Identification and Characterization of Novel <em>Salmonella</em> Mobile Elements Involved in the Dissemination of Genes Linked to Virulence and Transmission

<div><p>The genetic diversity represented by >2,500 different <em>Salmonella</em> serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of <em>Salmonella</em> mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in <em>Salmonella</em>, we used prediction algorithms to screen for mobile elements in 16 sequenced <em>Salmonella</em> genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different <em>Salmonella</em> serovars and contained a virulence gene array that had not been previously identified. A <em>Salmonella</em> Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal <em>Salmonella</em> serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in <em>Salmonella</em>, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among <em>Salmonella</em> serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.</p> </div

Linear representations of three phage genomes identified to have virulence genes.

<p>At the top is phage PhInv-1b in <i>S.</i> Inverness, the middle is phage PhUga-3 in <i>S.</i> Uganda and in the bottom is PhGam-1 in <i>S.</i> Gaminara. Genes were color coded according to function as follows: virulence (red), replication (yellow), phage structural genes (green), metabolism (purple), and hypothetical protein (grey).</p

Phylogeny inferred with maximum likelihood of the IncI1 replicon.

<p>Maximum likelihood phylogeny conducted with IncI1 sequences for plasmids found in this study and currently available sequences. Analysis was conducted with RAxML. Two clades were observed (clade I and clade II), with clade I containing the <i>Salmonella</i> plasmids analyzed here.</p

Isolates positive for type IVb pili, and/or IncI1-IncFIB replicons.

1<p>Isolates used for invasion assay are marked with ^a, isolates with completed whole genome sequences are marked with ^b, isolates were IncFIB and IncI1 replicons were identified by PCR and sequencing are marked with ^c (which could indicate the presence of both an IncI1 and an IncFIB plasmids or the presence of a cointegrated plasmid).</p

Characteristics of SGI2 variants.

1<p>SGI2 and SGI2.6 share the same type I restriction modification system.</p>2<p>SGI2.1 and SGI2.5 share the same type II restriction modification system.</p><p>RM: restriction modification system.</p

Plasmids predicted and validated in this study.

<p>Plasmids predicted and validated in this study.</p

Comparison, made using the Blast algorithm, of SPI-7 in <i>S.</i> Typhi, ICES1 identified in this study, and previously sequenced ICE.

<p>At the top is SPI-7 of <i>S.</i> Typhi with its three main regions (i.e., type IVb pilus operon, SopEΦ phage and capsular operon). Black arrows indicate the position of the primers used to validate the presence of the type IVb pilus operon in ICES1 in <i>S.</i> Rubislaw, <i>S.</i> Inverness and <i>S.</i> Urbana. Blue arrows are the coding regions and grey shaded are regions that present homology.</p

Circular representation of IncI1-IncFIB cointegrated plasmids.

<p>Circular representations of IncI1-IncFIB plasmids identified in <i>Salmonella</i> serovars Urbana pR8-2977 (inner circle), Inverness pR8-3668 (second circle) and Mississippi pA4-633 (third circle). Plasmids were aligned against the Urbana plasmid. Gene cluster (1) represents insertions in the accessory region in plasmids in serovars Mississippi and Inverness relative to the <i>S</i>. Urbana plasmid. Gene cluster (2) represents insertions in plasmids in serovars Mississippi and Inverness relative to the <i>S</i>. Urbana plasmid. For plasmid in serovar Rubislaw pA4-653, there is a 90 kb insertion (3) that differentiates this plasmid from other plasmids (outer cluster of genes), and encodes several putative virulence genes, arrows indicate the insertion location for this region in plasmid pA4-653. Genes were color coded according to function as follows: virulence (red), plasmid transfer (blue), transposition/IS (yellow), replication (green), plasmid stability (turquoise), metabolism (purple), phage origin proteins (brown), antibiotic production (black), hypothetical proteins (grey).</p

Circular representation of plasmid pS5-403-1 identified in <i>Salmonella</i> Montevideo.

<p>(A) Circular representation of pS5-403-1. Circles from inside to outside: the inner circle represents the GC%, the second circle is pS5403-1, a 53 kb plasmid that encodes antimicrobial resistance genes, the outer circle represents the shared backbone with p271A plasmid in <i>E. coli</i>. The curly bracket represents a cluster of genes in a transposon insertion in p271A that includes the NDM-β-lactamase. (B) Cluster of genes representing a comparison of a class 1 integron in pS5-403-1 homologous to an integron inserted in pSN254 (plasmid in <i>S.</i> Newport). Genes were color coded according to function as follows: resistance (pink), plasmid transfer (blue), transposition/IS (orange), replication (green), plasmid stability (turquoise), metabolism (brown), and hypothetical proteins (grey).</p

Comparison, made using the Blast algorithm, of PSP3-like and P22-like phages.

<p>(A) Comparison of six phages that resemble PSP3; phages were identified in serovars Uganda, Johannesburg, Adelaide, Gaminara, Montevideo, and Urbana. Coding regions are represented as blue arrows and regions of homology are shaded in grey. (B) Comparison of six phages that resemble P22, identified in serovars Wandsworth, Mississippi, Rubislaw, Uganda, Johannesburg, and Montevideo. Coding regions are represented as orange arrows and regions of homology are shaded in grey.</p