Presence and expression of typhoid-associated virulence factors among NTS serovars.

<p>(A) PCR analysis was used to confirm CGH results and to determine the distribution of three typhoid-associated genes in 35 clinical isolates from the 12 NTS serovars. PCR amplicons of 353-bp, 294-bp, and 335-bp indicate the presence of <i>cdtB</i>, <i>hlyE</i> and <i>tcfA</i>, respectively. Tested isolates are numbered from 1–35 according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058449#pone.0058449.s001" target="_blank">Table S1</a>, with the isolates that were characterized in mice and subjected to CGH analysis in bold. <i>S.</i> Typhi CT18 (CT18) and <i>S.</i> Typhimurium SL1344 (SL1344) were used as positive and negative controls, respectively. (B) Reverse transcription-PCR was applied to examine expression of <i>cdtB</i>, <i>hlyE, taiA</i> and <i>tcfA</i> genes in serovars Schwarzengrund, Montevideo, 9,12:l,v:- and Bredeney. Bacterial RNA was extracted from <i>Salmonella</i> cultures grown to late logarithmic phase in LB, followed by treatment with DNase I and reverse transcription. cDNA was used as template for PCR amplification using the primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058449#pone.0058449.s002" target="_blank">Table S2</a>. <i>Salmonella</i> RNA without a reverse transcriptase treatment (-RT) and purified gDNA were used as negative and positive controls, respectively.</p

Intracellular growth of invasive and enteritis strains in epithelial cells.

<p>Clinical isolates (N = 41) from blood and stool sources were grown to late logarithmic phase in LB medium and used to infect epithelial HeLa cells at a MOI of ∼100∶1. Intracellular replication (ratio between recovered CFU at 24 h/CFU at 2 h p.i.) is shown in relation to the stool isolate of each serovar. In <i>S</i>. Typhimurium, replication is presented relative to median value of the stool isolates (isolate 88359). Indicated values present the mean and the SEM of at least 4 independent infections.</p

Pathogenicity of iNTS strains in mice.

<p>Twelve groups of 4–5 female C3H\HeN mice were challenged i.p. with ∼5×10³ CFU of bacteremic strains from serovars Schwarzengrund (isolate number 124983), 9,12:l,v:- (94293), Bredeney (96115), Choleraesuis (90958), Dublin (74007), Enteritidis (122205), Hadar (121851), Heidelberg (78646), Montevideo (111072), Newport (91532), Typhimurium (103259), and Virchow (103033). Survival of the mice during 40 days post-infection is shown (A). At end-points (as shown in A), harvested organs were homogenized and serial dilutions were plated onto XLD-agar plates for CFU count. Bacterial load in each mouse is represented as CFU/organ by individual dots in the liver (B), spleen (C), ileum (D), cecum (E), and colon (F). Geometric mean for each serovar in the different sites is shown by a horizontal line.</p

Invasion of bacteremic and enteritis strains into human epithelial cells.

<p>Clinical isolates from 12 NTS serovars from blood (invasive) and stool (gastrointestinal) sources were grown to late logarithmic phase in LB medium and used to infect HeLa cells at a MOI of ∼100∶1. The invasion of each strain (source and isolate number are indicated below each bar) is shown in relation to the invasion of the stool isolate in each serovar. In <i>S</i>. Typhimurium, invasion is presented relative to median value of the stool isolates (isolate 93561). Indicated values present the mean and the standard error of the mean (SEM; represented by the error bars) of at least 4 independent infections. ND, no data, as the source of isolate 4311–10781 from serovar Dublin is not known.</p

Persistent infection of iNTS strains in the mouse model.

<p>Following challenge with ∼5×10³ CFU of the bacteremic strains, fecal pellets were collected from the C3H\HeN mice at the indicated time points during 40 days (or until the animal was sacrificed). Pellets were weighed, homogenized in PBS and plated onto XLD-agar plates to determine the number of CFU/g stool. Shedding of <i>S.</i> Choleraesuis (D) and <i>S.</i> Dublin (E) is shown only until 3 days p.i., as the mice had to be euthanized. Dots represent independent CFU counts in pellets of individual mice.</p

Distribution of virulence genes across NTS bacteremic isolates.

<p>The presence-absence of genes associated with <i>Salmonella</i> virulence were determined in five <i>S</i>. Typhimurium stool isolates (115043, 88359, 93561, 98001, 130100) and five blood isolates (103259, 111682, 116449, 93130, 98666) in addition to 11 invasive strains from serovars Schwarzengrund (124983), 9,12:l,v:- (94293), Bredeney (96115), Choleraesuis (90958), Dublin (74007), Enteritidis (122205), Hadar (121851), Heidelberg (78646), Montevideo (111072), Newport (91532) and Virchow (103033). Where not stated otherwise, presence was determined by CGH using the <i>Salmonella</i> STv7E microarray. <i>a</i>, presence was determined or confirmed by southern blot; <i>b</i>, presence was determined or confirmed by PCR. A plus sign indicates the presence of the gene; white blocks indicate an absence; a variable presence among the blood or stool isolates of <i>S</i>. Typhimurium is shown by the number of positive isolates/ total number of isolates (N = 5); ND, no data (CGH was not conclusive).</p

Core, variable and absent genes in iNTS strains.

<p>Microarray-based comparative genomic hybridization was used to determine the presence and absence of the <i>Salmonella</i> ORFs represented on the <i>S. enterica</i> STv7E microarray. After excluding redundant probes (corresponding to the same ORF) and ambiguous predictions, we were able to assign 4548 <i>Salmonella</i> genes into three groups. A set of 3233 common genes was determined to be present in all of the bacteremic strains and defined as the core genome of iNTS (left panel). 1200 genes with varying distribution were missing from, at least, one serovar and were considered as the variable iNTS genome (center panel). 115 genes were absent from all the 16 blood strains analyzed in this study (right panel). Functional classification of the genes is shown and assigned using GenProtEC (<a href="http://genprotec.mbl.edu/" target="_blank">http://genprotec.mbl.edu/</a>).</p

Survival of invasive and enteritis strains in macrophages.

<p><i>Salmonella</i> isolates from blood and stool sources from serovars Typhimurium (A), Schwarzengrund (B), 9,12:l,v:-(C), Montevideo (D) and Bredeney (E) were grown to stationary phase in LB medium and used to infect RAW2464.7 cells at a MOI of ∼1. Survival is shown in relation to the stool isolate of each serovar. In <i>S</i>. Typhimurium, survival is presented relative to median value of the stool isolates (isolate 93561). Indicated values present the mean and the SEM of at least 4 independent infections.</p