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

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

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

Using a Control to Better Understand Phyllosphere Microbiota

<div><p>An important data gap in our understanding of the phyllosphere surrounds the <i>origin</i> of the many microbes described as phyllosphere communities. Most sampling in phyllosphere research has focused on the collection of microbiota without the use of a control, so the opportunity to determine which taxa are actually driven by the biology and physiology of plants as opposed to introduced by environmental forces has yet to be fully realized. To address this data gap, we used plastic plants as inanimate controls adjacent to live tomato plants (phyllosphere) in the field with the hope of distinguishing between bacterial microbiota that may be endemic to plants as opposed to introduced by environmental forces. Using 16S rRNA gene amplicons to study bacterial membership at four time points, we found that the vast majority of all species-level operational taxonomic units were shared at all time-points. Very few taxa were unique to phyllosphere samples. A higher taxonomic diversity was consistently observed in the control samples. The high level of shared taxonomy suggests that environmental forces likely play a very important role in the introduction of microbes to plant surfaces. The observation that very few taxa were unique to the plants compared to the number that were unique to controls was surprising and further suggests that a subset of environmentally introduced taxa thrive on plants. This finding has important implications for improving our approach to the description of core phytobiomes as well as potentially helping us better understand how foodborne pathogens may become associated with plant surfaces.</p></div

Shared and Unique OTUs for Phyllosphere and Control Environments.

<p>Shared and Unique OTUs for Phyllosphere and Control Environments.</p

Unique OTUs in Control and Phyllosphere Environments.

<p>Unique OTUs in Control and Phyllosphere Environments.</p

Network relationships in Control and Phyllosphere.

<p>Computing Spearman’s correlation coefficients with associated P values, (P< 0.05) after correction using FDR for all pairwise relationships of genera in control and phyllosphere samples (using Cytoscape v3x for visualization <a href="http://www.Cytoscape.org" target="_blank">www.Cytoscape.org</a>), 21 pairwise correlations were shared (C) between control and phyllosphere samples. A total of 37 correlations were unique to phyllosphere (B) and 23 correlations were unique to controls (A). Correlations unique to the phyllosphere appear increased among members of Bacilli, and Betaproteobacteria, including genera such as <i>Ralstonia</i>, <i>Staphylococcus</i>, and <i>Arthrobacter</i>. For example, <i>Ralstonia</i> has many significant relationships in phyllosphere samples but none in controls.</p