Different Assay Conditions for Detecting the Production and Release of Heat-Labile and Heat-Stable Toxins in Enterotoxigenic Escherichia coli Isolates

Abstract

Enterotoxigenic Escherichia coli (ETEC) produce heat-labile (LT) and/or heat-stable enterotoxins (ST). Despite that, the mechanism of action of both toxins are well known, there is great controversy in the literature concerning the in vitro production and release of LT and, for ST, no major concerns have been discussed. Furthermore, the majority of published papers describe the use of only one or a few ETEC isolates to define the production and release of these toxins, which hinders the detection of ETEC by phenotypic approaches. Thus, the present study was undertaken to obtain a better understanding of ST and LT toxin production and release under laboratory conditions. Accordingly, a collection of 90 LT-, ST-, and ST/LT-producing ETEC isolates was used to determine a protocol for toxin production and release aimed at ETEC detection. For this, we used previously raised anti-LT antibodies and the anti-ST monoclonal and polyclonal antibodies described herein. The presence of bile salts and the use of certain antibiotics improved ETEC toxin production/release. Triton X-100, as chemical treatment, proved to be an alternative method for toxin release. Consequently, a common protocol that can increase the production and release of LT and ST toxins could facilitate and enhance the sensitivity of diagnostic tests for ETEC using the raised and described antibodies in the present work.

Diarrheagenic Escherichia coli

“…Previous observations demonstrated that the amounts of LT produced and/or secreted by ETEC are dramatically different among strains and clinical isolates 332, 351, 354, 355, 356. The presence of single nucleotide changes in the etx operon regulatory region may be found and, at least for some of them, are associated with different transcriptional and translational activity among wild ETEC strains 332 .…”

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

“…No animal model was employed in the present work. The hybridomas used as template for scFv development were previously obtained [ 17 , 18 ] for LT monoclonal antibody (mAb) and for ST mAb, respectively. All experiments were conducted in agreement with the Ethical Principles in Animal Research, adopted by the Brazilian College of Animal Experimentation, and they were approved by the Ethical Committee for Animal Research of Butantan Institute (314/06).…”

“…The plasmid vector pET28a was obtained from Novagen (USA) and the pGEM-T Easy Vector System kit from Promega (USA). Bacterial isolates used in this study consisted of strains previously defined as ETEC by the presence of LT and/or ST encoding-gene, as well as the production of the respective toxins [ 18 ]. Also, ETEC H10407 (O78:H11) and 3321–4 (O153:H45) were employed as ST/LT-producing and ST-producing prototypes, respectively [ 19 , 20 ].…”

<b>Caracterização fenotípica e genotípica de <i>Escherichia coli</i> diarreiogênicas isoladas de psitacídeos de pequeno porte criados como \"pets\":</b> análise dos riscos zoonóticos

“…coli enterotoxigênica (ETECs) é associada à diarreia infantil com óbito de mais de 300.000 crianças com menos de cinco anos, em regiões endêmicas de paí-ses em desenvolvimento. É considerada, ainda, a principal causa da diarreia dos viajantes, afetando turistas e militares que circulam em regiões endêmicas (ROCHA et al, 2013).…”

“…Constituída por estirpes que infectam homens e animais, este patotipo é caracterizado pela produção das enterotoxinas termo-lábil (LT) e/ou enterotoxina termoestável (ST), ambas codificadas em plasmídeos (ROCHA et al, 2013 , 2013). A toxina LT apresenta estrutura e função semelhantes à toxina colérica e pode ser classificada em LT-I, expressa em cepas patogênicas, tanto para humanos, como para animais, e LT-II, encontrada principalmente em isolados animais e raramente em humanos (FOCACCIA, 2005).…”