Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains

Identification of a VAh-specific genetic region associated with prophage AH4 that is induced after mitomycin C treatment.

<p>(Panel A) Mauve multiple genome alignment of the prophage A4 region from AH strains MN98-04 and AL97-91 with VAh strain ML09-119, depicting the two upstream ORFs (in red) and two within-prophage regions (shown by lack of Mauve alignment) that are VAh-associated. (Panel B) Induced phage DNAs were subjected to 454 pyrosequencing and were reference mapped against the AH4 prophage region of the <i>A. hydrophila</i> Ml09-119 genome. Each predicted ORF is indicated as an arrow, and the four VAH-associated ORFs are depicted in red. </p

Schematic organization of the four different types of O-antigen biosynthesis gene cluster present within the genome of the 11 <i>A. hydrophila</i> isolates sequenced in this study.

<p>All of the genes on the cluster are transcribed in the same direction. All VAh strains along with RAh strain TN97-08 shared the ML09-119-type O-antigen biosynthesis gene cluster. This cluster encodes proteins predicted to be involved in the biosynthesis of the nucleotide sugars D-rhamnose, D-mannose, D-Fucose, and 3-acetamido-3, 6-dideoxy-d-galactose (D-Fuc<i>p</i>3NAc). The AL97-91-type cluster (that was also shared with MN98-04) encodes genes predicted to be required for S-layer biosynthesis and transport in addition to O-antigen biosynthesis. Genes that encode conserved proteins with similar functions are marked with the same color. The number displayed next to the maps indicates the nucleotide positions on the respective contig from each strain. The designation of each of the genes presented on the schematic map of the AL06-01, ML09-119, AL06-06 and AL97-91 O-antigen clusters are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080943#pone-0080943-t002" target="_blank">Tables 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080943#pone-0080943-t004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080943#pone-0080943-t005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080943#pone-0080943-t006" target="_blank">6</a>, respectively.</p

Evolutionary relationships of 37 <i>A. hydrophila</i> taxa based on <i>gyrB</i> gene sequences (out of a larger dataset of 107 <i>A. hydrophila</i><i>gyrB</i> sequences).

<p>The evolutionary history was inferred using the Maximum Parsimony method. Tree #1 out of 67 most parsimonious trees (length = 218) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths calculated using the average pathway method and are in the units of the number of changes over the whole sequence. </p

Genetic elements involved in <i>myo-</i>inositol utilization in VAh strains.

<p>The schematic organization depicts the cluster of genes involved in <i>myo</i>-inositol utilization in epidemic <i>A. hydrophila</i> ML09-119. The presence of a functional <i>myo</i>-inositol utilization pathway in VAh strains was confirmed by their ability to grow on <i>myo</i>-inositol as a sole carbon source.</p

Mauve Alignment of the O-antigen cluster from 16 different <i>A. hydrophila</i> isolates.

<p>Segments with a similar color indicate homologous regions. The alignment is on scale based on the size of the O-antigen cluster of <i>A. hydrophila</i> ATCC 7966 which is 44 kb in length. All of the EAh strains including RAh strain TN97-08 shared the ML09-119-type O-antigen biosynthesis gene cluster.</p

Pan and core-genome plot of 11 different <i>A. hydrophila</i> isolates.

<p>The red and blue lines indicated the number of genes within the core and pan-genomes, respectively. The <i>A. hydrophila</i> core genome contained 3,511 core genes whereas the <i>A. hydrophila</i> pan-genome contained 6,856 genes. Note that the addition of other VAh strains after <i>A. hydrophila</i> ML09-119 did not significantly increase the number of new gene families, which was in agreement with the highly clonal nature of VAh strains. </p