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 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

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

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

Average genome size, guanine-cytosine (GC) content, and number of contigs and average number of extra-chromosomal genes, virulence, disease and defense genes and plasmids of enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype (<i>stx</i>/<i>eae</i> negative) <i>Escherichia coli</i> O103 strains of bovine and human origin.

<p>Average genome size, guanine-cytosine (GC) content, and number of contigs and average number of extra-chromosomal genes, virulence, disease and defense genes and plasmids of enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype (<i>stx</i>/<i>eae</i> negative) <i>Escherichia coli</i> O103 strains of bovine and human origin.</p

Proportional branch transformed phylogenetic tree^† of 75 strains of enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype (<i>stx</i>/<i>eae</i> negative) <i>Escherichia coli</i> O103 of bovine and human origin using FigTree 1.4.

<p>^†Numbers on the branches correspond to bootstrap values.</p

Comparative genomics reveals differences in mobile virulence genes of <i>Escherichia coli</i> O103 pathotypes of bovine fecal origin

<div><p><i>Escherichia coli</i> O103, harbored in the hindgut and shed in the feces of cattle, can be enterohemorrhagic (EHEC), enteropathogenic (EPEC), or putative non-pathotype. The genetic diversity particularly that of virulence gene profiles within O103 serogroup is likely to be broad, considering the wide range in severity of illness. However, virulence descriptions of the <i>E</i>. <i>coli</i> O103 strains isolated from cattle feces have been primarily limited to major genes, such as Shiga toxin and intimin genes. Less is known about the frequency at which other virulence genes exist or about genes associated with the mobile genetic elements of <i>E</i>. <i>coli</i> O103 pathotypes. Our objective was to utilize whole genome sequencing (WGS) to identify and compare major and putative virulence genes of EHEC O103 (positive for Shiga toxin gene, <i>stx</i>1, and intimin gene, <i>eae;</i> n = 43), EPEC O103 (negative for <i>stx</i>1 and positive for <i>eae</i>; n = 13) and putative non-pathotype O103 strains (negative for <i>stx</i> and <i>eae;</i> n = 13) isolated from cattle feces. Six strains of EHEC O103 from human clinical cases were also included. All bovine EHEC strains (43/43) and a majority of EPEC (12/13) and putative non-pathotype strains (12/13) were O103:H2 serotype. Both bovine and human EHEC strains had significantly larger average genome sizes (<i>P</i> < 0.0001) and were positive for a higher number of adherence and toxin-based virulence genes and genes on mobile elements (prophages, transposable elements, and plasmids) than EPEC or putative non-pathotype strains. The genome size of the three pathotypes positively correlated (R² = 0.7) with the number of genes carried on mobile genetic elements. Bovine strains clustered phylogenetically by pathotypes, which differed in several key virulence genes. The diversity of <i>E</i>. <i>coli</i> O103 pathotypes shed in cattle feces is likely reflective of the acquisition or loss of virulence genes carried on mobile genetic elements.</p></div

Multiple genome comparison of representative strains of enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype (<i>stx</i>/<i>eae</i> negative) <i>Escherichia coli</i> O103 strains of bovine and human origin using BLAST Ring Image Generator (BRIG) v0.95.

<p>^†The nucleotide sequence (45,325 bp) of the locus of enterocyte effacement (LEE) pathogenicity island (GenBank accession no.: AF071034.1) was mapped for comparison of LEE between target strains.</p