Phylogenetics and Differentiation of <em>Salmonella</em> Newport Lineages by Whole Genome Sequencing

<div><p><em>Salmonella</em> Newport has ranked in the top three <em>Salmonella</em> serotypes associated with foodborne outbreaks from 1995 to 2011 in the United States. In the current study, we selected 26 <em>S</em>. Newport strains isolated from diverse sources and geographic locations and then conducted 454 shotgun pyrosequencing procedures to obtain 16–24 × coverage of high quality draft genomes for each strain. Comparative genomic analysis of 28 <em>S</em>. Newport strains (including 2 reference genomes) and 15 outgroup genomes identified more than 140,000 informative SNPs. A resulting phylogenetic tree consisted of four sublineages and indicated that <em>S</em>. Newport had a clear geographic structure. Strains from Asia were divergent from those from the Americas. Our findings demonstrated that analysis using whole genome sequencing data resulted in a more accurate picture of phylogeny compared to that using single genes or small sets of genes. We selected loci around the <em>mutS</em> gene of <em>S</em>. Newport to differentiate distinct lineages, including those between <em>invH</em> and <em>mutS</em> genes at the 3′ end of <em>Salmonella</em> Pathogenicity Island 1 (SPI-1), <em>ste</em> fimbrial operon, and Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) associated-proteins (<em>cas</em>). These genes in the outgroup genomes held high similarity with either <em>S</em>. Newport Lineage II or III at the same loci. <em>S</em>. Newport Lineages II and III have different evolutionary histories in this region and our data demonstrated genetic flow and homologous recombination events around <em>mutS</em>. The findings suggested that <em>S</em>. Newport Lineages II and III diverged early in the serotype evolution and have evolved largely independently. Moreover, we identified genes that could delineate sublineages within the phylogenetic tree and that could be used as potential biomarkers for trace-back investigations during outbreaks. Thus, whole genome sequencing data enabled us to better understand the genetic background of pathogenicity and evolutionary history of <em>S</em>. Newport and also provided additional markers for epidemiological response.</p> </div

Parsimony phylogenetic tree for <i>cas</i> genes.

<p>We constructed this parsimony tree with 100,000 iterations by TNT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055687#pone.0055687-Goloboff1" target="_blank">[38]</a> based on concatenated sequences of the <i>cas</i> genes. This dendrogram indicated that <i>cas</i> genes of Lineages II and III were originated from distinct sources.</p

Characteristics of <i>Salmonella</i> Newport strains used in the study.

*<p>AMC = Amoxicillin/Clavulanic Acid, AMP = Ampicillin, FOX = Cefoxitin, AXO = Ceftriaxone, CHL = Chloramphenicol, GEN = Gentamicin, KAN = Kanamycin, STR = Streptomycin, SUL = Sulfamethoxazole or Sulfisoxazole, TET = Tetracycline, TIO = Ceftiofur.</p>#<p>These two samples were received from Eastern Shore of Virginia in 2007. Isolates may have been collected earlier than 2007.</p

Characteristics of genes/open reading frames (ORFs) between <i>relA</i> and <i>mazG</i> genes of <i>S</i>. Newport SL254 and SL317.

<p>We listed the detailed information of genes between <i>relA</i> and <i>mazG</i> genes. <i>S</i>. Newport SL254 and SL317 were selected. Our data indicated the genomic diversity of this region between Lineages II and III. Interestingly, ORF SNSL254_A3176 and SNSL317_A4073 were found adjoining together in <i>S</i>. Typhi CT18. The existence of <i>ste</i> fimbrial operon might enable Lineage II strains to infect variable hosts. The genes in both <i>S</i>. Newport SL254 and SL317 are ordered top to bottom as their synteny on bacterial chromosome from 5′ to 3′.</p

Pulsed Field Gel Electrophoresis (PFGE) profile digested with <i>Xba</i>I.

<p>We performed PFGE analysis of 24 <i>S</i>. Newport strains (without two environmental farm isolates) isolated from diverse sources and geographic locations. PFGE profiles divided these strains into two major clusters with different groupings compared with the phylogenetic tree based on whole genome wide SNPs.</p

Characteristics of genes/open reading frames (ORFs) between <i>invH</i> and <i>mutS</i> genes in Gene Cluster 1 of <i>S</i>. Newport SL254 and Gene Cluster 2 of strain from chicken_MO.

<p>Differences between Gene Cluster 1 and 2 demonstrated the mosaic genomic structure around <i>mutS</i> gene. Transposase and integrase were found in both sequences, indicating that both of them could be the hot spots for recombination events. The genes in both <i>S</i>. Newport SL254 and strain from chicken_MO are ordered top to bottom as their synteny on bacterial chromosome from 5′ to 3′.</p