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

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

<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

Table_2_Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis.xlsx

<p>In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.</p

Image_2_Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis.TIF

<p>In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.</p