The Salmonella pathogenicity island 13 contributes to pathogenesis in streptomycin pre-treated mice but not in day-old chickens
© Elder et al. 2016
Received: 29 February 2016
Accepted: 3 April 2016
Published: 2 May 2016
The Erratum to this article has been published in Gut Pathogens 2016 8:35
Salmonella enterica serovar Enteritidis (S. Enteritidis) is a human and animal pathogen that causes gastroenteritis characterized by inflammatory diarrhea and occasionally an invasive systemic infection. Salmonella pathogenicity islands (SPIs) are horizontally acquired genomic segments known to contribute to Salmonella pathogenesis. The objective of the current study was to determine the contribution of SPI-13 to S. Enteritidis pathogenesis.
We deleted the entire SPI-13 (∆SPI-13) from the genome of S. Enteritidis CDC_2010K_0968 strain isolated from a human patient during the 2010 egg-associated outbreak in the US. The kinetics of infection of the wild-type parent and the ∆SPI-13 were compared in orally challenged day-old chickens and streptomycin pre-treated mice. The degree of intestinal inflammation and the survival of mutant strain within the avian (HD11) and murine (RAW264.7) macrophages were also determined.
The deletion of the SPI-13 resulted in impaired infection kinetics of S. Enteritidis in streptomycin pre-treated mice which was characterized by significantly lower (P < 0.05) viable counts in the ceca, liver and spleen, impaired ability to induce intestinal inflammation and reduced survival within murine macrophages. Conversely, there were no significant differences in the infection kinetics of ∆SPI-13 in day-old chickens in any of the organs tested and the survival of ∆SPI-13 within chicken macrophages remained unaltered.
The results of this study show that SPI-13 contributes to the pathogenesis of S. Enteritidis in streptomycin pre-treated mice but not in day-old chickens and raises the possibility that SPI-13 may play a role in pathogenesis and the host adaptation/restriction of Salmonella serovars.
Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) is a major food-borne pathogen that causes inflammatory diarrhea in immunocompetent patients, however poor immune response or co-infection with malaria or HIV may result in invasive infections with severe systemic illness [1, 2]. The Salmonella pan-genome has 23 annotated genomic islands [3, 4] that are referred to as Salmonella pathogenicity islands (SPIs). Of these, SPI-1 and SPI-2 have been extensively characterized. The current paradigm is that SPI-1 is required for invasion of the epithelial cells in the intestinal tract whereas SPI-2 is required for survival in macrophages and systemic spread (Reviewed in ). However, the majority of the other SPIs are poorly characterized and their contribution to the biology of Salmonella in general and pathogenesis in particular remains unclear.
The sequences downstream of tRNA genes are hotspots for recombination in bacteria, it is therefore not surprising that SPI-13 appears to have undergone multiple recombination events in the evolution of the genus Salmonella. Evidence of recombination events at this locus include insertion/deletion of segments of SPI-13 resulting in distinct SPI-13 sequences observed between S. enterica and S. bongori, as well as differences between subspecies and serovars within S. enterica. Salmonella bongori shares very few SPI-13 genes with S. Enteritidis and strains representing the subspecies indica, salamae, diarizonae, arizonae and houtenae also lack many SPI-13 genes . The differences in gene content between S. enterica subspecies could be related to host adaptation as S. enterica subspecies enterica is associated with warm-blooded host while S. bongori and the other Salmonella subspecies are associated with cold-blooded hosts. Interestingly, there are also SPI-13 gene content differences within subspecies enterica serovars that seem to be related to host adaptation. Serovars Typhi, Paratyphi A and Sendai, that cause typhoid-like disease and are human-adapted, have a different SPI-13 gene composition compared to the majority of the non-typhoidal Salmonella (NTS) serovars with broad host range [6, 7]. Klebseilla pneumoniae and Yersinia pestis have homologs to some of the genes in SPI-13 which also suggests that SPI-13 is not monophyletic.
Since the original identification of SPI-13 in an avian host-adapted S. Gallinarum, few negative selection screens have been conducted using non-host restricted NTS serovars in which several SPI-13 genes were identified. Chaudhuri et al.  screened a library containing pools of >7700 mutants of S. Typhimurium in orally infected chickens, pigs, and calves and reported that insertion mutations in up to fifteen genes of SPI-13 resulted in negative selection of these mutants in the intestines of these hosts. Moreover, few SPI-13 mutants of S. Typhimurium were negatively selected in internal organs in intra-peritoneally [8, 9] and orally inoculated mice . To date, only one study reported use of S. Enteritidis as a model organism in which insertion mutations in seven SPI-13 genes (SEN2961–SEN2964, SEN2972, SEN2976–SEN2977) resulted in negative selection in intra-peritoneally inoculated mice . It is important to note that the negative selection of a mutant during such large-scale in vivo screening assays could result from number of underlying factors. These include direct impact of the mutation on the pathogen-host cell interaction or indirectly due to in vivo competitive growth defects or because of impaired metabolic fitness or merely due stochastic loss of a mutant from a population. Moreover, the negative selection-screening assays employed in different studies are often limited to a single organ and a single time point and only few have used natural route of infection. Thus, although negative selection of SPI-13 mutants reported in the published studies raises a possibility that SPI-13 may have a role in pathogenesis of Salmonella, this has not been conclusively demonstrated.
The specific objective of this study was to directly demonstrate the role of SPI-13 in S. Enteritidis pathogenesis by constructing S. Enteritidis mutant lacking the entire SPI-13 and determining the effects of absence of SPI-13 on kinetics of infection (i.e. intestinal colonization and invasion, modulation of gut inflammation and internal organ colonization). To dissect the role of SPI-13 in pathogenesis, we used two biologically relevant animal models, which included: (1) streptomycin pre-treated mouse (an established model for human intestinal disease) and (2) day-old chickens (the reservoir host and a major source of human infection). To the best of our knowledge, this is the first study showing the direct evidence that SPI-13 contributes to intestinal pathogenesis of S. Enteritidis in streptomycin pre-treated mice. The results of this study also point towards the possibility that SPI-13 is likely involved in host- adaptation and propagation of S. Enteritidis in gastrointestinal environment of mice, but not in chickens.
Bacterial strains and growth media
The S. Enteritidis CDC_2010K_0968 strain, isolated from a human patient in Ohio during the 2010 egg-associated outbreak , was used as the wild-type (WT) parent strain for constructing following mutants: (1) a k/o mutant of SPI-13 (ΔSPI-13), (2) a k/o mutant of SPI-14 (ΔSPI-14), (3) a k/o mutant of both SPI-13 and SPI-14 (ΔSPI-13/14) and, (4) a kanamycin resistant derivative of the WT strain (WT KanR). The ΔSPI-14 and ΔSPI-13/14 mutants were included as control strains to compare and confirm the role of SPI-13 in pathogenesis of S. Enteritidis in streptomycin pre-treated mouse model (see below). The kanamycin resistant derivative of the reference strain P125109 (UK KanR) was also included for comparison of infection kinetics in streptomycin pre-treated mice . The λ-Red recombinase system was used to construct ΔSPI-13, ΔSPI-14, ΔSPI-13/14 and KanR WT strains following procedures described previously . For the ΔSPI-13 strain the entire SPI-13 was replaced with the kanamycin resistance cassette encoded on the pKD4 plasmid amplified with the forward primer, SPI13KOFw (5′-TATAAACGGATGCGTGATCATAATAAAGGCAGTAATAGTAAGTTTTAACAGTGTAGGCTGGAGCTGCTTC-3′), and reverse primer, SPI13KORv (5′-CGCTACAGGTCAGACGGCGCGGAGCTAATGTTTTTTAACGAGGCTTTATCATATGAATATCCTCCTTAG-3′). The ΔSPI-14 strain was generated by replacement of SPI-14 with the kanamycin resistant cassette using the forward primer, SPI14KOFw (5′-TTTTAAGATATATTGAATTATCAGATGCTCCATTCAAATGAGAGACGAGAGTGTAGGCTGGAGCTGCTTC-3′), and the reverse primer SPI14KORv (5′-TGCATAACATGGATAAAATGGGTAGTCATGCTAGCGAGATAAGACAATGACATATGAATATCCTCCTTAG-3′). The ΔSPI-13/14 mutant was constructed by replacing SPI-13 in the previously constructed ΔSPI-14 mutant with the chloramphenicol resistance cassette using the primers listed above for SPI-13 k/o. For the construction of WT KanR and UK KanR strains, the kanamycin resistance cassette was amplified using the forward primer, att_tn7KOFw (5′-AGCGCAGGTAGGCGTAGCACCTCTTAGTCGCTCTTCAGCCACCATAGAGAGTGTAGGCTGGAGCTGCTTC-3′), and reverse primer, att_tn7KORv (5′-GGCCGTCGATAGACGGCCTTTTTTTGTGCGCCGTGACAGGCGCTGTTCTTATATGAATATCCTCCTTA-3′). This cassette was inserted at the tn7 attachment site (att_tn7, found between nucleotides 3,939,373 and 3,939,408) which lies in the intergenic region between SEN3674 and glmS in the S. Enteritidis strain P125109 genome (NCBI GenBank accession: NC_011294). Unless otherwise noted, S. Enteritidis and E. coli strains were grown in Luria–Bertani (LB) broth (Difco, USA) overnight (16 h) at 37 °C with shaking at 200 rpm. When appropriate, the medium was supplemented with 1.6 % (w/v) Bacto Agar (Difco), carbenicillin (Cb, 100 μg/ml), chloramphenicol (Cm, 20 μg/ml) and kanamycin (Km, 50 μg/ml).
Mouse infection experiments
All animal experiments were performed following the protocols approved by Institutional Animal Care and Usage Committee (IACUC). The streptomycin pre-treated mouse model was used as previously described . For each experiment, 7–8 week-old, female C57BL/6 mice were acquired from Harlan laboratories, USA. Food was withheld for 4 h prior to orogastric administration of 20 mg of streptomycin (Sigma Aldrich, USA) in 100 µl of sterile H2O. At 24 h post streptomycin treatment, food was withheld for 4 h. Subsequently, mice were orogastrically challenged with ~7 log10 CFU WT parent (WT KanR) strain or one of the ΔSPI-13, ΔSPI-14, or ΔSPI-13/14 mutant strains. At days 5 and 7 post-infection (p.i.), three mice were sacrificed. The liver, spleen, and cecum were collected for direct plate counts and section of the cecum was also collected for histopathological analysis (see below). Intracellular counts in the cecum were determined by washing the tissues 3× in sterile PBS to remove as much of the intestinal contents and extracellular bacteria as possible and then incubating for 30 min at 37 °C with gentamicin (200 µg/ml) to kill any remaining extracellular bacteria. The sections were washed with sterile PBS to remove residual gentamicin, homogenized, then treated with 0.5 % Triton-X 100 to lyse epithelial cells and release intracellular bacteria. Samples from the liver, spleen, and cecum were weighed, homogenized in sterile maximum recovery diluent (MRD, Difco), serially diluted and directly plated on XLT-4 media (Difco) with 50 μg/ml kanamycin when appropriate. Only in the cases when no colonies were recovered from samples by direct plating of organ homogenates were samples enriched; 500 µl of homogenate was used to inoculate 10 ml tetrathionate broth (TTB, Neogen) prepared according to manufacturer’s directions. Enrichment cultures were incubated at 37 °C for 24 h prior to plating on XLT-4 media followed by incubation for 24 h at 37 °C. If TTB enrichment cultures were negative after 24 h, they were plated again at 48 h. If a sample was positive after 24 or 48 h of enrichment they were assigned the limit of detection for this experiment as the lowest CFU/g observed for that specific organ across the experiment. If samples were negative at both time points 0 CFU/g was assigned for statistical analysis. Organ homogenates were directly plated on XLT-4 media (Difco) supplemented with 50 μg/ml kanamycin when appropriate. Samples found negative by direct plating were enriched as described above.
Cecal sections from mice were embedded in paraffin wax and stained with hematoxylin and eosin (H&E). The sections were analyzed independently by two individuals blinded to the sample IDs to assign total inflammation scores as described previously with minor modifications . Briefly, the score for percent submucosal edema (% SE) was calculated from the proportion of the diameter of mucosa made up by the space between the tunica muscularis and the epithelial layer using following scores: 0 for no detectable edema, 1 for detectable edema (0–10 % of the diameter of the cross section of the intestinal mucosa and submucosa), 2 for moderate edema (11–40 %), and 3 for severe edema (>40 %). Infiltration of the lamina propia by polymorphonuclear (PMN) cells was scored according to number of PMNs in the lamina propia per 10 high power (400×) fields by using following scores: 0 for normal (<5), 1 for slight infiltration (6–20), 2 moderate (21–60), 3 for high infiltration (61–100), and 4 for severe (>100). Reduction in number of goblet cells were scored based on the number of goblet cells per high power field; 0 for normal (>28), 1 for slight reduction (11–28), 2 for moderate reduction (1–10), and 3 for severe reduction (<1). Integrity of the intestinal epithelium was scored as follows: 0 no detectable loss of integrity, 1 for epithelial desquamation, 2 for epithelial erosion and 3 for epithelial ulceration. Finally, a total combined score was calculated. The combined score of 0 was considered to indicate no inflammation, 1, 2 indicated minimal inflammation, 3, 4 indicated slight inflammation, 5–8 indicated significant inflammation, and 9–13 indicated severe inflammation.
Chicken infection experiments
Specific-pathogen-free (SPF) White Leghorn eggs were acquired from Sunrise farms (Catskill, NY) and incubated for 18 days in the egg incubator (Ova-Easy 190 Advance Cabinet Incubator, Brinsea Products Inc, Titusville, FL) before transferring to a hatcher (1550 hatcher-GQF, GQF manufacturer Co, Savannah, GA) for 3 days following manufacturer’s instructions. Newly hatched SPF chicks were transferred and housed in HEPA-filtered isolator cages. Chickens were orally challenged with 8.5 log10 CFU of ΔSPI-13 mutant and the WT parent strain, respectively at day-1 of age . Subsequently, four chickens were sacrificed at days-1, 3, 5, 7, 14 and 28 p.i. except on 5 and 28 days p.i. when three birds were sacrificed. The spleen, liver, duodenum, jejunum, ileum, and ceca were collected for direct plate counts. For the determination of viable bacterial counts in the small intestine, segments from the duodenum, jejunum and ileum were pooled for both intracellular and total counts. A portion of ceca and each segment of the small intestine were processed for determination of intracellular counts similar to as described for mouse experiments.
Intra-macrophage survival assays
RAW264.7 mouse macrophages (ATCC) were grown in D-MEM medium (Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, Sigma Aldrich, USA), 2 mM l-glutamine, and 20 μg/ml gentamicin at 37 °C with 5 % CO2. HD11 chicken macrophages were grown in IMDM medium (Gibco, USA) supplemented with 10 % FBS at 42 °C with 5 % CO2. Gentamicin-protection assays were performed as described previously . Briefly, for each experiment, RAW264.7 or HD11 cells were seeded in three 48-well plates each with 1.25 × 105 cells per well and grown for 16–24 h. Overnight (16 h) cultures of S. Enteritidis strains were sub-cultured in fresh LB medium and grown for ~3 h at 37 °C with shaking at 200 rpm until OD600 of 0.6 was achieved. Bacterial strains were pelleted by centrifugation at 13,000g for 2 min, washed three times with sterile PBS and resuspended in pre-warmed D-MEM (RAW264.7) or IMDM (HD11) to obtain 2.5 × 106 CFU/100 μl. Subsequently, 100 μl of each S. Enteritidis strain was added to each well (multiplicity of infection of ~20), infections were synchronized by spinning the plates at 250g for 5 min. Cell infection was allowed to proceed for 30 min followed by three washings with PBS and gentamicin treatment (200 μg/ml) for 30 min to kill extracellular bacteria. Next, the cells from plate-1 were washed 3× with sterile PBS and lysed with 0.5 % Triton-X 100. The numbers of intracellular bacteria were enumerated via direct plate counts which served as a measure of total uptake of each bacterial strain. In plate-2 and plate-3, the media was replaced with the lower concentration of gentamicin (20 μg/ml) followed by incubation for 2 h (plate-2) and 20 h (plate-3). At 2 and 20 h post infection, cells from respective plates were processed identically as described for plate-1. Percent survival/replication of S. Enteritidis strains at 2 and 20 h was determined as follows: CFU at 2 or 20 h/total uptake CFU × 100. Each strain was tested in duplicates in at least three independent experiments.
Statistically significant (P < 0.05) differences in mean log10 CFU counts in each organ were determined using Student’s t test for comparing the two means in the preliminary mouse experiments (for determination of dose and comparing WT strains) and the chicken experiment. A one-way ANOVA with Tukey’s post hoc was used to compare means and identify statistically significant differences between the four groups in subsequent mouse experiment. Mean percent survival in mouse and chicken mouse macrophage survival assays were also compared using Student’s t test. Mean inflammation score for each parameter as wells as mean combined inflammation score were compared for the entire experiment using two-way ANOVA with Tukey’s post hoc analysis to identify statistically significant differences (P < 0.05).
Results and discussion
SPI-13 contributes to colonization of the intestine and internal organs, and induction of acute cecal inflammation by S. Enteritidis in streptomycin pre-treated mice
NTS infection in the conventional laboratory mice typically manifests as a systemic disease resembling typhoid in humans (Reviewed in ). In contrast, pre-treatment with high dose of streptomycin (20 mg) 24 h prior to infection results in reduced diversity of the microflora in the gastrointestinal tract. This in turn, allows Salmonella to extensively colonize the intestinal tract resulting in gastro-intestinal disease that closely resembles human intestinal disease which is characterized by acute gut inflammation and diarrhea (Reviewed in ). Thus, streptomycin pre-treated mouse model has been extensively characterized and widely adapted as an improved model to assess the role of Salmonella genetic factors in causing enterocolitis .
SPI-13 contributes to survival in mouse macrophages
Salmonella Enteritidis SPI-13 does not significantly impair the intestinal colonization and organ colonization in chickens
SPI-13 does not contribute to the survival of S. Enteritidis in chicken macrophages
In summary, our results show that SPI-13 contributes to the pathogenesis of S. Enteritidis which is characterized by decreased colonization of the cecum, reduced inflammation and reduced colonization of the spleen and liver in streptomycin pre-treated mice. The decreased colonization may be at least in part due to the decreased survival in murine macrophages that could limit dissemination to the internal organs. In contrast, we did not observe significant difference in the pathogenesis of ΔSPI-13 mutant in chicken host and there was no defect in the growth and/or survival of the mutant in chicken macrophages. These data raise several interesting questions why SPI-13 is required for full virulence of S. Enteritidis in murine host, but does not appear to be imporant in its reservoir host, chickens? Is it is likely that SPI-13 is a host-specific pathogenicity island? and if true, is this host-specificity is linked to the potential role of SPI-13 in S. Enteritidis metabolism which may impact metabolic fitness of S. Enteritidis in specific hosts? It is intriguing because broad-host range Salmonella serovars are known to have diverse metabolism to support optimal growth in potentially nutrient-limited conditions encountered in different hosts or in the external environment . One plausible hypothesis is that SPI-13 may be important for S. Enteritidis’s ability to utilize monoamines and/or hexuronates in hosts such as streptomycin pre-treated mouse where these nutrients may serve as primary source of energy in vivo. In contrast, this nutrient demand of S. Enteritidis in chicken host is compensated with other sources making SPI-13 dispensable in this reservoir host.
Finally, several Salmonella serovars such as Typhi, Paratyphi A and Sendai and at least few NTS serotypes, have a different SPI-13 gene composition compared to the majority of the other NTS serovars with broad host range [6, 7]. More specifically, the first six genes within SPI-13 are completely absent in S. Typhi and S. Paratyphi whereas S. Seftenberg and S. Infantis have large insertions at the upstream end of SPI-13. This suggest that SPI-13 has a modular architechture [6, 7]. However it is currently unknown if this genetic diversity within SPI-13 also contributes to differential virulence of Salmonella in different hosts and if it is associated with differential of metabolic dependence of Salmonella in different hosts. The modular architechture of SPI-13 is intriguing. We chose to use the most inclusive description of SPI-13 for defining SPI-13 in the current study. However, work is in progress to more rigorously define the island and determine which genes within this island specifically contribute to the pathogenesis and metabolism of S. Enteritidis.
Conceived and designed experiments: JE, DS. Performed experiments: JE, KC, NP, GH. Analyzed the data: JE, DS. All authors read and approved the final manuscript.
We gratefully acknowledge the technical assistance of Ngan Mai and Zachary Bailey. We thank Dr. Subramaniam Srikumaran for generously providing RAW264.7 cells.
The authors declare that they have no competing interests.
This project was funded in part with the funds the Agricultural Animal Health Program, College of Veterinary Medicine, Washington State University. Jacob Elder was supported by Poncin Trust Fellowship.
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- Ao TT, Feasey NA, Gordon MA, Keddy KH, Angulo FJ, Crump JA. Global burden of invasive nontyphoidal Salmonella disease, 2010. Emerg Infect Dis. 2015;21(6):941–9.View ArticlePubMed CentralGoogle Scholar
- Lokken KL, Mooney JP, Butler BP, Xavier MN, Chau JY, Schaltenberg N, Begum RH, Muller W, Luckhart S, Tsolis RM. Malaria parasite infection compromises control of concurrent systemic non-typhoidal Salmonella infection via IL-10-mediated alteration of myeloid cell function. PLoS Pathog. 2014;10(5):e1004049. doi:10.1371/journal.ppat.1004049.View ArticlePubMedPubMed CentralGoogle Scholar
- Fookes M, Schroeder GN, Langridge GC, Blondel CJ, Mammina C, Connor TR, Seth-Smith H, Vernikos GS, Robinson KS, Sanders M, et al. Salmonella bongori provides insights into the evolution of the Salmonellae. PLoS Pathog. 2011;7:e1002191. doi:10.1371/journal.ppat.1002191.View ArticlePubMedPubMed CentralGoogle Scholar
- Hayward MR, Jansen VA, Woodward MJ. Comparative genomics of Salmonella enterica serovars Derby and Mbandaka, two prevalent serovars associated with different livestock species in the UK. BMC Genom. 2013;14:365.View ArticleGoogle Scholar
- Hansen-Wester I, Hensel M. Salmonella pathogenicity islands encoding type III secretion systems. Microbes Infect. 2001;3:549–59.View ArticlePubMedGoogle Scholar
- Shah DH, Lee M-J, Park J-H, Lee J-H, Eo S-K, Kwon J-T, Chae J-S. Identification of Salmonella Gallinarum virulence genes in a chicken infection model using PCR-based signature-tagged mutagenesis. Microbiol (Read Engl). 2005;151:3957–68.View ArticleGoogle Scholar
- Desai PT, Porwollik S, Long F, Cheng P, Wollam A, Bhonagiri-Palsikar V, Hallsworth-Pepin K, Clifton SW, Weinstock GM, McClelland M. Evolutionary genomics of Salmonella enterica subspecies. MBio. doi:10.1128/mBio.00579-12.
- Chaudhuri RR, Morgan E, Peters SE, Pleasance SJ, Hudson DL, Davies HM, Wang J, van Diemen PM, Buckley AM, Bowen AJ, et al. Comprehensive assignment of roles for Salmonella Typhimurium genes in intestinal colonization of food-producing animals. PLoS Genet. 2013;9:e1003456. doi:10.1371/journal.pgen.1003456.View ArticlePubMedPubMed CentralGoogle Scholar
- Haneda T, Ishii Y, Danbara H, Okada N. Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection. FEMS Microbiol Lett. 2009;297:241–9.View ArticlePubMedGoogle Scholar
- Chakraborty S, Chaudhuri D, Balakrishnan A, Chakravortty D. Salmonella methylglyoxal detoxification by STM3117-encoded lactoylglutathione lyase affects virulence in coordination with Salmonella pathogenicity island 2 and phagosomal acidification. Microbiol (Read Engl). 2014;160:1999–2017.View ArticleGoogle Scholar
- Silva CA, Blondel CJ, Quezada CP, Porwollik S, Andrews-Polymenis HL, Toro CS, Zaldívar M, Contreras I, McClelland M, Santiviago CA. Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium. Infect Immun. 2012;80:839–49.View ArticlePubMedPubMed CentralGoogle Scholar
- Allard MW, Luo Y, Strain E, Pettengill J, Timme R, Wang C, Li C, Keys CE, Zheng J, Stones R, et al. On the evolutionary history, population genetics and diversity among isolates of Salmonella Enteritidis PFGE pattern JEGX01.0004. PLoS One. 2013;8:e55254. doi:10.1371/journal.pone.0055254.View ArticlePubMedPubMed CentralGoogle Scholar
- Thomson NR, Clayton DJ, Windhorst D, Vernikos G, Davidson S, Churcher C, Quail MA, Stevens M, Jones MA, Watson M, et al. Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. Genome Res. 2008;18:1624–37.View ArticlePubMedPubMed CentralGoogle Scholar
- Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci. 2000;97:6640–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Suar M, Jantsch J, Hapfelmeier S, Kremer M, Stallmach T, Barrow PA, Hardt W-D. Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model. Infect Immun. 2006;74:632–44.View ArticlePubMedPubMed CentralGoogle Scholar
- Addwebi TM, Call DR, Shah DH. Contribution of Salmonella Enteritidis virulence factors to intestinal colonization and systemic dissemination in 1-day-old chickens. Poult Sci. 2014;93:871–81.View ArticlePubMedGoogle Scholar
- Blondel CJ, Jiménez JC, Leiva LE, Alvarez SA, Pinto BI, Contreras F, Pezoa D, Santiviago CA, Contreras I. The type VI secretion system encoded in Salmonella pathogenicity island 19 is required for Salmonella enterica serotype Gallinarum survival within infected macrophages. Infect Immun. 2013;81:1207–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Watson KG, Holden DW. Dynamics of growth and dissemination of Salmonella in vivo. Cell Microbiol. 2010;12(10):1389–97.View ArticlePubMedGoogle Scholar
- Kaiser P, Diard M, Stecher B, Hardt W-D. The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen’s virulence factors, and the host’s mucosal immune response. Immunol Rev. 2012;245:56–83.View ArticlePubMedGoogle Scholar
- Bohnhoff M, Miller CP. Enhanced susceptibility to Salmonella infection in streptomycin-treated mice. J Infect Dis. 1962;111:117–27.View ArticlePubMedGoogle Scholar
- Wisner ALS, Desin TS, Koch B, Lam P-KS, Berberov EM, Mickael CS, Potter AA, Köster W. Salmonella enterica subspecies enterica serovar Enteritidis Salmonella pathogenicity island 2 type III secretion system: role in intestinal colonization of chickens and systemic spread. Microbiol (Read Engl). 2010;156:2770–81.View ArticleGoogle Scholar
- Shi L, Adkins JN, Coleman JR, Schepmoes AA, Dohnkova A, Mottaz HM, Norbeck AD, Purvine SO, Manes NP, Smallwood HS, et al. Proteomic analysis of Salmonella enterica serovar Typhimurium isolated from RAW 264.7 macrophages identification of a novel protein that contributes to the replication of serovar Typhimurium inside macrophages. J Biol Chem. 2006;281:29131–40.View ArticlePubMedGoogle Scholar
- Hautefort I, Thompson A, Eriksson-Ygberg S, Parker ML, Lucchini S, Danino V, Bongaerts RJM, Ahmad N, Rhen M, Hinton JCD. During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cell Microbiol. 2008;10:958–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Eriksson S, Lucchini S, Thompson A, Rhen M, Hinton JCD. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol Microbiol. 2003;47:103–18.View ArticlePubMedGoogle Scholar
- Pujol C, Grabenstein JP, Perry RD, Bliska JB. Replication of Yersinia pestis in interferon gamma-activated macrophages requires ripA, a gene encoded in the pigmentation locus. Proc Natl Acad Sci USA. 2005;102(36):12909–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Torres R, Swift RV, Chim N, Wheatley N, Lan B, Atwood BR, Pujol C, Sankaran B, Bliska JB, Amaro RE, et al. Biochemical, structural and molecular dynamics analyses of the potential virulence factor RipA from Yersinia pestis. PLoS One. 2011;6(9):e25084. doi:10.1371/journal.pone.0025084.View ArticlePubMedPubMed CentralGoogle Scholar
- Sasikaran J, Ziemski M, Zadora PK, Fleig A, Berg IA. Bacterial itaconate degradation promotes pathogenicity. Nat Chem Biol. 2014;10:371–7.View ArticlePubMedGoogle Scholar
- Michelucci A, Cordes T, Ghelfi J, Pailot A, Reiling N, Goldmann O, Binz T, Wegner A, Tallam A, Rausell A, et al. Immune-responsive gene 1 protein links metabolism to immunity by catalyzing itaconic acid production. Proc Natl Acad Sci. 2013;110:7820–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Marcus R, Varma JK, Medus C, Boothe EJ, Anderson BJ, Crume T, Fullerton KE, Moore MR, White PL, Lyszkowicz E, et al. Re-assessment of risk factors for sporadic Salmonella serotype Enteritidis infections: a case-control study in five FoodNet sites, 2002–2003. Epidemiol Infect. 2007;135:84–92.View ArticlePubMedGoogle Scholar
- Batz MB, Hoffmann S, Morris J, Glenn J. Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. J Food Prot. 2012;75:1278–91.View ArticlePubMedGoogle Scholar
- Shah DH, Zhou X, Kim H-Y, Call DR, Guard J. Transposon Mutagenesis of Salmonella enterica serovar Enteritidis identifies genes that contribute to invasiveness in human and chicken cells and survival in egg albumen. Infect Immun. 2012;80:4203–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao Y, Jansen R, Gaastra W, Arkesteijn G, van der Zeijst BA, van Putten JP. Identification of Genes Affecting Salmonella enterica serovar Enteritidis infection of chicken macrophages. Infect Immun. 2002;70:5319–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Becker D, Selbach M, Rollenhagen C, Ballmaier M, Meyer TF, Mann M, Bumann D. Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature. 2006;440(7082):303–7.View ArticlePubMedGoogle Scholar
- McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature. 2001;413(6858):852–6.View ArticlePubMedGoogle Scholar