A Comparison of Shiga-Toxin 2 Bacteriophage from Classical Enterohemorrhagic Escherichia coli Serotypes and the German E. coli O104:H4 Outbreak Strain

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

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PCR Detection of Enterohemorrhagic Escherichia coli O145 in Food by Targeting Genes in the E. coli O145 O-Antigen Gene Cluster and the Shiga Toxin 1 and Shiga Toxin 2 Genes

Foodborne Pathogens and Disease ◽

10.1089/fpd.2008.0254 ◽

2009 ◽

Vol 6 (5) ◽

pp. 605-611 ◽

Cited By ~ 42

Author(s):

Pina M. Fratamico ◽

Chitrita DebRoy ◽

Takahisa Miyamoto ◽

Yanhong Liu

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Shiga Toxin ◽

Pcr Detection ◽

Enterohemorrhagic Escherichia Coli ◽

E Coli ◽

Antigen Gene ◽

O Antigen ◽

Shiga Toxin 2

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Human Microbiota-Secreted Factors Inhibit Shiga Toxin Synthesis by Enterohemorrhagic Escherichia coli O157:H7

Infection and Immunity ◽

10.1128/iai.01048-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 783-790 ◽

Cited By ~ 74

Author(s):

Thibaut de Sablet ◽

Christophe Chassard ◽

Annick Bernalier-Donadille ◽

Marjolaine Vareille ◽

Alain P. Gobert ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Factor ◽

Shiga Toxin ◽

Enterohemorrhagic Escherichia Coli ◽

Lytic Cycle ◽

E Coli ◽

Human Microbiota ◽

Shiga Toxin 2 ◽

Uremic Syndrome ◽

Serious Disease

ABSTRACT Escherichia coli O157:H7 is a food-borne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome, especially in children. The main virulence factor responsible for the more serious disease is the Shiga toxin 2 (Stx2), which is released in the gut after oral ingestion of the organism. Although it is accepted that the amount of Stx2 produced by E. coli O157:H7 in the gut is critical for the development of disease, the eukaryotic or prokaryotic gut factors that modulate Stx2 synthesis are largely unknown. In this study, we examined the influence of prokaryotic molecules released by a complex human microbiota on Stx2 synthesis by E. coli O157:H7. Stx2 synthesis was assessed after growth of E. coli O157:H7 in cecal contents of gnotobiotic rats colonized with human microbiota or in conditioned medium having supported the growth of complex human microbiota. Extracellular prokaryotic molecules produced by the commensal microbiota repress stx 2 mRNA expression and Stx2 production by inhibiting the spontaneous and induced lytic cycle mediated by RecA. These molecules, with a molecular mass of below 3 kDa, are produced in part by Bacteroides thetaiotaomicron, a predominant species of the normal human intestinal microbiota. The microbiota-induced stx 2 repression is independent of the known quorum-sensing pathways described in E. coli O157:H7 involving SdiA, QseA, QseC, or autoinducer 3. Our findings demonstrate for the first time the regulatory activity of a soluble factor produced by the complex human digestive microbiota on a bacterial virulence factor in a physiologically relevant context.

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Chromosomal Dynamism in Progeny of Outbreak-Related Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Applied and Environmental Microbiology ◽

10.1128/aem.72.3.1900-1909.2006 ◽

2006 ◽

Vol 72 (3) ◽

pp. 1900-1909 ◽

Cited By ~ 43

Author(s):

Martina Bielaszewska ◽

Rita Prager ◽

Wenlan Zhang ◽

Alexander W. Friedrich ◽

Alexander Mellmann ◽

...

Keyword(s):

Escherichia Coli ◽

Enterohemorrhagic Escherichia Coli ◽

Outbreak Strain ◽

E Coli ◽

Shiga Toxin 2 ◽

Uremic Syndrome ◽

Sporadic Cases ◽

Different Strains ◽

K 12

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx 2) in the outbreak strain or with the loss of this gene. The two stx 2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx 2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.

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Enterohemorrhagic Escherichia coli Colony Check Assay for the Identification of Serogroups O26, O45, O103, O111, O121, O145, and O157 Colonies Isolated on Plating Media

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-555 ◽

2014 ◽

Vol 77 (7) ◽

pp. 1212-1218 ◽

Cited By ~ 1

Author(s):

BURTON BLAIS ◽

MYLÈNE DESCHÊNES ◽

GEORGE HUSZCZYNSKI ◽

MARTINE GAUTHIER

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

High Throughput Screening ◽

Enterohemorrhagic Escherichia Coli ◽

Test Results ◽

Bacterial Strains ◽

Enrichment Broth ◽

Substrate Solution ◽

E Coli ◽

Assay Performance

A simple immunoenzymatic enterohemorrhagic Escherichia coli (EHEC) colony check (ECC) assay was developed for the presumptive identification of priority EHEC colonies isolated on plating media from enrichment broth cultures of foods. With this approach, lipopolysaccharide extracted from a colony is spotted on the grid of a polymyxin-coated polyester cloth strip, and bound E. coli serogroup O26, O45, O103, O111, O121, O145, and O157 antigens are subsequently detected by sequential reactions with a pool of commercially available peroxidase-conjugated goat antibodies and tetramethylbenzidine substrate solution. Each strip can accommodate up to 15 colonies, and test results are available within 30 min. Assay performance was verified using colonies from a total of 73 target EHEC isolates covering the range of designated priority serogroups (all of which were reactive), 41 nontarget E. coli isolates including several nontarget Shiga toxin–producing E. coli serogroups (all unreactive), and 33 non–E. coli strains (all unreactive except two bacterial strains possessing O-antigenic structures in common with those of the priority EHEC). The ECC assay was reactive with target colonies grown on several types of selective and nonselective plating media designed for their cultivation. These results support the use of the ECC assay for high-throughput screening of colonies isolated on plating media for detecting priority EHEC strains in foods.

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Outbreak of Shiga Toxin–Producing Escherichia coli (STEC) O104:H4 Infection in Germany Causes a Paradigm Shift with Regard to Human Pathogenicity of STEC Strains

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-452 ◽

2012 ◽

Vol 75 (2) ◽

pp. 408-418 ◽

Cited By ~ 151

Author(s):

LOTHAR BEUTIN ◽

ANNETT MARTIN

Keyword(s):

Escherichia Coli ◽

Hemolytic Uremic Syndrome ◽

Shiga Toxin ◽

Outbreak Strain ◽

Fenugreek Seeds ◽

E Coli ◽

Aggregative Adherence ◽

Uremic Syndrome ◽

Enterocyte Effacement ◽

The Way

An outbreak that comprised 3,842 cases of human infections with enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 occurred in Germany in May 2011. The high proportion of adults affected in this outbreak and the unusually high number of patients that developed hemolytic uremic syndrome makes this outbreak the most dramatic since enterohemorrhagic E. coli (EHEC) strains were first identified as agents of human disease. The characteristics of the outbreak strain, the way it spread among humans, and the clinical signs resulting from EAHEC infections have changed the way Shiga toxin–producing E. coli strains are regarded as human pathogens in general. EAHEC O104:H4 is an emerging E. coli pathotype that is endemic in Central Africa and has spread to Europe and Asia. EAHEC strains have evolved from enteroaggregative E. coli by uptake of a Shiga toxin 2a (Stx2a)–encoding bacteriophage. Except for Stx2a, no other EHEC-specific virulence markers including the locus of enterocyte effacement are present in EAHEC strains. EAHEC O104:H4 colonizes humans through aggregative adherence fimbrial pili encoded by the enteroaggregative E. coli plasmid. The aggregative adherence fimbrial colonization mechanism substitutes for the locus of enterocyte effacement functions for bacterial adherence and delivery of Stx2a into the human intestine, resulting clinically in hemolytic uremic syndrome. Humans are the only known natural reservoir known for EAHEC. In contrast, Shiga toxin–producing E. coli and EHEC are associated with animals as natural hosts. Contaminated sprouted fenugreek seeds were suspected as the primary vehicle of transmission of the EAHEC O104:H4 outbreak strain in Germany. During the outbreak, secondary transmission (human to human and human to food) was important. Epidemiological investigations revealed fenugreek seeds as the source of entry of EAHEC O104:H4 into the food chain; however, microbiological analysis of seeds for this pathogen produced negative results. The survival of EAHEC in seeds and the frequency of human carriers of EAHEC should be investigated for a better understanding of EAHEC transmission routes.

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Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00937-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 19

Author(s):

Andreas Bauwens ◽

Lisa Kunsmann ◽

Helge Karch ◽

Alexander Mellmann ◽

Martina Bielaszewska

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Shiga Toxin ◽

Membrane Vesicles ◽

Polymyxin B ◽

Outer Membrane Vesicles ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

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Enterohemorrhagic Escherichia coli O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades

PLoS ONE ◽

10.1371/journal.pone.0191834 ◽

2018 ◽

Vol 13 (1) ◽

pp. e0191834 ◽

Cited By ~ 2

Author(s):

Shinichiro Hirai ◽

Eiji Yokoyama ◽

Taku Wakui ◽

Taichiro Ishige ◽

Masaki Nakamura

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Enterohemorrhagic Escherichia Coli ◽

Escherichia Coli O157 ◽

Chiba Prefecture ◽

Shiga Toxin 2

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Contribution of the Twin Arginine Translocation System to the Virulence of Enterohemorrhagic Escherichia coli O157:H7

Infection and Immunity ◽

10.1128/iai.71.9.4908-4916.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 4908-4916 ◽

Cited By ~ 70

Author(s):

Nathalie Pradel ◽

Changyun Ye ◽

Valérie Livrelli ◽

Jianguo Xu ◽

Bernard Joly ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Immunoblot Analysis ◽

Vero Cells ◽

Soft Agar ◽

Enterohemorrhagic Escherichia Coli ◽

E Coli ◽

Twin Arginine Translocation ◽

Tat System ◽

K 12

ABSTRACT Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB 1 genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA1 and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02937-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3144-3150 ◽

Cited By ~ 140

Author(s):

Martina Bielaszewska ◽

Rita Prager ◽

Robin Köck ◽

Alexander Mellmann ◽

Wenlan Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Gene Loss ◽

Pulsed Field Gel Electrophoresis ◽

Enterohemorrhagic Escherichia Coli ◽

Toxin Gene ◽

Shiga Toxins ◽

E Coli

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 stx 2 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.

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