken meat was identified as the source of infection. From 1997 to 2004, S. Enteritidis was the most frequently identified serovar in Uruguay, accounting for more than 50% of the strains received each year at the National Salmonella Center and for more than 85% of the strains isolated from humans (3). After 2005, there was a dramatic reduction in the number of S. Enteritidis outbreaks, and this year was considered the end of the epidemic. Over the last 3 years, S. Typhimurium has become the serovar most frequently associated with isolated cases of food poisoning, and S. Derby and S. Panama have been sporadically isolated. Nevertheless, S. Enteritidis is still the serovar most frequently associated with outbreaks in the country.S. Enteritidis frequently colonizes the alimentary tracts of chickens without causing disease. However, it can produce a systemic infection in young chicks that may further lead to the infection of egg contents (13, 51). With the aim of knowing the prevalence of S. Enteritidis infection in poultry, we designed and conducted a countrywide serological and microbiological survey of chicken flocks and commercially available eggs from 2000 to 2002, and the results are presented here. An unexpected result of the survey was a higher prevalence of S. Derby than S. Enteritidis in eggs, particularly because while the latter was identified as the etiological agent of the epidemic there were no reports of human infections with S. Derby in the same period of time. This suggested a low capacity of S. Derby isolates to infect humans; thus, we performed a genomic comparison of the two serovars to search for genetic differences that could be the basis of such marked differences in epidemiological behavior. We found that S. Derby lacks several genomic regions related to virulence, suggesting that these regions could be involved in the capacity of Salmonella to cause food-borne disease.     

Genetic Diversity and Evolution of Salmonella enterica Serovar Enteritidis Strains with Different Phage Types

Phage typing has been used for the epidemiological surveillance of Salmonella enterica serovar Enteritidis for over 2 decades. However, knowledge of the genetic and evolutionary relationships between phage types is very limited, making differences difficult to interpret. Here, single nucleotide polymorphisms (SNPs) identified from whole-genome comparisons were used to determine the relationships between some S. Enteritidis phage types (PTs) commonly associated with food-borne outbreaks in the United States. Emphasis was placed on the predominant phage types PT8, PT13a, and PT13 in North America. With >89,400 bp surveyed across 98 S. Enteritidis isolates representing 14 distinct phage types, 55 informative SNPs were discovered within 23 chromosomally anchored loci. To maximize the discriminatory and evolutionary partitioning of these highly homogeneous strains, sequences comprising informative SNPs were concatenated into a single combined data matrix and subjected to phylogenetic analysis. The resultant phylogeny allocated most S. Enteritidis isolates into two distinct clades (clades I and II) and four subclades. Synapomorphic (shared and derived) sets of SNPs capable of distinguishing individual clades/subclades were identified. However, individual phage types appeared to be evolutionarily disjunct when mapped to this phylogeny, suggesting that phage typing may not be valid for making phylogenetic inferences. Furthermore, the set of SNPs identified here represents useful genetic markers for strain differentiation of more clonal S. Enteritidis strains and provides core genotypic markers for future development of a SNP typing scheme with S. Enteritidis.     

The Global Regulator ArcA Controls Resistance to Reactive Nitrogen and Oxygen Intermediates in Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis is a major cause of food-borne diseases associated with consumption of shell eggs. Clinical isolates of S. enterica serovar Enteritidis exhibit a wide spectrum of virulence in mice. A highly virulent isolate (SE2472) was previously shown to be more resistant in vitro than other clinical isolates to acidified sodium nitrite (ASN), a generator of reactive nitrogen and oxygen intermediates (RNI/ROI). SE2472 is also more resistant to S-nitrosoglutathione (GSNO) and hydrogen peroxide (H(2)O(2)) than an ASN-susceptible isolate of S. enterica serovar Enteritidis (SE8743). To investigate the molecular basis for the RNI/ROI resistance of S. enterica serovar Enteritidis, we transformed a genomic DNA library of SE2472 into SE8743. A plasmid clone conferred upon SE8743 enhanced resistance to ASN, GSNO, and H(2)O(2). The DNA insert in the clone encoded ArcA, a global regulator. An arcA mutant of SE2472 was constructed and was found to be more susceptible to GSNO and hydrogen peroxide but not more susceptible to ASN than wild-type SE2472. The susceptibility of the arcA mutant to GSNO and H(2)O(2) was complemented by a plasmid harboring arcA. The coding sequence of the arcA gene in SE2472 and the coding sequence of the arcA gene in SE8743 were identical, suggesting that the difference in resistance to RNI/ROI maybe due to the activity of genes regulated by ArcA. No significant difference in virulence between the wild type and the arcA mutant of SE2472 was observed in mice. These observations show that arcA is essential for resistance of S. enterica serovar Enteritidis to nitrosative and oxidative stress. However, additional genetic loci may contribute to the resistance to RNI/ROI and unusually high virulence for mice of SE2472.     

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.     

In Vivo Genetic Analysis Indicates That PhoP-PhoQ and the Salmonella Pathogenicity Island 2 Type III Secretion System Contribute Independently to Salmonella enterica Serovar Typhimurium Virulence

Many virulence factors are required for Salmonella enterica serovar Typhimurium to replicate intracellularly and proliferate systemically within mice. In this work, we have carried out genetic analyses in vivo to determine the functional relationship between two major virulence factors necessary for systemic infection by S. enterica serovar Typhimurium: the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) and the PhoP-PhoQ two-component regulatory system. Although previous work suggested that PhoP-PhoQ regulates SPI-2 TTSS gene expression in vitro, in vivo competitive analysis of mutant strains indicates that these systems contribute independently to S. typhimurium virulence. Our results also suggest that mutation of phoP may compensate partially for defects in the SPI-2 TTSS by deregulating SPI-1 TTSS expression. These results provide an explanation for previous reports showing an apparent functional overlap between these two systems in vitro.     

Nitric Oxide and Apoptosis Induced in Peyer's Patches by Attenuated Strains of Salmonella enterica Serovar Enteritidis

Nitric oxide (NO) is a toxic molecule of the immune system which contributes to the control of microbial pathogens. Additional functions of NO in innate and adaptive immunity have recently been described; these functions include the modulation of the cytokine response of lymphocytes and the regulation of immune cell apoptosis. In addition to direct microbicidal actions, NO has immunoregulatory effects relevant to the control of infections. In turn, infected macrophages and macrophage-regulating lymphocytes may undergo apoptosis during infection by Salmonella spp. In this work we investigated the ability of attenuated strains of Salmonella enterica serovar Enteritidis with different protective capacities to induce intestinal inducible nitric oxide synthase (iNOS) and apoptosis in Peyer's patches (PP) in mice. Results showed that the intestinal iNOS activity correlated with increased apoptosis in PP. Furthermore, the ability to induce intestinal NO production and apoptosis within the first few hours after immunization seemed to correlate with the protective capacity of mutant E/1/3 of S. enterica serovar Enteritidis. It was found that nonprotective mutant C/2/2, which was unable to induce intestinal NO production, also failed to induce apoptosis in PP. Moreover, aminoguanidine treatment at the time of immunization resulted in inhibition of the NO production and apoptosis induced by protective mutant E/1/3 and completely abolished protection against challenge. These results suggest that the induction of iNOS in the intestinal mucosa by attenuated mutant E/1/3 of S. enterica serovar Enteritidis at the time of immunization is necessary to generate a protective immune response.     

Comparison of Inflammation, Organ Damage, and Oxidant Stress Induced by Salmonella enterica Serovar Muenchen Flagellin and Serovar Enteritidis Lipopolysaccharide

Gram-negative sepsis is related to the activation of interconnected inflammatory cascades in response to bacteria and their products. Recent work showed that flagellin, the monomeric subunit of bacterial flagella, triggers innate immune responses mediated by Toll-like receptor 5. Here, we compared the effects of Salmonella enterica serovar Enteritidis lipopolysaccharide (LPS) and recombinant Salmonella enterica serovar Muenchen flagellin administered intravenously (100 microg) to mice. Flagellin and LPS both elicited a prototypical systemic inflammatory response, with increased levels of tumor necrosis factor alpha, gamma interferon, interleukin 6 and 10, and nitrate in plasma. Flagellin induced a widespread oxidative stress, evidenced by an increase in malondialdehyde and a decrease in reduced glutathione in most organs, as well as liver (increased plasma aminotransferases), but not renal, injury. Alternatively, LPS resulted in a less severe oxidative stress and triggered renal, but not liver, damage. Sequestration of polymorphonuclear neutrophils (increased myeloperoxidase activity) in the lungs was observed with both toxins, while only LPS recruited neutrophils in the gut. In additional experiments, the simultaneous administration of small doses of LPS and flagellin (10 microg) induced a synergistic enhancement of the production of proinflammatory cytokines. Our data support a novel concept implicating flagellin as a mediator of systemic inflammation, oxidant stress, and organ damage induced by gram-negative bacteria.     

Refined Live Attenuated Salmonella enterica Serovar Typhimurium and Enteritidis Vaccines Mediate Homologous and Heterologous Serogroup Protection in Mice

Invasive nontyphoidal Salmonella (NTS) infections constitute a major health problem among infants and toddlers in sub-Saharan Africa; these infections also occur in infants and the elderly in developed countries. We genetically engineered a Salmonella enterica serovar Typhimurium strain of multilocus sequence type 313, the predominant genotype circulating in sub-Saharan Africa. We evaluated the capacities of S. Typhimurium and Salmonella enterica serovar Enteritidis ΔguaBA ΔclpX live oral vaccines to protect mice against a highly lethal challenge dose of the homologous serovar and determined protection against other group B and D serovars circulating in sub-Saharan Africa. The vaccines S. Typhimurium CVD 1931 and S. Enteritidis CVD 1944 were immunogenic and protected BALB/c mice against 10,000 50% lethal doses (LD₅₀) of S. Typhimurium or S. Enteritidis, respectively. S. Typhimurium CVD 1931 protected mice against the group B serovar Salmonella enterica serovar Stanleyville (91% vaccine efficacy), and S. Enteritidis CVD 1944 protected mice against the group D serovar Salmonella enterica serovar Dublin (85% vaccine efficacy). High rates of survival were observed when mice were infected 12 weeks postimmunization, indicating that the vaccines elicited long-lived protective immunity. Whereas CVD 1931 did not protect against S. Enteritidis R11, CVD 1944 did mediate protection against S. Typhimurium D65 (81% efficacy). These findings suggest that a bivalent (S. Typhimurium and S. Enteritidis) vaccine would provide broad protection against the majority of invasive NTS infections in sub-Saharan Africa.     

Genetic Analysis of Non-Hydrogen Sulfide-Producing Salmonella enterica Serovar Typhimurium and S. enterica Serovar Infantis Isolates in Japan

Whole-genome sequencing of non-H₂S-producing Salmonella enterica serovar Typhimurium and S. enterica serovar Infantis isolates from poultry meat revealed a nonsense mutation in the phsA thiosulfate reductase gene and carriage of a CMY-2 β-lactamase. The lack of production of H₂S might lead to the incorrect identification of S. enterica isolates carrying antimicrobial resistance genes.     

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH(2)-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells.     

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways.     

First Report of Human Infection with Salmonella enterica Serovar Apapa Resulting from Exposure to a Pet Lizard

We present the first documented human case of Salmonella enterica serovar Apapa infection, isolated concurrently from a hospital inpatient and a pet lizard. The isolates were identical by biochemical profiling and pulsed-field gel electrophoresis. This rare serotype is known to be associated with reptiles. The current practice for avoiding reptile-associated infections is reviewed.     

Role of Nod1 in Mucosal Dendritic Cells during Salmonella Pathogenicity Island 1-Independent Salmonella enterica Serovar Typhimurium Infection

Recent advances in immunology have highlighted the critical function of pattern-recognition molecules (PRMs) in generating the innate immune response to effectively target pathogens. Nod1 and Nod2 are intracellular PRMs that detect peptidoglycan motifs from the cell walls of bacteria once they gain access to the cytosol. Salmonella enterica serovar Typhimurium is an enteric intracellular pathogen that causes a severe disease in the mouse model. This pathogen resides within vacuoles inside the cell, but the question of whether cytosolic PRMs such as Nod1 and Nod2 could have an impact on the course of S. Typhimurium infection in vivo has not been addressed. Here, we show that deficiency in the PRM Nod1, but not Nod2, resulted in increased susceptibility toward a mutant strain of S. Typhimurium that targets directly lamina propria dendritic cells (DCs) for its entry into the host. Using this bacterium and bone marrow chimeras, we uncovered a surprising role for Nod1 in myeloid cells controlling bacterial infection at the level of the intestinal lamina propria. Indeed, DCs deficient for Nod1 exhibited impaired clearance of the bacteria, both in vitro and in vivo, leading to increased organ colonization and decreased host survival after oral infection. Taken together, these findings demonstrate a key role for Nod1 in the host response to an enteric bacterial pathogen through the modulation of intestinal lamina propria DCs.Recognition of microbes is a critical step in the initiation of the host immune response against infection. Indeed, detection of microbe-associated molecular patterns by germ line-encoded receptors such as Toll-like receptors (18) and Nod-like receptors (NLRs) (8) is an early event that leads to inflammatory responses through the production of cytokines and chemokines. Nod1 and Nod2 are cytosolic proteins of the NLR family that detect distinct substructures from bacterial peptidoglycan (8). Whereas Nod2 detects muramyl dipeptide (12, 16), a motif common to gram-negative and gram-positive bacteria, Nod1 senses meso-diaminopimelic acid-containing peptidoglycan (3, 11), which is more commonly found in gram-negative bacteria. In macrophages and dendritic cells (DC), triggering of Nod1 and Nod2 induces proinflammatory cytokines and costimulatory molecules (21). In addition, synergistic effects of Nod ligands with Toll-like receptor ligands in myeloid cells have been reported (9, 29). Nod1 has been shown to regulate the colonization of mice by Helicobacter pylori (31), and Nod2 affects the pathogenicity of Listeria monocytogenes (19) and Mycobacterium tuberculosis (6) in mice models. However, no studies have been conducted on the impact of Nod1 and Nod2 on the in vivo infection process of the specific enteric pathogen, Salmonella enterica serovar Typhimurium.Salmonella enterica is a gram-negative bacterium of the Enterobacteriaceae family. S. enterica serovar Typhimurium is a mouse pathogen that provokes a typhoid-like syndrome in orally infected mice, with colonization of the deeper organs, including the liver and spleen (5). S. Typhimurium is capable of entering intestinal epithelial cells using a unique mechanism involving a type 3 secretion system, Salmonella pathogenicity island 1 (Spi1) (10), and resides in a vacuole within infected cells via a mechanism dependent on a second type 3 secretion system, Spi2 (27). Hence, the bacteria are able to avoid killing and spread throughout the infected host by invading immune cells. The intracellular lifestyle of Salmonella is in line with a possible implication of Nod proteins during the course of the infection. However, to date, no study has been conducted in vivo to determine the role of Nod1 and Nod2 after oral infection with S. Typhimurium.Spi1 is critical for the invasiveness of the bacteria in epithelial cells and is thought to be responsible for the main route of entry of the bacteria through Peyer''s patches (13, 30). Strikingly, bacteria deficient for Spi2 are completely avirulent whereas Spi1 mutants are still capable of inducing the disease (26). Recently, myeloid cells from the intestinal lamina propria have been shown to sample the luminal contents of the gut, including intact bacteria. This mechanism is crucial for gut homeostasis but provides a portal of entry for S. Typhimurium and explains the persistent virulence of Spi1-deficient bacteria (4, 24, 30).In the present study we show that Nod1 deficiency leads to increased susceptibility to Spi1 deficient-S. Typhimurium but not the wild-type (WT) strain, suggesting a critical role for Nod1 in myeloid cells from the intestinal lamina propria for defense against S. Typhimurium infection in vivo. Accordingly, using bone marrow-chimeric mice, we have been able to locate the defect in in vivo hematopoietic cells. Indeed, Nod1 deficient animals show increased S. Typhimurium in the lamina propria DC subpopulation and an impaired cellular response after infection with Spi1-deficient bacteria. Additionally, we observed an impaired response of Nod1-deficient DCs toward the bacteria. Taken together, our findings uncover a surprising role of Nod1 in lamina propria DCs in the control of S. Typhimurium infection in vivo.     

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis.