<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>

ABSTRACT The bacterial pathogens enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae are major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), while V. cholerae produces cholera toxin (CT). In this study, we determine...

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Autores principales:	Yasmin Ara Begum, Hanna A. Rydberg, Kaisa Thorell, Young-Keun Kwak, Lei Sun, Enrique Joffré, Firdausi Qadri, Åsa Sjöling
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	ETEC Vibrio cholerae diarrhea enterotoxin quantification Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f5dcaff36ec54405a0a988728cba58f5
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spelling	oai:doaj.org-article:f5dcaff36ec54405a0a988728cba58f52021-11-15T15:22:01Z<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>10.1128/mSphere.00517-172379-5042https://doaj.org/article/f5dcaff36ec54405a0a988728cba58f52018-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00517-17https://doaj.org/toc/2379-5042ABSTRACT The bacterial pathogens enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae are major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), while V. cholerae produces cholera toxin (CT). In this study, we determined the occurrence and bacterial doses of the two pathogens and their respective toxin expression levels directly in liquid diarrheal stools of patients in Dhaka, Bangladesh. By quantitative culture and real-time quantitative PCR (qPCR) detection of the toxin genes, the two pathogens were found to coexist in several of the patients, at concentrations between 102 and 108 bacterial gene copies per ml. Even in culture-negative samples, gene copy numbers of 102 to 104 of either ETEC or V. cholerae toxin genes were detected by qPCR. RNA was extracted directly from stool, and gene expression levels, quantified by reverse transcriptase qPCR (RT-qPCR), of the genes encoding CT, LT, STh, and STp showed expression of toxin genes. Toxin enzyme-linked immunosorbent assay (ELISA) confirmed active toxin secretion directly in the liquid diarrhea. Analysis of ETEC isolates by multiplex PCR, dot blot analysis, and genome sequencing suggested that there are genetic ETEC profiles that are more commonly found as dominating single pathogens and others that are coinfectants with lower bacterial loads. The ETEC genomes, including assembled genomes of dominating ETEC isolates expressing LT/STh/CS5/CS6 and LT/CS7, are provided. In addition, this study highlights an emerging important ETEC strain expressing LT/STp and the novel colonization factor CS27b. These findings have implications for investigations of pathogenesis as well as for vaccine development. IMPORTANCE The cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenic E. coli (ETEC) and Vibrio cholerae directly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.Yasmin Ara BegumHanna A. RydbergKaisa ThorellYoung-Keun KwakLei SunEnrique JoffréFirdausi QadriÅsa SjölingAmerican Society for MicrobiologyarticleETECVibrio choleraediarrheaenterotoxinquantificationMicrobiologyQR1-502ENmSphere, Vol 3, Iss 1 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	ETEC Vibrio cholerae diarrhea enterotoxin quantification Microbiology QR1-502
spellingShingle	ETEC Vibrio cholerae diarrhea enterotoxin quantification Microbiology QR1-502 Yasmin Ara Begum Hanna A. Rydberg Kaisa Thorell Young-Keun Kwak Lei Sun Enrique Joffré Firdausi Qadri Åsa Sjöling <italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
description	ABSTRACT The bacterial pathogens enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae are major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), while V. cholerae produces cholera toxin (CT). In this study, we determined the occurrence and bacterial doses of the two pathogens and their respective toxin expression levels directly in liquid diarrheal stools of patients in Dhaka, Bangladesh. By quantitative culture and real-time quantitative PCR (qPCR) detection of the toxin genes, the two pathogens were found to coexist in several of the patients, at concentrations between 102 and 108 bacterial gene copies per ml. Even in culture-negative samples, gene copy numbers of 102 to 104 of either ETEC or V. cholerae toxin genes were detected by qPCR. RNA was extracted directly from stool, and gene expression levels, quantified by reverse transcriptase qPCR (RT-qPCR), of the genes encoding CT, LT, STh, and STp showed expression of toxin genes. Toxin enzyme-linked immunosorbent assay (ELISA) confirmed active toxin secretion directly in the liquid diarrhea. Analysis of ETEC isolates by multiplex PCR, dot blot analysis, and genome sequencing suggested that there are genetic ETEC profiles that are more commonly found as dominating single pathogens and others that are coinfectants with lower bacterial loads. The ETEC genomes, including assembled genomes of dominating ETEC isolates expressing LT/STh/CS5/CS6 and LT/CS7, are provided. In addition, this study highlights an emerging important ETEC strain expressing LT/STp and the novel colonization factor CS27b. These findings have implications for investigations of pathogenesis as well as for vaccine development. IMPORTANCE The cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenic E. coli (ETEC) and Vibrio cholerae directly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.
format	article
author	Yasmin Ara Begum Hanna A. Rydberg Kaisa Thorell Young-Keun Kwak Lei Sun Enrique Joffré Firdausi Qadri Åsa Sjöling
author_facet	Yasmin Ara Begum Hanna A. Rydberg Kaisa Thorell Young-Keun Kwak Lei Sun Enrique Joffré Firdausi Qadri Åsa Sjöling
author_sort	Yasmin Ara Begum
title	<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
title_short	<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
title_full	<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
title_fullStr	<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
title_full_unstemmed	<italic toggle="yes">In Situ</italic> Analyses Directly in Diarrheal Stool Reveal Large Variations in Bacterial Load and Active Toxin Expression of Enterotoxigenic <named-content content-type="genus-species">Escherichia coli</named-content> and <named-content content-type="genus-species">Vibrio cholerae</named-content>
title_sort	<italic toggle="yes">in situ</italic> analyses directly in diarrheal stool reveal large variations in bacterial load and active toxin expression of enterotoxigenic <named-content content-type="genus-species">escherichia coli</named-content> and <named-content content-type="genus-species">vibrio cholerae</named-content>
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/f5dcaff36ec54405a0a988728cba58f5
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