Wenlan Zhang

Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms

Shiga toxin (Stx)-producing Escherichia coli (STEC) from patients with hemolytic-uremic syndrome (HUS), patients with diarrhea without HUS, or asymptomatic subjects were genotyped to assess associations between stx2 variants and clinical manifestations of infection. Neither stx2d nor stx2e was found in 268 STEC isolates from patients with HUS. Of 262 STEC isolates from patients with diarrhea, stx(2d) was found in 41 (15.6%; P<.000001), and stx2e was found in 12 (4.6%; P=.0004). The stx2c genotype frequency was similar among isolates from patients with HUS (3.7%) and diarrhea (5.0%). The frequencies of stx2c, stx2d, and stx2e among 96 STEC isolates from asymptomatic subjects were comparable to those among isolates from patients with diarrhea. None of the 626 STEC isolates contained stx2f. All stx2d-positive or stx2e-positive STEC isolates were eae negative and originated from subjects older than those with STEC isolates with stx2c. stx2c-positive STEC isolates can cause HUS, but the presence of stx2d or stx2e may predict a milder disease with a minimal risk of HUS.

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.

Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study

BACKGROUND In an ongoing outbreak of haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany (with some cases elsewhere in Europe and North America), 810 cases of the syndrome and 39 deaths have occurred since the beginning of May, 2011. We analysed virulence profiles and relevant phenotypes of outbreak isolates recovered in our laboratory. METHODS We analysed stool samples from 80 patients that had been submitted to the National Consulting Laboratory for Haemolytic Uraemic Syndrome in Münster, Germany, between May 23 and June 2, 2011. Isolates were screened with standard PCR for virulence genes of Shiga-toxin-producing E coli and a newly developed multiplex PCR for characteristic features of the outbreak strain (rfb(O104), fliC(H4), stx(2), and terD). Virulence profiles of the isolates were determined with PCR targeting typical virulence genes of Shiga-toxin-producing E coli and of other intestinal pathogenic E coli. We sequenced stx with Sanger sequencing and measured Shiga-toxin production, adherence to epithelial cells, and determined phylogeny and antimicrobial susceptibility. FINDINGS All isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx(2), iha, lpf(O26), lpf(O113)) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum β-lactamase phenotype absent in HUSEC041. INTERPRETATION Augmented adherence of the strain to intestinal epithelium might facilitate systemic absorption of Shiga toxin and could explain the high progression to haemolytic uraemic syndrome. This outbreak demonstrates that blended virulence profiles in enteric pathogens, introduced into susceptible populations, can have extreme consequences for infected people. FUNDING German Federal Ministry of Education and Research, Network Zoonoses.

Genetic Diversity of Intimin Genes of Attaching and Effacing Escherichia coli Strains

ABSTRACT In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-η (eta), eae-ι (iota), and eae-κ (kappa). The fourth was identical to the recently described eae-ζ (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-α, intimin-β, intimin-γ1, intimin-γ2, intimin-ε, and intimin-θ alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3′ region (starting at bp 2112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: α and ζ; β and κ; γ1 and γ2/θ; and ε and η. Calculation of homoplasy ratios of sequences of the 5′ region of eae (positions 1 to 2111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.

Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans

ABSTRACT By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant “Stx1c” and developed stxB1 restriction fragment length polymorphism and stx1c-specific PCR strategies to determine the frequency and distribution of stx1c among 212 STEC strains isolated from humans. stx1c was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx1c was most frequently (in 23 STEC strains [63.9%]) associated with stx2d, but 12 (33.3%) of the 36 STEC strains possessed stx1c only. A single STEC strain possessed stx1c together with stx2 and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx1c-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx1c-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx1c variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx1c among clinical STEC isolates and their association with human disease in prospective studies.

Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

ABSTRACT Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

Genetic Structure and Distribution of the Colibactin Genomic Island among Members of the Family Enterobacteriaceae

ABSTRACT A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

ABSTRACT The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx2-harboring bacteriophages, stx2 transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased stx2-harboring phage induction and Stx2 production in outbreak isolates (P values of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P > 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P ≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulated stx2 transcription, respectively (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induced stx2-harboring phages nor increased stx2 transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

Phenotypic and Genotypic Analyses of Enterohemorrhagic Escherichia coli O145 Strains from Patients in Germany

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H−). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLPH28 pattern harbored eae γ, whereas the three strains with the fliC-RFLPH25 pattern possessed eae β. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.

Prevalence, Virulence Profiles, and Clinical Significance of Shiga Toxin–Negative Variants of Enterohemorrhagic Escherichia coli O157 Infection in Humans

BACKGROUND Escherichia coli O157, of the H7 clone, exists in humans and in the environment as Shiga toxin (Stx)-positive and Stx-negative variants. Stx production by infecting organisms is considered to be a critical requirement for the development of hemolytic uremic syndrome (HUS), which occurs in approximately 15% of E. coli O157-infected patients. It is unknown if loss of the stx gene during the early stage of an enterohemorrhagic E. coli infection prevents HUS, or if absence of the stx gene from E. coli O157 reduces or ablates virulence. METHODS We determined the frequency of stx-positive and stx-negative E. coli O157 isolates in stool samples obtained from patients who experienced sporadic cases of diarrhea or HUS, as well as the frequency in samples obtained during outbreaks, and investigated the clinical course of the disease. RESULTS Among E. coli O157 isolates obtained from samples related to sporadic cases of diarrhea, stx-negative strains accounted for 4%. The proportion of stx-negative strains was significantly higher among sorbitol-fermenting, nonmotile E. coli O157 isolates (12.7%) than among non-sorbitol-fermenting E. coli O157:H7 or nonmotile isolates (0.8%; P<.001). stx-Negative sorbitol-fermenting E. coli O157 isolates were also observed in samples related to 3 HUS outbreaks and 1 outbreak of diarrhea caused by sorbitol-fermenting, nonmotile enterohemorrhagic E. coli O157; additionally, they were the only pathogens that were isolated in 2 other outbreaks of diarrhea without HUS. CONCLUSIONS Strains of stx-negative E. coli O157 isolated from stool samples of patients are either inherently stx-negative strains that cause mostly uncomplicated diarrhea, or strains that descended from enterohemorrhagic E. coli O157 by the loss of the stx gene during infection; the latter strains may still cause severe disease.