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

Extensive Gene Amplification as a Mechanism for Piperacillin-Tazobactam Resistance in Escherichia coli.

Although the TEM-1 β-lactamase (BlaTEM-1) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to β-lactam/β-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only blaTEM-1 One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher β-lactamase activity and BlaTEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in blaTEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in blaTEM-1 gene dosage, allowing BlaTEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the blaTEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the blaTEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.IMPORTANCE Understanding how bacteria acquire resistance to antibiotics is essential for treating infected patients effectively, as well as preventing the spread of resistant organisms. In this study, a clinical isolate of E. coli was identified that dedicated more than 15% of its genome toward tandem amplification of a ~10-kb resistance module, allowing it to escape antibiotic-mediated killing. Our research is significant in that it provides one possible explanation for clinical isolates that exhibit discordant behavior when tested for antibiotic resistance by different phenotypic methods. Our research also shows that GRM amplification is difficult to detect by short-read WGS technologies. Analysis of raw long-read sequence data was required to confirm GRM amplification as a mechanism of antibiotic resistance. MBio 2018 Apr 24; 9(2):e00583-18

Obscured phylogeny and possible recombinational dormancy in Escherichia coli

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>is one of the best studied organisms in all of biology, but its phylogenetic structure has been difficult to resolve with current data and analytical techniques. We analyzed single nucleotide polymorphisms in chromosomes of representative strains to reconstruct the topology of its emergence.</p> <p>Results</p> <p>The phylogeny of <it>E. coli </it>varies according to the segment of chromosome analyzed. Recombination between extant <it>E. coli </it>groups is largely limited to only three intergroup pairings.</p> <p>Conclusions</p> <p>Segment-dependent phylogenies most likely are legacies of a complex recombination history. However, <it>E. coli </it>are now in an epoch in which they no longer broadly share DNA. Using the definition of species as organisms that freely exchange genetic material, this recombinational dormancy could reflect either the end of <it>E. coli </it>as a species, or herald the coalescence of <it>E. coli </it>groups into new species.</p

Intestinal damage in enterohemorrhagic Escherichia coli infection.

Enterohemorrhagic Escherichia coli (EHEC) infection leads to marked intestinal injury. Sigmoid colon obtained from two children during EHEC infection exhibited abundant TUNEL-positive cells. To define which bacterial virulence factors contribute to intestinal injury the presence of Shiga toxin-2 (Stx2), intimin and the type III secretion system were correlated with symptoms and intestinal damage. C3H/HeN mice were inoculated with Stx2-producing (86-24) and non-producing (87-23) E. coli O157:H7 strains and 86-24 mutants lacking eae, encoding intimin (strain UMD619) or escN regulating the expression of type III secretion effectors (strain CVD451). Severe symptoms developed in mice inoculated with 86-24 and 87-23. Few mice inoculated with the mutant strains developed severe symptoms. Strain 86-24 exhibited higher fecal bacterial counts, followed by 87-23, whereas strains UMD619 and CVD451 showed minimal fecal counts. More TUNEL-positive cells were found in proximal and distal colons of mice inoculated with strain 86-24 compared with strains 87-23 and CVD451 (p </= 0.01) or UMD619 (p < 0.05, proximal colon, p < 0.01, distal colon). The results show that strains 86-24 and 87-23 exhibited better colonic persistence and more symptoms, presumably due to the presence of intimin and type III secretion effectors. Extensive intestinal mucosal cell death was related to the presence of Stx2

Lineage and Genogroup-Defining Single Nucleotide Polymorphisms of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP)-based typing panel was developed that redundantly identified 11 genogroups that span six of the eight lineages recently described for E. coli O157:H7 (J. L. Bono, T. P. Smith, J. E. Keen, G. P. Harhay, T. G. McDaneld, R. E. Mandrell, W. K. Jung, T. E. Besser, P. Gerner-Smidt, M. Bielaszewska, H. Karch, M. L. Clawson, Mol. Biol. Evol. 29:2047–2062, 2012) and additionally defined subgroups within four of those lineages. This assay was applied to 530 isolates from human and bovine sources. The SNP-based lineage groups were concordant with previously identified E. coli O157:H7 genotypes identified by other methods and were strongly associated with carriage of specific Stx genes. Two SNP lineages (Ia and Vb) were disproportionately represented among cattle isolates, and three others (IIa, Ib, and IIb) were disproportionately represented among human clinical isolates. This 48-plex SNP assay efficiently and economically identifies biologically relevant lineages within E. coli O157:H7