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

Molecular Characterization of the Predominant Influenza A(H1N1)pdm09 Virus in Mexico, December 2011–February 2012

<div><p>When the A(H1N1)pdm09 pandemic influenza virus moved into the post-pandemic period, there was a worldwide predominance of the seasonal influenza A(H3N2) and B viruses. However, A(H1N1)pdm09 became the prevailing subtype in the 2011–2012 influenza season in Mexico and most of Central America. During this season, we collected nasopharyngeal swabs of individuals presenting with influenza-like illness at our institution in Mexico City. Samples were tested for seasonal A(H3N2) and B influenza viruses, as well as A(H1N1)pdm09 by real-time reverse transcription–polymerase chain reaction. Of 205 samples tested, 46% were positive to influenza, all of them A(H1N1)pdm09. The clinical characteristics of patients showed a similar pattern to the 2009 pandemic cases. Using next generation sequencing, we obtained whole genome sequences of viruses from 4 different patients, and in 8 additional viruses we performed partial Sanger sequencing of the HA segment. Non-synonymous changes found in the Mexican isolates with respect to the prototype isolate H1N1 (A/California/04/2009) included HA S69T, K163R and N260D unique to 2012 Mexican and North American isolates and located within or adjacent to HA antigenic sites; HA S143G, S185T, A197T and S203T previously reported in viruses from the 2010–2011 season, located within or adjacent to HA antigenic sites; and HA E374K located in a relevant site for membrane fusion. All Mexican isolates had an oseltamivir-sensitive genotype. Phylogenetic analysis with all 8 influenza gene segments showed that 2012 Mexican sequences formed a robust, distinct cluster. In all cases, 2012 Mexican sequences tended to group with 2010–2011 Asian and European sequences, but not with 2009 Mexican sequences, suggesting a possible recent common ancestor between these latter regions and the 2012 Mexican viruses. It remains to be defined if these viral changes represent an important antigenic drift that would enable viral immune evasion and/or affect influenza vaccine effectiveness.</p> </div

Plasmids predicted and validated in this study.

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

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

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

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

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

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

Genetic distances between Mexican 2012 isolates and viruses from all over the world.

<p>On the right side of the plot the average distance between the Mexican 2012 sequences and the sequences included for each geographical transmission zone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050116#pone.0050116-WHO3" target="_blank">[13]</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050116#pone-0050116-g003" target="_blank">Figure 3</a> is shown, for each influenza gene segment. On the left a histogram is shown with the distribution of the 20 closest sequences to the Mexican 2012 cluster for each viral segment. The four 2012 Mexican viruses sequenced by NGS are considered. A single virus from Central America and Caribbean and one from Central Asia were omitted from the distance graphs. SEA, South Eastern Asia; SWE, South Eastern Europe; EE, Eastern Europe; CA, Central Asia; OCP, Oceania, Micronesia and Polynesia; NA, North America; SA, Tempered South America; MX09, Mexican sequences from 2009 to 2011.</p