Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology.
ABSTRACT: 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.
Project description:Whereas the O104:H4 enterohemorrhagic <i>Escherichia coli</i> (EHEC) outbreak strain from 2011 expresses aggregative adherence fimbriae of subtype I (AAF/I), its close relative, the O104:H4 enteroaggregative <i>Escherichia coli</i> (EAEC) strain 55989, encodes AAF of subtype III. Tight adherence mediated by AAF/I in combination with Shiga toxin 2 production has been suggested to result in the outbreak strain's exceptional pathogenicity. Furthermore, the O104:H4 outbreak strain adheres significantly better to cultured epithelial cells than archetypal EAEC strains expressing different AAF subtypes. To test whether AAF/I expression is associated with the different virulence phenotypes of the outbreak strain, we heterologously expressed AAF subtypes I, III, IV, and V in an AAF-negative EAEC 55989 mutant and compared AAF-mediated phenotypes, incl. autoaggregation, biofilm formation, as well as bacterial adherence to HEp-2 cells. We observed that the expression of all four AAF subtypes promoted bacterial autoaggregation, though with different kinetics. Disturbance of AAF interaction on the bacterial surface via addition of ?-AAF antibodies impeded autoaggregation. Biofilm formation was enhanced upon heterologous expression of AAF variants and inversely correlated with the autoaggregation phenotype. Co-cultivation of bacteria expressing different AAF subtypes resulted in mixed bacterial aggregates. Interestingly, bacteria expressing AAF/I formed the largest bacterial clusters on HEp-2 cells, indicating a stronger host cell adherence similar to the EHEC O104:H4 outbreak strain. Our findings show that, compared to the closely related O104:H4 EAEC strain 55989, not only the acquisition of the Shiga toxin phage, but also the acquisition of the AAF/I subtype might have contributed to the increased EHEC O104:H4 pathogenicity.
Project description:The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum ?-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic E scherichia c oli (EAHEC).
Project description:Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (blaCTX-M, blaTEM, blaSHV).The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli.While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.
Project description:In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in Germany. In 2013, E. coli O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis. All E. coli O104:H4 isolates tested were positive for stx2a, aatA, and terD but were negative for escV. All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies.
Project description:The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H-, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H-, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.
Project description:BACKGROUND: A large-scale Escherichia coli O104:H4 outbreak occurred in Germany from May to July 2011, causing numerous cases of hemolytic-uremic syndrome (HUS) and deaths. Genomes of ten outbreak isolates and a historical O104:H4 strain isolated in 2001 were sequenced using different new generation sequencing platforms. Phylogenetic analyses were performed using various approaches which either are not genome-wide or may be subject to errors due to poor sequence alignment. Also, detailed pathogenicity analyses on the 2001 strain were not available. FINDINGS: We reconstructed the phylogeny of E. coli using the genome-wide and alignment-free feature frequency profile method and revealed the 2001 strain to be the closest relative to the 2011 outbreak strain among all available E. coli strains at present and confirmed findings from previous alignment-based phylogenetic studies that the HUS-causing O104:H4 strains are more closely related to typical enteroaggregative E. coli (EAEC) than to enterohemorrhagic E. coli. Detailed re-examination of pathogenicity-related virulence factors and secreted proteins showed that the 2001 strain possesses virulence factors shared between typical EAEC and the 2011 outbreak strain. CONCLUSIONS: Our study represents the first attempt to elucidate the whole-genome phylogeny of the 2011 German outbreak using an alignment-free method, and suggested a direct line of ancestry leading from a putative EAEC-like ancestor through the 2001 strain to the 2011 outbreak strain.
Project description:<label>BACKGROUND</label>Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. The strain displays "stacked-brick" aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.<label>METHODOLOGY/PRINCIPAL FINDINGS</label>E. coli O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS) were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1%) patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (P?0.01). This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (P ?=?0.001).<label>CONCLUSIONS/SIGNIFICANCE</label>The pAA plasmid can be lost by E. coli O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.
Project description:In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC(O104), wzx(O104), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104:H4) locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR(O104:H4) PCR (99.06% specificity).
Project description:In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.
Project description:The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.