Dataset Information

Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli.

ABSTRACT: The aim of this study was to establish isothermal multiple self-matching initiated amplification (IMSA) and cross-priming amplification (CPA) methods to detect heat-stable I enterotoxin (STa)-producing enterotoxigenic Escherichia coli (ETEC). To avoid cross-contamination of aerosols, a closed independent isothermal amplification tube (IAT) was used to perform the assays. Optimal amplification conditions for IMSA and CPA were selected for specificity and sensitivity, respectively, and for clinical relevance. Both IMSA and CPA assays could specifically recognize all 3-STa positive strains in which they fluoresced green under UV light, but not in the 11 non-STa strains. The results of the sensitivity analysis indicated that the detection limit of the IMSA assay was 1.5 ×102 CFU, comparable to real-time PCR, but 10-fold more sensitive than CPA and LAMP. Further evaluation of the detection methods of swine diarrhea samples demonstrated that both could successfully identify the DNA of STa-producing ETEC in clinical specimens, consistent with LAMP and qPCR methods. The results demonstrated that the IMSA and CPA methods had high specificity and sensitivity with rapid detection of ETEC, so having great potential in clinical applications.

SUBMITTER: Zhai J

PROVIDER: S-EPMC7173923 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli.

Zhai Junjun J Yan Zhang Z Ping Feng F Lei Qu Q Chen Xuelong X Chen Xuelong X Qi Yanping Y

PloS one 20200421 4

The aim of this study was to establish isothermal multiple self-matching initiated amplification (IMSA) and cross-priming amplification (CPA) methods to detect heat-stable I enterotoxin (STa)-producing enterotoxigenic Escherichia coli (ETEC). To avoid cross-contamination of aerosols, a closed independent isothermal amplification tube (IAT) was used to perform the assays. Optimal amplification conditions for IMSA and CPA were selected for specificity and sensitivity, respectively, and for clinica ...[more]

PMID: 32315305

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Project description:Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage.

Project description:Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST+) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.

Dataset Information

Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli.

Publications

Establishment and application of isothermal amplification techniques for the detection of heat-stable I enterotoxin of enterotoxigenic Escherichia coli.

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