Dataset Information

Establishment and application of isothermal multiple-self-matching-initiated amplification (IMSA) in detecting Type II heat-labile enterotoxin of Escherichia coli.

ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) constitutes a major cause of diarrhea in young children and animals, particularly in poor regions of the world, as well the traveler's diarrhea in adult individuals. Type II heat-labile enterotoxin (LT-II) from ETEC can cause profuse watery diarrhea, posing a potential threat to public health and animal husbandry. In the present study, isothermal multiple-self-matching-initiated amplification (IMSA) was established to rapidly detect LT-II producing ETEC. The specificity and sensitivity were assessed, and clinical samples were tested. The established IMSA method had good specificity for the detection of LT-II gene with a limit of detection of 25 CFU/mL, i.e. 2 times higher than that of real-time PCR and other two isothermal amplifications (loop-mediated isothermal amplification, LAMP and cross-primer isothermal amplification, CPA). Meanwhile, in 103 clinical Escherichia coli strains isolated from diarrhea samples, 9 strains with LT-II+ gene were detected (8.73%), corroborating real-time PCR, LAMP and CPA data. Therefore, the IMSA technology applied for the detection of LT-II producing ETEC has a good application prospect for screening clinical samples in primary medical units or common laboratories.

SUBMITTER: Liu W

PROVIDER: S-EPMC6497269 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Establishment and application of isothermal multiple-self-matching-initiated amplification (IMSA) in detecting Type II heat-labile enterotoxin of Escherichia coli.

Liu Wenxin W Yuan Chaowen C Zhang Liguo L Feng Yufei Y

PloS one 20190502 5

Enterotoxigenic Escherichia coli (ETEC) constitutes a major cause of diarrhea in young children and animals, particularly in poor regions of the world, as well the traveler's diarrhea in adult individuals. Type II heat-labile enterotoxin (LT-II) from ETEC can cause profuse watery diarrhea, posing a potential threat to public health and animal husbandry. In the present study, isothermal multiple-self-matching-initiated amplification (IMSA) was established to rapidly detect LT-II producing ETEC. T ...[more]

PMID: 31048928

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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:Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.

Project description:Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

Dataset Information

Establishment and application of isothermal multiple-self-matching-initiated amplification (IMSA) in detecting Type II heat-labile enterotoxin of Escherichia coli.

Publications

Establishment and application of isothermal multiple-self-matching-initiated amplification (IMSA) in detecting Type II heat-labile enterotoxin of Escherichia coli.

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