Relationship between outer membrane protein and lipopolysaccharide profiles and serotypes of enterotoxigenic Escherichia coli isolated in Brazil

Nishimura, Lucilia S.; Ferreira, L. C. S.; Pacheco, Amílcar; Guth, Beatriz E. C.

doi:10.1111/j.1574-6968.1996.tb08489.x

Cited by 17 publications

(11 citation statements)

References 24 publications

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“…Moreover, the observed homogeneity of OMP and LPS profiles among ETEC strains of the same serotype but isolated at different periods of time in São Paulo, suggested that only few clones were disseminated in our population (Nishimura et al 1996). The use of random amplification of polymorphic DNA (RAPD) confirmed these observations, and also revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods (Pacheco et al 1997).…”

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Enterotoxigenic Escherichia coli - an overview

Guth

2000

Mem. Inst. Oswaldo Cruz

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Enterotoxigenic Escherichia coli is an important cause of traveler's diarrhea and diarrheal illnesses in children in the developing world. In this presentation we will focus on the main virulence attributes of this pathogenic category of E. coli, and discuss the evolution of studies conducted in our laboratory. The importance of ETEC as a major cause of diarrhea was recognized in the late 60's. Since then, a large body of information on the epidemiology and pathogenesis of this group of bacteria has been accumulated.ETEC strains are a worldwide cause of acute diarrheal disease in both human and animals, responsible for a high rate of infantile mortality in the developing countries. They are also an important agent of diarrhea among travelers from industrialized countries visiting tropical or subtropical areas of the world.Two virulence attributes that characterize ETEC are the colonization of the small intestine surface and the production of enterotoxins that induce a net secretion of electrolytes and water into the gut lumen.The enterotoxins produced by these organisms belong to two major classes of heat-labile and heatstable toxins (LT and ST, respectively), that differed in regard to structure, antigenicity and mechanisms of action, usually related to cyclic nucleotides (Acheson 1992, Spangler 1992, Sears & Kaper 1996. LT, which is closely related to cholera toxin (CT), is an oligomeric protein of 85 kDa composed by five identical B subunits, arranged in a ring and in one A subunit. The B subunits bind strongly to the intestinal receptor GM1, while the A subunit is responsible for the enzymatic activity of the toxin, and is proteolitically cleaved to yield A 1 and A 2 peptides joined by a disulfide bond. After binding to the host cell membranes, LT enters into the cell through an endocytic process and the A 1 peptide ADP-ribosylates the alpha subunit of the GTPbinding protein Gs, leading to activation of adenylate cyclase in the enterocyte and accumulation of cyclic AMP. The intracellular accumulation of cAMP elicits secretion by crypt cells and decrease absorption by villus tip cells. In this family of toxins two distinct classes were described LT-I and LT-II, that although very similar present some differences specially in the B subunit.In contrast to LTs, the STs are small, monomeric toxins of approximately 5 kDa, and two different classes were also distinguished based on biological and chemical properties: ST-I and ST-II. ST-I is methanol soluble and active in the infant mouse model, and differences in the aminoacid sequence of the toxin led to the identification of two genetic variants ST-Ih and ST-Ip, described initially in association with strains isolated from humans and pigs, respectively. However, subsequent studies showed that both variants can be produced by human ETEC strains, although ST-Ih seems to prevailed. ST-II, which is methanol insoluble and causes a positive response on pig and rat intestinal loops, is associated primarily with diarrheal disease in pigs, but some human ETEC strains expre...

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confidence: 65%

Enterotoxigenic Escherichia coli - an overview

Guth

2000

Mem. Inst. Oswaldo Cruz

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“…In spite of this serotype diversity, some flagellar antigens are serogroup specific and are often associated with the toxigenic phenotypes LT-I (heat labile toxin I)/ST-I (heat stable toxin I) and ST-I, which are highly associated with diarrhea (10,12,16). We performed serotyping of ETEC samples with a previously unknown O:H serotype by fliC RFLP and somatic antigen serotyping (data not shown).…”

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confidence: 99%

Can the fliC PCR-Restriction Fragment Length Polymorphism Technique Replace Classic Serotyping Methods for Characterizing the H Antigen of Enterotoxigenic Escherichia coli Strains?

2006

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In this study, we performed fliC PCR-restriction fragment length polymorphism (RFLP) to investigate whether this technique would be better than classic serotyping for the characterization of the H antigen in enterotoxigenic Escherichia coli (ETEC) strains. We showed that the fliC genes from ETEC strains can be characterized by restriction analysis of their polymorphism. Only one allele of the fliC gene from ETEC strains was found for each flagellar antigen, with the exception of H21. Nonmotile strains could also be characterized using this molecular technique. Moreover, determination of the somatic antigen was guided by the identification of the flagellar antigen from previously unknown serotypes of ETEC strains by PCR-RFLP, thus reducing the number of anti-antigen O sera used. The PCR-RFLP technique proved to be faster than classic serotyping for the characterization of the E. coli H antigen, taking 2 days to complete instead of the 7 or more days using classic serotyping. In conclusion, the H molecular typing for Enterobacteriaceae members may become an important epidemiological tool for the characterization of the H antigen of E. coli pathotypes. The PCR-RFLP technique is capable of guiding the determination of the H antigen and could partially replace seroagglutination. With the determination of the molecular profiles of alleles from strains obtained in epidemiological studies, new patterns will be described for ETEC strains or other E. coli pathotypes, thus permitting widespread use of this technique to characterize fliC genes and determine the H antigen of E. coli strains.

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“…Outer membrane protein extracts were prepared according to the method described by Nishimura et al 12 Briefly, bacteria were cultured in tryptic soy broth (TSB, Difco Laboratories, Detroit, MI), centrifuged and washed in 10 mM HEPES pH 7.5 (Difco Laboratories) with 2 mM MgCl 2 , and then disrupted by 3 cycles of ultrasound (Branson Sonifier 450, Branson Ultrasonics Corporation, Danbury, CT). The supernatant was ultracentrifuged, the pellet resuspended in 10 mM Tris-EDTA pH 8.0, and the proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Outer Membrane Protein Extract Preparationsmentioning

confidence: 99%