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• Rocha, LB.
• Ozaki, CY.
• Horton, DS. P. Q.
• Menezes, CA.
• Silva, A
• Fernandes, I
• Magnoli, FC.
• Vaz, TM. I.
• Guth, BE. C.
• Piazza, RM. F.
• [+] on Google Scholar
• Rocha, LB.
• Ozaki, CY.
• Horton, DS. P. Q.
• Menezes, CA.
• Silva, A
• Fernandes, I
• Magnoli, FC.
• Vaz, TM. I.
• Guth, BE. C.
• Piazza, RM. F.
• [+] on PubMed
• Rocha, LB.
• Ozaki, CY.
• Horton, DS. P. Q.
• Menezes, CA.
• Silva, A
• Fernandes, I
• Magnoli, FC.
• Vaz, TM. I.
• Guth, BE. C.
• Piazza, RM. F.

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Toxins 2013, 5(12), 2384-2402; doi:10.3390/toxins5122384

Article

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

Letícia B. Rocha ^1,†, Christiane Y. Ozaki ^1,2,†, Denise S. P. Q. Horton ^1,* , Caroline A. Menezes ^1,3, Anderson Silva ¹, Irene Fernandes ⁴, Fabio C. Magnoli ⁵, Tania M. I. Vaz ⁶, Beatriz E. C. Guth ⁷ and Roxane M. F. Piazza ^1,*

¹ Bacteriology Laboratory, Butantan Institute, São Paulo, SP 05503-900, Brazil ² Seroepidemiology and Immunology Laboratory, São Paulo Tropical Medicine Institute, São Paulo, SP 05403-000, Brazil ³ Fleury-Medicine and Health, São Paulo, SP 04344-903, Brazil ⁴ Immunopathology Laboratory, Butantan Institute, São Paulo, SP 05503-900, Brazil ⁵ Immunochemistry Laboratory, Butantan Institute, São Paulo, SP 05503-900, Brazil ⁶ Bacteriology Section, Adolfo Lutz Institute, São Paulo, SP 01246-000, Brazil ⁷ Department of Microbiology, Immunology, Parasitology, Escola Paulista de Medicina, Federal University of São Paulo, SP 04923-062, Brazil ^† These authors contributed equally to this work.

* Authors to whom correspondence should be addressed.

Received: 13 September 2013; in revised form: 19 November 2013 / Accepted: 21 November 2013 / Published: 2 December 2013

(This article belongs to the Special Issue Advances in Toxin Detection)

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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.

Keywords: ETEC; heat-labile toxin; heat-stable toxin; production; release; detection

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