CRISPR profiles obtained for 12 strains and two genomes of <i>S. enterica</i> serotype Paratyphi A.

<p>The spacer sequences and direct repeat (DR) sequences are indicated. The set of strains and genomes has been reported elsewhere <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a>. n” is the number of strains harboring each profile. The primers used for serotype Paratyphi A-specific amplification (PA-F and PA-R) and for amplification of the entire CRISPR1 sequence (A1 and A2) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a> are indicated in different colors.</p

Structure of the CRISPR/Cas system from <i>S. enterica</i> serotypes Typhi and Paratyphi A.

<p>Two CRISPR loci (CRISPR1 and CRISPR2) flank the CRISPR-associated sequences (cas). The <i>cas</i> genes, spacers, direct repeats, leader sequences are represented by gray arrows, colored diamonds, black diamonds, and light gray boxes marked L, respectively. The genomic orientation from <i>iap</i> to <i>ygcF</i> has been maintained to ensure consistency with previous studies <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Touchon1" target="_blank">[28]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Liu1" target="_blank">[29]</a>. The probable transcriptional orientation of the CRISPR loci, as extrapolated from studies in <i>E. coli </i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Pul1" target="_blank">[30]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Pougach1" target="_blank">[31]</a>, is indicated by light blue dashed arrows.</p

EvaGreen real-time PCR assay.

<p>(A) Amplification and melting curves for 43 serotype Typhi isolates tested in duplicate with the TY-F and TY-R primers, yielding a mean Ct value of 22.6±1.1 and a single melting curve peak at 82.5–83°C. The two negative controls (<i>S. enterica</i> serotype Paratyphi A 1K and sterile water), also tested in duplicate, appear as flat lines. (B) Amplification and melting curves for 37 serotype Paratyphi A isolates tested in duplicate with the PA-F and PA-R primers, yielding a mean Ct value of 23.1±1.1 and a melting curve peak at 85–85.5°C. The two negative controls (<i>S. enterica</i> serotype Typhi Ty2 and sterile water), also tested in duplicate, appear as flat lines.</p

CRISPR profiles obtained for the 18 strains and two genomes of <i>S. enterica</i> serotype Typhi.

<p>The spacer sequences and direct repeat (DR) sequences are indicated. The set of strains and genomes has been reported elsewhere <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a>. “n” is the number of strains harboring each profile. Haplotypes <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Roumagnac1" target="_blank">[19]</a> are indicated, to illustrate the genetic diversity of the strains studied. ND, not determined. The primers used for serotype Typhi-specific amplification (TY-F and TY-R) and those for amplification of the entire CRISPR2 sequence (B1 and B2) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd.0002671-Fabre1" target="_blank">[15]</a> are indicated in different colors.</p

Strategy used to design TY-R, the reverse primer for the serotype Typhi-specific PCR assay.

<p>The DR sequence upstream from EntB0/EntB0var1 is indicated in black letters. The spacers EntB0 and EntB0var1 are indicated in blue letters. The nucleotides belonging to the template for primer TY-R are represented by letters of larger size. The sequence of TY-R is indicated in red, with the deliberate mismatch in green. Mismatch positions between TY-R and the templates of serotypes Emek (EntB0) and Enteritidis (EntB0var1) are underlined.</p

Results of conventional PCR assays targeting <i>S. enterica</i> serotypes Typhi and Paratyphi A.

<p>¹ The details of the strains are provided in Supplemental <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002671#pntd-0002671-t001" target="_blank">Table 1</a>.</p><p>²+, amplicon of the expected size; −, no amplicon of the expected size.</p

<i>Shigella</i> serotypes identified in the study.

<p>*Not including the four rough (auto-agglutinable) and the two non-agglutinable isolates</p><p><i>Shigella</i> serotypes identified in the study.</p

Distribution of organism identified in children with watery or bloody diarrhea by age group.

<p>Distribution of organism identified in children with watery or bloody diarrhea by age group.</p

Cecal persistence and colonization levels of <i>S.</i> Senftenberg isolates in infected chicks.

<p>Number of CFU of <i>Salmonella</i> Senftenberg rifampicin-resistant strains per gram of cecum of chicks orally infected with 10⁵ CFU. (A) Enumeration was performed up to 33 days post-infection for strains SS304, SS308, SS270, SS267, and 26 days p.i for strains SS17, SS291, SS209, SS13, SS48, SS47, SS278, SS41. (B) Cecal load of chicks inoculated with four selected strains (SS308, SS209, SS304, SS278) in a second experiment up to 26 days p.i. The data presented correspond to the mean of positive and negative cecum samples ± SEM.</p

Measurement of proximal cecal villus height and crypt depth of control chicks and animals infected with persistent SS308 and non-persistent SS304 strains of <i>S.</i> Senftenberg at 5 and 26 days post-infection (infection at 1 day of age).

a,b<p>The mean of proximal cecal villus heights and crypt depths ±SD. Different letters in the same line indicate a significant difference with the Mann -Whitney test (P≤0.05).</p