scholarly journals STEC-EPEC Oligonucleotide Microarray: A New Tool for Typing Genetic Variants of the LEE Pathogenicity Island of Human and Animal Shiga Toxin–Producing Escherichia coli (STEC) and Enteropathogenic E. coli (EPEC) Strains

2006 ◽  
Vol 52 (2) ◽  
pp. 192-201 ◽  
Author(s):  
Patricia Garrido ◽  
Miguel Blanco ◽  
Mercedes Moreno-Paz ◽  
Carlos Briones ◽  
Ghizlane Dahbi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Variants ◽  
Pathogenicity Island ◽  
Oligonucleotide Microarray ◽  
Chromosomal Dna ◽  
Emerging Pathogens ◽  
E Coli ◽  
Enterocyte Effacement ◽  
Gene 4

Abstract Background: Shiga toxin–producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are important emerging pathogens that can cause a severe and sometimes fatal illness. Differentiation of eae, tir, espA, espD, and espB gene variants of the locus of enterocyte effacement (LEE) pathogenicity island represents an important tool for typing in routine diagnostics as well as in pathogenesis, epidemiologic, clonal, and immunologic studies. Methods: Type-specific oligonucleotide microarrays and a PCR scheme were designed and constructed for the detection and typing of genetic variants of the LEE genes. Oligonucleotide probes were tested for their specificity against the corresponding type strain by microarray hybridization using fluorescent DNA, either PCR-amplified (single, multiplex, long-range), chromosomal, or amplified chromosomal DNA. Results: The PCR scheme and the oligonucleotide microarray allowed us to distinguish 16 variants (α1, α2, β1, β2, γ1, γ2/θ, δ/κ, ε, ζ, η, ι, λ, μ, ν, ξ, ο) of the eae gene, 4 variants (α1, β1, γ1, γ2/θ) of the tir gene, 4 variants (α1, β1, β2, γ1) of the espA gene, 3 variants (α1, β1, γ1) of the espB gene, and 3 variants (α1, β1, γ1) of the espD gene. We found a total of 12 different combinations of tir, espA, espB, and espD genes among the 25 typed strains. Conclusions: The PCR scheme and the oligonucleotide microarray described are effective tools to rapidly screen multiple virulence genes and their variants in E. coli strains isolated from human and animal infections. The results demonstrate the great genetic diversity among LEE genes of human and animal STEC and EPEC strains.

scholarly journals The Gene tia, Harbored by the Subtilase-Encoding Pathogenicity Island, Is Involved in the Ability of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli Strains To Invade Monolayers of Epithelial Cells

2017 ◽  
Vol 85 (12) ◽  
Author(s):  
Roslen Bondì ◽  
Paola Chiani ◽  
Valeria Michelacci ◽  
Fabio Minelli ◽  
Alfredo Caprioli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Cultured Cells ◽  
Pathogenicity Island ◽  
Content Type ◽  
Cell Monolayers ◽  
E Coli ◽  
Enterocyte Effacement ◽  
K 12

ABSTRACT Locus of enterocyte effacement (LEE)-negative Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are human pathogens that lack the LEE locus, a pathogenicity island (PAI) involved in the intimate adhesion of LEE-positive strains to the host gut epithelium. The mechanism used by LEE-negative STEC strains to colonize the host intestinal mucosa is still not clear. The cell invasion determinant tia, previously described in enterotoxigenic E. coli strains, has been identified in LEE-negative STEC strains that possess the subtilase-encoding pathogenicity island (SE-PAI). We evaluated the role of the gene tia, present in these LEE-negative STEC strains, in the invasion of monolayers of cultured cells. We observed that these strains were able to invade Caco-2 and HEp-2 cell monolayers and compared their invasion ability with that of a mutant strain in which the gene tia had been inactivated. Mutation of the gene tia resulted in a strong reduction of the invasive phenotype, and complementation of the tia mutation with a functional copy of the gene restored the invasion activity. Moreover, we show that the gene tia is overexpressed in bacteria actively invading cell monolayers, demonstrating that tia is involved in the ability to invade cultured monolayers of epithelial cells shown by SE-PAI-positive E. coli, including STEC, strains. However, the expression of the tia gene in the E. coli K-12 strain JM109 was not sufficient, in its own right, to confer to this strain the ability to invade cell monolayers, suggesting that at least another factor must be involved in the invasion ability displayed by the SE-PAI-positive strains.

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

2012 ◽  
Vol 75 (2) ◽  
pp. 408-418 ◽  
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.

scholarly journals Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

2012 ◽  
Vol 47 (No. 6) ◽  
pp. 149-158 ◽  
Author(s):  
J. Osek ◽  
P. Gallien
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Rflp Analysis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Chromosomal Dna ◽  
E Coli ◽  
Shiga Toxin Genes ◽  
Porcine Origin

Fourteen Escherichia coli O157 strains isolated from cattle and pigs in Poland and in Germany were investigated, using PCR, for the genetic markers associated with Shiga toxin-producing E. coli (STEC). Only two strains, both of cattle origin, were positive for the fliC (H7) gene and could be classified as O157 : H7. Nine isolates had stx shiga toxin genes, either stx1 (1 strain), stx2 (4 isolates) or both (4 strains). The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that all but one stx2-positive bacteria possessed the stx2c Shiga toxin gene type and one stx2 STEC isolate had the stx2d virulence factor sub-type. The eaeA (intimin) gene was found in 9 strains (8 isolates from cattle and one strain from pigs); all of them harboured the genetic marker characteristic of the gamma intimin variant. The translocated intimin receptor (tir) gene was detected in 7 isolates tested and among them only one tir-positive strain was recovered from pigs. The ehly E. coli enterohemolysin gene was amplified in all but one strains obtained from cattle and only in one isolate of porcine origin. The genetic relatedness of the analysed E. coli O157 strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with XbaI. Two distinct but related RFLP pattern clusters were observed: one with 9 strains (8 isolates of bovine origin and one strain obtained from pigs) and the other one comprises the remaining 5 E. coli isolates (4 of porcine origin and one strain recovered from cattle). The results suggest that pigs, besides cattle, may be a reservoir of E. coli O157 strains potentially pathogenic to humans. Moreover, epidemiologically unrelated isolates of the O157 serogroup, recovered from different animal species, showed a clonal relationship as demonstrated by the RFLP analysis.

scholarly journals Quorum-Sensing Escherichia coli Regulator A: a Regulator of the LysR Family Involved in the Regulation of the Locus of Enterocyte Effacement Pathogenicity Island in Enterohemorrhagic E. coli

2002 ◽  
Vol 70 (6) ◽  
pp. 3085-3093 ◽  
Author(s):  
Vanessa Sperandio ◽  
Caiyi C. Li ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Iii Secretion ◽  
Pathogenicity Island ◽  
The Other ◽  
E Coli ◽  
Type Iii ◽  
Enterocyte Effacement ◽  
K 12 ◽  
Lysr Family

ABSTRACT The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the E. coli genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E. coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E. coli regulator A (QseA). We observed that QseA activated transcription of ler and therefore of the other LEE genes. An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans. Similar results were also observed with a qseA mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.

scholarly journals Virulence Properties of Shiga Toxin-Producing Escherichia coli (STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves

1998 ◽  
Vol 36 (6) ◽  
pp. 1604-1607 ◽  
Author(s):  
L. H. Wieler ◽  
Anja Schwanitz ◽  
Elke Vieler ◽  
Barbara Busse ◽  
H. Steinrück ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Diagnostic Value ◽  
Adhesive Properties ◽  
E Coli ◽  
Virulence Potential ◽  
High Prevalence ◽  
Enterocyte Effacement ◽  
Diarrheic Calves ◽  
Virulence Properties

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [n = 38] and O118:H− [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containinghly EHEC (hly from enterohemorrhagicE. coli). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hly EHEC, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.

scholarly journals Is Shiga Toxin-Producing Escherichia coli O45 No Longer a Food Safety Threat? The Danger is Still Out There

2020 ◽  
Vol 8 (5) ◽  
pp. 782 ◽  
Author(s):  
Yujie Zhang ◽  
Yen-Te Liao ◽  
Xiaohong Sun ◽  
Vivian C.H. Wu
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Evolutionary Relationship ◽  
Severe Illness ◽  
Next Generation Sequencing Technology ◽  
E Coli ◽  
Related Information ◽  
Foodborne Outbreaks ◽  
Enterocyte Effacement

Many Shiga toxin-producing Escherichia coli (STEC) strains, including the serogroups of O157 and most of the top six non-O157 serotypes, are frequently associated with foodborne outbreaks. Therefore, they have been extensively studied using next-generation sequencing technology. However, related information regarding STEC O45 strains is scarce. In this study, three environmental E. coli O45:H16 strains (RM11911, RM13745, and RM13752) and one clinical E. coli O45:H2 strain (SJ7) were sequenced and used to characterize virulence factors using two reference E. coli O45:H2 strains of clinical origin. Subsequently, whole-genome-based phylogenetic analysis was conducted for the six STEC O45 strains and nine other reference STEC genomes, in order to evaluate their evolutionary relationship. The results show that one locus of enterocyte effacement pathogenicity island was found in all three STEC O45:H2 strains, but not in the STEC O45:H16 strains. Additionally, E. coli O45:H2 strains were evolutionarily close to E. coli O103:H2 strains, sharing high homology in terms of virulence factors, such as Stx prophages, but were distinct from E. coli O45:H16 strains. The findings show that E. coli O45:H2 may be as virulent as E. coli O103:H2, which is frequently associated with severe illness and can provide genomic evidence to facilitate STEC surveillance.

scholarly journals Molecular Characterization of a Serotype O121:H19 Clone, a Distinct Shiga Toxin-Producing Clone of Pathogenic Escherichia coli

2002 ◽  
Vol 70 (12) ◽  
pp. 6853-6859 ◽  
Author(s):  
Cheryl L. Tarr ◽  
Teresa M. Large ◽  
Chris L. Moeller ◽  
David W. Lacher ◽  
Phillip I. Tarr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Allelic Variation ◽  
Virulence Gene ◽  
Gene Profiling ◽  
Enzyme Electrophoresis ◽  
Sequencing Analysis ◽  
Chromosomal Dna ◽  
Waterborne Outbreak ◽  
E Coli

ABSTRACT Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int-ε) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx 2), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands—large chromosomal DNA segments present in the O157:H7 genome—were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.

scholarly journals Low-Density Macroarray Targeting Non-Locus of Enterocyte Effacement Effectors (nle Genes) and Major Virulence Factors of Shiga Toxin-Producing Escherichia coli (STEC): a New Approach for Molecular Risk Assessment of STEC Isolates

2009 ◽  
Vol 76 (1) ◽  
pp. 203-211 ◽  
Author(s):  
Marie Bugarel ◽  
Lothar Beutin ◽  
Patrick Fach
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genomic Islands ◽  
Low Density ◽  
Healthy Children ◽  
E Coli ◽  
Health Authorities ◽  
Public Health Authorities ◽  
Enterocyte Effacement ◽  
High Level

ABSTRACT Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes stx 1, stx 2, eae, and ehxA and six different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli (EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H−, O55:H7, O123:H11, O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2, nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear signature of STEC strains with high virulence for humans.

Glucose Decreases Virulence Gene Expression of Escherichia coli O157:H7

2012 ◽  
Vol 75 (4) ◽  
pp. 748-752 ◽  
Author(s):  
V. DELCENSERIE ◽  
G. LaPOINTE ◽  
T. CHARASLERTRANGSI ◽  
A. RABALSKI ◽  
M. W. GRIFFITHS
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Quorum Sensing ◽  
Shiga Toxin ◽  
Virulence Gene ◽  
Escherichia Coli O157 ◽  
Virulence Gene Expression ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Enterocyte Effacement

Escherichia coli O157:H7 is responsible for a human toxico-infection that can lead to severe complications such as hemolytic uremic syndrome. Inside the intestine, E. coli O157:H7 forms typical attaching-effacing lesions and produces Shiga toxins. The genes that are responsible for these lesions are located in a pathogenicity island called the locus of enterocyte effacement (LEE). LEE gene expression is influenced by quorum sensing through the luxS system. In this study, the effect of glucose on the expression of several genes from LEE, on the expression of Shiga toxin genes, and on the expression of luxS was assessed with real-time, reverse transcription PCR. All concentrations of glucose (from 0.1 to 1%) were able to down-regulate genes from the LEE operon. A slight down-regulation of genes implicated in Shiga toxin expression was also observed but was significant for low doses of glucose (0.1 to 0.5%) only. A slight but significant increase in luxS expression was observed with 1% glucose. This confirms that in addition to quorum sensing, the presence or absence of nutrients such as glucose has an impact on the down- or upregulation of LEE-encoded virulence genes by the bacterium. The influence of glucose on the virulence of E. coli O157:H7 has received little attention, and these results suggest that glucose can have an important effect on the virulence of E. coli O157:H7.

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