Evaluation of experimental conditions for quantification of LT produced by human derived enterotoxigenic Escherichia coli strains

Abstract

The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.

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 .…”

“…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 could facilitate and enhance the sensitivity of diagnostic tests for ETEC 355 . Afterwards, those monoclonal antibodies were rebuilt resulting in single chain fragment variable (scFv) fragments.…”

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

“…Polymyxin B has been long used for LT release [ 21 , 23 , 30 , 31 ], but some authors have demonstrated that triton X-100 treatment shows a superior performance in LT release [ 32 ]. Nevertheless, Lasaro et al [ 33 ] found that polymyxin B recovered 25% of LT produced by strain H10407, where it was more effective than triton X-100, which recovered less than 15% of the toxin. The effect of urea has also been described for LT release [ 34 ], and in our study, the results of urea treatment showed no reproducibility (data not shown).…”

“…The effect of urea has also been described for LT release [ 34 ], and in our study, the results of urea treatment showed no reproducibility (data not shown). Although some authors [ 33 , 35 ] consider that “sonic disruption” is the most efficient condition, this method is laborious and impractical in routine use and in dealing with a large collection of bacterial isolates. In our tests, treatment with triton X-100 was the common chemical treatment for the release of both toxins and therefore chosen to be added directly to bacterial culture growth, confirming that production/release of toxins is indeed affected by chemicals in vitro .…”

Quantifying and Engineering Mucus Adhesion of Probiotics

“…Escherichia coli Nissle 1917 (EcN) is an extensively studied probiotic sold over-the-counter as Mutaflor® to treat chronic inflammatory and infectious intestinal diseases and is used significantly in the development of microbial therapeutics 16,[64][65][66][67][68][69] . Though there are conflicting reports whether EcN can persist in human intestines 68,70 , it has been shown to colonize the porcine intestinal tract 71 .…”