Genomic organization and role of SPI-13 in nutritional fitness of Salmonella

Salmonella pathogenicity island 13 (SPI-13) contributes to the virulence of Salmonella. The majority of the SPI-13 genes encode proteins putatively involved in bacterial metabolism, however, their functions largely remain uncharacterized. It is currently unknown if SPI-13 contributes to metabolic fitness of Salmonella and, if so, what are the metabolic substrates for the protein encoded by genes within SPI-13. We employed Phenotype Microarray (Biolog, USA) to compare the metabolic properties of SPI-13 deficient mutant (ΔSPI-13) and the WT parent strain of non-typhoidal Salmonella enterica sub sp. enterica serovar Enteritidis (S. Enteritidis). The results of Phenotype Microarray revealed that SPI-13 is required for efficient utilization of two micronutrients, namely, d-glucuronic acid (DGA) and tyramine (TYR), as sole sources of carbon and/or nitrogen. By systematic deletion of the individual gene(s), we identified specific genes within SPI-13 that are required for efficient utilization of DGA (SEN2977-80) and TYR (SEN2967 and SEN2971-72) as sole nutrient sources. The results show that SPI-13 mediated DGA and TYR metabolic pathways afford nutritional fitness to S. Enteritidis. Comparative genomics analysis of the SPI-13 locus from 247 Salmonella strains belonging to 57 different serovars revealed that SPI-13 genes specifically involved in the metabolism of DGA and TYR are highly conserved in Salmonella enterica. Because DGA and TYR are naturally present as metabolic byproducts in the gastrointestinal tract and other host tissues, we propose a metabolic model that shows that the role of SPI-13 mediated DGA and TYR metabolism in the nutritional fitness of Salmonella is likely linked to nutritional virulence of this pathogen.     

One-Step Identification of Five Prominent Chicken Salmonella Serovars and Biotypes

Based on bacterial genomic data, we developed a one-step multiplex PCR assay to identify Salmonella and simultaneously differentiate the two invasive avian-adapted S. enterica serovar Gallinarum biotypes Gallinarum and Pullorum, and the most frequent, specific, and asymptomatic colonizers of chickens, serovars Enteritidis, Heidelberg, and Kentucky.     

Multilocus variable-number tandem-repeat analysis for subtyping Salmonella enterica serovar Gallinarum

Salmonella enterica serovar Gallinarum causes a severe systemic disease, fowl typhoid, primarily in chickens and turkeys, and it remains a disease of worldwide significance. Multilocus variable-number tandem-repeat analysis (MLVA) has proved to be very useful for subtyping other Salmonella serovars. We describe the development of a simple MLVA assay for S. enterica serovar Gallinarum that is comparable with pulsed-field gel electrophoresis (PFGE) in resolution. The genome sequence of S. enterica serovar Gallinarum strain 287/91 was analysed for potential variable-number tandem repeats (VNTRs) and then polymerase chain reaction assays were developed to assess the variability of the loci. Four VNTR markers were selected and used in a multiplex fragment analysis assay. The stability of the VNTR markers was assessed by conducting in vitro passage experiments with two strains (95 clones per strain) over a 30-day period. A MLVA of 68 strains of S. enterica serovar Gallinarum based on the four VNTR loci distinguished 26 allelic profiles. The MLVA assay showed a Simpson's diversity index of 0.918, whereas PFGE analysis produced 23 patterns and had a diversity index of 0.874. Most importantly, the MLVA further discriminated strains having the same PFGE pattern. The MLVA assay is a highly discriminatory genotyping method for S. enterica serovar Gallinarum. Therefore, MLVA can be a useful addition to routine PFGE analysis for molecular epidemiological investigation of fowl typhoid.     

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.     

Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nal^r strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.     

Presence of F-like OriT base-pair sequence on the virulence plasmids of Salmonella serovars Gallinarum,Enteritidis, and Typhimurium,but absent in those of Choleraesuis and Dublin

The 90 kb virulence plasmids of Salmonella biosers Gallinarum and Pullorum (expressed as serovar Gallinarum) are non-conjugative. They were, however, found to be readily mobilized by IncFI and IncFIV plasmids, but not by conjugative IncA, IncB = C, IncH, IncL and IncM R plasmids. No virulence plasmids of serovars Choleraesuis, Dublin, Enteritidis, and Typhimurium were mobilized by any of these conjugative plasmids. The 90 kb virulence plasmid of Gallinarum was shown to hybridize to the Tra genes region of IncFI and IncFIV plasmids, suggesting it contains some F Tra genes region. It also hybridized with 75% homology to the virulence plasmid of Typhimurium, and, in decreasing order, to those of Dublin, Enteritidis, and Choleraesuis. A probe made of 319 basepairs of the F OriT region hybridized with approximately 45% homology to the virulence plasmid of Gallinarum, 63% to that of Typhimurium and 50% to that of Enteritidis. The probe, however, failed to hybridize to the plasmid DNAs of IncA, IncB = C, IncH, IncL and IncM, and to the virulence plasmids of Choleraesuis and Dublin.     

Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways

We have determined the complete genome sequences of a host-promiscuous Salmonella enterica serovar Enteritidis PT4 isolate P125109 and a chicken-restricted Salmonella enterica serovar Gallinarum isolate 287/91. Genome comparisons between these and other Salmonella isolates indicate that S. Gallinarum 287/91 is a recently evolved descendent of S. Enteritidis. Significantly, the genome of S. Gallinarum has undergone extensive degradation through deletion and pseudogene formation. Comparison of the pseudogenes in S. Gallinarum with those identified previously in other host-adapted bacteria reveals the loss of many common functional traits and provides insights into possible mechanisms of host and tissue adaptation. We propose that experimental analysis in chickens and mice of S. Enteritidis–harboring mutations in functional homologs of the pseudogenes present in S. Gallinarum could provide an experimentally tractable route toward unraveling the genetic basis of host adaptation in S. enterica.     

ERIC-PCR genotyping of field isolates of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella Gallinarum (SG) and Salmonella Pullorum (SP) have been classified as biovars belonging to Salmonella enterica subsp. enterica serovar Gallinarum. Genetic diversity among isolates of the same biovar can be detected by DNA fingerprinting techniques which are useful in epidemiological investigations. In this study, we applied the PCR amplification of Enterobacterial Repetitive Intergenic Consensus sequences (ERIC-PCR) to analyse 45 strains of SG and SP, most of which were isolated from diseased poultry of different Brazilian regions over a period of 27 years until 2014. The ERIC-genotypes obtained were used to describe the epidemiological relationship amongst the strains. Our findings showed that there were six ERIC-patterns for SG strains at 80% similarity. In addition, some of the SG isolates recovered from different regions and years clustered with 100% similarity, suggesting that transfer of genotypes between these regions has taken place. The commercial rough vaccine strain 9R showed a unique profile. Meanwhile, more genetic diversity was observed among SP strains where ten ERIC-patterns were also formed at 80% similarity.     

Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep

The colonization of intestinal and systemic tissues by Salmonella enterica serovars with different host specificities was determined 7 days after inoculation of 1 to 2-month-old lambs. Following oral inoculation, S. enterica serovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites following intravenous inoculation was similar. The magnitude of intestinal invasion was evaluated in ovine ligated ileal loops in vivo. Serovars Dublin and Gallinarum and the broad-host-range Salmonella serovar Typhimurium were recovered in comparable numbers from ileal mucosa 3 h after loop inoculation, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Microscopic analysis of intestinal mucosae infected with serovars Typhimurium and Dublin showed dramatic morphological changes and infiltration of inflammatory cells, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest that Salmonella serovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the host's intestinal inflammatory response may contribute to but do not determine the specificity of serovar Abortosovis for sheep. Intestinal invasion by serovar Abortusovis was significantly reduced after mutation of invH but was not reduced following curing of the virulence plasmid, suggesting that the Salmonella pathogenicity island 1 influences but the virulence plasmid genes do not influence the ability of serovar Abortusovis to invade the intestinal mucosa in sheep.     

The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S. Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpV and vgrG) revealed that SPI-19 T6SS contributes to S. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.     

Integrative Analysis of Salmonellosis in Israel Reveals Association of Salmonella enterica Serovar 9,12:l,v:? with Extraintestinal Infections,Dissemination of Endemic S. enterica Serovar Typhimurium DT104 Biotypes,and Severe Underreporting of Outbreaks

Salmonella enterica is the leading etiologic agent of bacterial food-borne outbreaks worldwide. This ubiquitous species contains more than 2,600 serovars that may differ in their host specificity, clinical manifestations, and epidemiology. To characterize salmonellosis epidemiology in Israel and to study the association of nontyphoidal Salmonella (NTS) serovars with invasive infections, 48,345 Salmonella cases reported and serotyped at the National Salmonella Reference Center between 1995 and 2012 were analyzed. A quasi-Poisson regression was used to identify irregular clusters of illness, and pulsed-field gel electrophoresis in conjunction with whole-genome sequencing was applied to molecularly characterize strains of interest. Three hundred twenty-nine human salmonellosis clusters were identified, representing an annual average of 23 (95% confidence interval [CI], 20 to 26) potential outbreaks. We show that the previously unsequenced S. enterica serovar 9,12:l,v:− belongs to the B clade of Salmonella enterica subspecies enterica, and we show its frequent association with extraintestinal infections, compared to other NTS serovars. Furthermore, we identified the dissemination of two prevalent Salmonella enterica serovar Typhimurium DT104 clones in Israel, which are genetically distinct from other global DT104 isolates. Accumulatively, these findings indicate a severe underreporting of Salmonella outbreaks in Israel and provide insights into the epidemiology and genomics of prevalent serovars, responsible for recurring illness.     

Evaluation of the Salmonella enterica Serovar Pullorum Pathogenicity Island 2 Mutant as a Candidate Live Attenuated Oral Vaccine

Salmonella enterica serovar Pullorum (S. Pullorum) is a highly adapted pathogen that causes pullorum disease (PD), an important systemic disease of poultry that causes severe economic losses in developing countries. In the interests of developing a safe and immunogenic oral vaccine, the efficacy of a Salmonella pathogenicity island 2 (SPI2)-deleted mutant of S. Pullorum (S06004ΔSPI2) was evaluated in chickens. S06004ΔSPI2 was severely less virulent than the parental wild-type strain S06004 as determined by the 50% lethal dose (LD₅₀) for 3-day-old chickens when injected intramuscularly. Two-day-old chickens immunized with a single oral dose of S06004ΔSPI2 showed no differences in body weight or clinical symptoms compared with those in the negative-control group. S06004ΔSPI2 bacteria were not isolated from livers or spleens of immunized chickens after a short period of time, and specific humoral and cellular immune responses were significantly induced. Immunized chickens were challenged with S. Pullorum strain S06004 and Salmonella enterica serovar Gallinarum (S. Gallinarum) strain SG9 at 10 days postimmunization (dpi), and efficient protection against the challenges was observed. None of the immunized chickens died, the clinical symptoms were slight and temporary following challenge in immunized chickens compared with those in the control group, and these chickens recovered by 3 to 5 dpi. Overall, these results demonstrate that S06004ΔSPI2 can be used as a live attenuated oral vaccine.     

Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.     

Molecular Characterization and Antimicrobial Susceptibility of Salmonella Isolates from Infections in Humans in Henan Province, China

We characterized 208 human Salmonella isolates from 2006 to 2007 and 27 human Salmonella enterica serovar Typhimurium isolates from 1987 to 1993 from Henan Province, China, by serotyping, by antimicrobial susceptibility testing, and, for the most common serovars, by pulsed-field gel electrophoresis (PFGE). The most common serovars among the 2006-2007 isolates were S. enterica serovar Typhimurium (27%), S. enterica serovar Enteritidis (17%), S. enterica serovar Derby (10%), S. enterica serovar Indiana (6%), and S. enterica serovar Litchfield (6%). A high percentage of the isolates were multiple-drug resistant, and 54% were resistant to both nalidixic acid and ciprofloxacin. Of these, 42% were resistant to a high level of ciprofloxacin (MIC > 4 μg/ml), whereas for the remaining isolates, the MICs ranged from 0.125 to 2 μg/ml. Five isolates (2%) were ceftiofur resistant and harbored bla_CTX-M14 or bla_CTX-M15. With the possible exception of the quinolones and cephalosporins, the 1987-1993 S. enterica serovar Typhimurium isolates were almost as resistant as the recent isolates. PFGE typing of S. enterica serovar Typhimurium showed that the most common cluster predominated over time. Two other clusters have emerged, and another cluster has disappeared.     

SopA inactivation or reduced expression is selected in intracellular Salmonella and contributes to systemic Salmonella infection

Pseudogenes are accumulated in host-restricted Salmonella enterica serovars, while pseudogenization is primarily regarded as a process that purges unnecessary genes from the genome. Here we showed that the inactivation of sopA, which encodes an effector of Salmonella Pathogenicity Island 1, in human-restricted S. enterica serovar Typhi (S. Ty) and Paratyphi A (S. PA) is under positive selection and aimed to reduce bacterial cytotoxicity toward host macrophages. Moreover, we found that the expression of sopA in Salmonella Typhimurium (S. Tm), a broad-host-range serovar which causes systemic disease in mice, was negatively regulated during mice infection and survival in murine macrophages. The sopA repression in S. Tm is mediated by IsrM, a small RNA absent from the genome of S. Ty and S. PA. Due to the lack of IsrM, sopA expression was unregulated in S. Ty and S. PA, which might have facilitated the convergent inactivation of sopA in these two serovars. In conclusion, our findings demonstrate that sopA inactivation or intracellular repression is the target of positive selection during the systemic infection caused by S. enterica serovars.     

Comparison of Typing Methods with a New Procedure Based on Sequence Characterization for Salmonella Serovar Prediction

As the development of molecular serotyping approaches is critical for Salmonella spp., which include >2,600 serovars, we performed an initial evaluation of the ability to identify Salmonella serovars using (i) different molecular subtyping methods and (ii) a newly implemented combined PCR- and sequencing-based approach that directly targets O- and H-antigen-encoding genes. Initial testing was performed using 46 isolates that represent the top 40 Salmonella serovars isolated from human and nonhuman sources, as reported by the U.S. Centers for Disease Control and Prevention and the World Health Organization. Multilocus sequence typing (MLST) was able to accurately predict the serovars for 42/46 isolates and showed the best ability to predict serovars among the subtyping methods tested. Pulsed-field gel electrophoresis (PFGE), ribotyping, and repetitive extragenic palindromic sequence-based PCR (rep-PCR) were able to accurately predict the serovars for 35/46, 34/46, and 30/46 isolates, respectively. Among the methods, S. enterica subsp. enterica serovars 4,5,12:i:−, Typhimurium, and Typhimurium var. 5− were frequently not classified correctly, which is consistent with their close phylogenetic relationship. To develop a PCR- and sequence-based serotyping approach, we integrated available data sources to implement a combination PCR-based O-antigen screening and sequencing of internal fliC and fljB fragments. This approach correctly identified the serovars for 42/46 isolates in the initial set representing the most common Salmonella serovars, as well as for 54/63 isolates representing less common Salmonella serovars. Our study not only indicates that different molecular approaches show the potential to allow for rapid serovar classification of Salmonella isolates, but it also provides data that can help with the selection of molecular serotyping methods to be used by different laboratories.     

Activation of apoptosis by Salmonella pathogenicity island-1 effectors through both intrinsic and extrinsic pathways in Salmonella-infected macrophages

BackgroundSalmonella enterica serovar Typhimurium, a non-typhoidal food-borne pathogen, causes acute enterocolitis, bacteremia, extraintestinal focal infections in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) contribute to invading into host cellular cytosol, residing in Salmonella-containing vacuoles for intracellular survival, and inducing cellular apoptosis. This study aimed to better understand the mechanism underlying apoptosis in Salmonella-infected macrophages.MethodsS. Typhimurium SL1344 was used to evaluate extrinsic and intrinsic apoptosis pathways in THP-1 monocyte-derived macrophages in response to Salmonella infection.ResultsActivated caspase-3-induced apoptosis pathways, including extrinsic (caspase-8-mediated) and intrinsic (caspase-9-mediated) pathways, in Salmonella-infected macrophages were verified. THP-1 cells with dysfunction of TLR-4 and TLR-5 and Salmonella SPI-1 and SPI-2 mutants were constructed to identify the roles of the genes associated with programmed cell death in the macrophages. Caspase-3 activation in THP-1 macrophages was induced by Salmonella through TLR-4 and TLR-5 signaling pathways. We also identified that SPI-1 structure protein PrgH and effectors SipB and SipD, but not SPI-2 structure protein SsaV, could induce apoptosis via caspase-3 activation and reduce the secretion of inflammation marker TNF-α in the Salmonella-infected cells. The two effectors also reduced the translocation of the p65 subunit of NF-κB into the nucleus and the expression of TNF-α, and then inflammation was diminished.ConclusionNon-typhoid Salmonella induced apoptosis of macrophages and thereby reduced inflammatory cytokine production through the expression of SPI-1. This mechanism in host–pathogen interaction may explain why Salmonella usually manifests as occult bacteremia with less systemic inflammatory response syndrome in the bloodstream infection of children.     

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.     

Repression of Flagella Is a Common Trait in Field Isolates of Salmonella enterica Serovar Dublin and Is Associated with Invasive Human Infections

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:−:−, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.