Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak

\u3ci\u3eFrancisella tularensis\u3c/i\u3e Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96- 3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that F. tularensis A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving F. tularensis genetic diversity and plasticity is needed

Reclassification of Wolbachia persica as Francisella persica comb. nov and emended description of the family Francisellaceae

The taxonomic status of the bacterium Wolbachia persica is described, and based on the evidence presented, transfer of this species to the genus Francisella as Francisella persica comb. nov. is proposed. This reclassification is supported by data generated from genomic comparisons of W. persica ATCC VR-331(T) (=FSC845(T)=DSM 101678(T)) to other near neighbours, including Francisella tularensis subsp. novicida. The full-length 16S rRNA gene sequence of strain ATCC VR-331(T) had 98.5 % nucleotide identity to the cognate gene in F. tularensis, with the highest similarity to subspecies novicida. Phylogenetic trees of full-length 16S rRNA gene, gyrA and recA sequences from species of the genera Wolbachia (class Alphaproteobacteria) and Francisella (class Gammaproteobacteria) indicated that W. persica ATCC VR-331(T) was most closely related to members of the genus Francisella and not Wolbachia. Local collinear blocks within the chromosome of strain ATCC VR-331(T) had considerable similarity with F. tularensis subsp. novicida, but not with any Wolbachia strain. The genomes of strain ATCC VR-331(T) and F. tularensis subsp. novicida Utah 112(T) (=ATCC 15482(T)) contained an average nucleotide identity mean of 88.72 % and median of 89.18 %. Importantly, the genome of strain ATCC VR-331(T) contained one Francisella Pathogenicity Island, similar to F. tularensis subsp. novicida, as well as the Francisella-specific gene fopA1 and F. tularensis-specific genes fopA2 and lpnA (also referred to as tul4). In contrast to the obligate intracellular genus Wolbachia, strain ATCC VR-331(T) and facultative intracellular Francisella can replicate in specialized cell-free media. Collectively, these results demonstrate that Wolbachia persica should be reclassified in the genus Francisella as Francisella persica comb. nov. The type strain of Francisella persica comb. nov. is ATCC VR-331(T) (=FSC845(T)=DSM 101678(T)). An emended description of the family Francisellaceae is also provided

<i>Francisella tularensis</i> Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

<div><p>Although <i>Francisella tularensis</i> is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the <i>tularensis</i> subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed <i>F</i>. <i>tularensis</i> type A genomes. In contrast with the <i>F</i>. <i>tularensis</i> A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced <i>F</i>. <i>tularensis</i> A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that <i>F</i>. <i>tularensis</i> A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving <i>F</i>. <i>tularensis</i> genetic diversity and plasticity is needed.</p></div

Whole genome mapping of <i>F</i>. <i>tularensis</i> subtype A.II strains WY-00W4114 and WY96-3418.

<p><i>Nco</i>I (A) and <i>Nhe</i>I (B) whole genome maps of <i>F</i>. <i>tularensis</i> WY-00W4114 (top linearized chromosome) compared to the corresponding theoretical <i>in silico</i> digestion of <i>F</i>. <i>tularensis</i> WY96-3418 (GenBank accession number CP000608, bottom linearized chromosome). Vertical lines within the genome maps denote the restriction endonuclease sites for <i>Nco</i>I (A) or <i>Nhe</i>I (B). Lines connecting the chromosomal restriction maps of WY-00W4114 and WY96-3418 and the adjacent unshaded genomic areas denote translocated regions.</p

Genome alignment of <i>F</i>. <i>tularensis</i> A.I and A.II strains.

<p>Chromosomal alignments of representative <i>F</i>. <i>tularensis</i> A.I strains SCHU S4, NE061598, FSC198, and TI0902 (A); pairwise genome alignment of the <i>F</i>. <i>tularensis</i> A.II strains WY-00W4114 and WY96-3418 (B); and multiple chromosomal alignment of the <i>F</i>. <i>tularensis</i> A.I and A.II strains sequenced to completion and shown in panels A and B <b>(C)</b>. The relative location of the <i>Francisella</i> pathogenicity island (FPI) in duplicated region 1 (DR1) and duplicated region 2 (DR2) is identified with a bar above the associated chromosomal region for each subtype. The progressiveMauve software tool was used to align the genomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124906#pone.0124906.ref021" target="_blank">21</a>].</p

Nucleotide polymorphisms in genome of WY-00W4114 compared to WY96-3418 and overall base pair size increase in WY-00W4114.

<p>Nucleotide polymorphisms in genome of WY-00W4114 compared to WY96-3418 and overall base pair size increase in WY-00W4114.</p

Description and nucleotide position of locally collinear blocks (LCBs) within A.II strains WY-00W4114 and WY96-3418.

<p>Description and nucleotide position of locally collinear blocks (LCBs) within A.II strains WY-00W4114 and WY96-3418.</p

Diagram illustrating GC skew within chromosomal topology map for <i>F</i>. <i>tularensis</i> A.I and A.II strains.

<p>The circular <i>F</i>. <i>tularensis</i> chromosome of subtype A.I strains are represented by SCHU S4 (A) and NE061598 (B), and WY96-3418 (C) and WY-00W4114 (D) represent the subtype A.II strains. The origin (<i>ori</i>) and termination (<i>ter</i>) region are denoted by a vertical black line at the top and bottom, respectively, of the corresponding chromosomal map. GC skew + (gray) and GC skew—(black) is shown in the outermost circle for each genome and the kilobase pair position is indicated in the innermost circle.</p

Diagram depicting large rearrangements of locally collinear blocks (LCBs) within <i>F</i>. <i>tularensis</i> A.II strains.

<p><i>F</i>. <i>tularensis</i> A.II strains WY-00W4114 and WY96-3418 chromosomal comparison showing related LCBs (A) and potential recombination events with a two-step parsimonious molecular process (B). Each LCB is represented with a different pattern and/or shading. Directionality of the LCBs is depicted with an arrow and is based on the reference strain WY96-3418 (GenBank accession number CP000608). Nucleotide positions are denoted in kilobase pairs by the corresponding genome.</p