Genetic Methods for the Detection of Microbial Pathogens. Identification of Enterotoxigenic Escherichia coli by DNA Colony Hybridization: Collaborative Study

1984 ◽  
Vol 67 (4) ◽  
pp. 801-807 ◽  
Author(s):  
Walter E Hill ◽  
William L Payne ◽  
◽  
R J Crouch ◽  
V M Davis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Collaborative Study ◽  
False Negative ◽  
Toxin Production ◽  
Microbial Pathogens ◽  
Enterotoxigenic Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chi Square ◽  
Recombinant Dna Techniques

Abstract Enteropathogenic Escherichia coli strains may produce a cholera-like, heat-labile enterotoxin (LT) as a virulence factor. The gene that codes for LT can be purified by recombinant DNA techniques and used as a genetic probe for DNA hybridization. These probes detect enterotoxigenic strains as well as strains that may not manifest toxin production but carry the genetic information to do so. In this study, 13 laboratories tested 3 known and 25 unknown (10 positive and 15 negative) cultures off. coli for the presence of the LT gene. The isolates had been tested and classified by the mouse Y-l adrenal cell test and an enzyme-linked immunosorbent assay. Cultures were spotted on nitrocellulose filters on MacConkey agar and incubated. Colonies were lysed in situ and their DNA was hybridized to 32P-labeled, purified LT gene DNA (provided to the collaborators). Positive colonies were identified by autoradiography. Of 325 samples, 315 (96.9%) were identified correctly and 10 were misclassified; there were 6 false negative and 4 false positive identifications. Chi-square values indicated that the method agreed with the previous classification and was equally efficient in distinguishing positive and negative samples (95.7 and 98.1%, respectively). The method has been adopted official first action.

DNA Colony Hybridization Method Using Synthetic Oligonucleotides to Detect Enterotoxigenic Escherichia coli: Collaborative Study

1986 ◽  
Vol 69 (3) ◽  
pp. 531-536
Author(s):  
Walter E Hill ◽  
Barry A Wentz ◽  
William L Payne ◽  
James A Jagow ◽  
Gerald Zon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Collaborative Study ◽  
Dna Probes ◽  
Enterotoxigenic Escherichia Coli ◽  
Hybridization Probe ◽  
Colony Hybridization ◽  
E Coli ◽  
Calf Thymus ◽  
Recombinant Dna Techniques

Abstract The genes that encode several of the enterotoxins produced by Escherichia coli have been cloned by recombinant DNA techniques. When the nucleotide sequence of these genes is determined, defined sequence oligonucleotides that include a part of these genes may be synthesized. A 22-base DNA hybridization probe was produced for each of 2 heatstable E. coli enterotoxin (ST) genes: STH, from strains originally isolated from humans; and STP, from strains first found in pigs. For this study, 32P end-labeled DNA probes, sonicated calf thymus DNA, and 3 known and 20 unknown (10 ST-positive and 10 ST-negative) strains were sent to each of 23 collaborators. Cultures were spotted onto an agar-based medium and grown into colonies, which were transferred by blotting to cellulose filters, lysed by alkali and steam, and used for DNA colony hybridization with the ST DNA probes. Strains containing an ST gene were recognized as dark spots on an autoradiogram. Of the 460 samples analyzed, 440 (95.7%) were correctly classified by the collaborators. The method has been adopted official first action.

Fluorescent antibody techniques have allowed for the direct identification and enumeration of individual bacteria in environmental samples without requiring prior growth in culture media (Bahlool and Schmidt 1980, Cloete and Steyn 1988, Macario et al. 1989). The technique involves the use of specific antibodies raised against surface markers of defined pure cultures that are either labelled directly with fluorescent dye molecules or via a fluorescent secondary antibody. This approach has yielded important insights into the spatial distribution of microorganisms, but it suffers from a number of disadvantages. For example, expression of the antigen may be influenced by environmental factors; false-positive and false-negative results may be obtained due to cross-reactivity or lack of reaction; non-specific binding of antibodies may result in high levels of background fluorescence; and production of specific antibodies requires a pure culture of the organism of interest (Cloete and de Bruyn Various recombinant DNA techniques have subsequently been developed that are independent of cultivation methods (Fig. 1). These techniques provide ways of detecting and quantifying specific phylogenetic groups of microbes on 16S rDNA sequences, and relevant structural genes provide ways of monitoring microbial populations of environmental and industrial systems. In addition to these tools, a number of emerging technologies such as the use of biomarker genes are being increasingly used to monitor with great precision and accuracy the behaviour of microbes in the environment.

2003 ◽  
pp. 218-219
Keyword(s):  
Recombinant Dna ◽  
Culture Media ◽  
Specific Binding ◽  
Fluorescent Antibody ◽  
False Negative ◽  
Cross Reactivity ◽  
Phylogenetic Groups ◽  
Specific Antibodies ◽  
Negative Results ◽  
Recombinant Dna Techniques

scholarly journals A Novel Deletion Mutation in pmrB Contributes to Concurrent Colistin Resistance in Carbapenem-Resistant Escherichia coli Sequence Type 405 of Clinical Origin

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ching-Hsun Wang ◽  
L. Kristopher Siu ◽  
Feng-Yee Chang ◽  
Yu-Kuo Tsai ◽  
Yi-Tsung Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Selective Pressure ◽  
Carbapenem Resistance ◽  
Sequence Type ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Recombinant Dna Techniques

ABSTRACT We report the first clinical Escherichia coli strain EC3000 with concomitant chromosomal colistin and carbapenem resistance. A novel in-frame deletion, Δ6-11 (RPISLR), in pmrB that contributes to colistin resistance was verified using recombinant DNA techniques. Although being less fit than the wild-type (WT) strain or EC3000 revertant (chromosomal replacement of WT pmrB in EC3000), a portion of serially passaged EC3000 strains preserving colistin resistance without selective pressure raises the concern for further spread.

Improvement of Escherichia coli strains overproducing lysine using recombinant DNA techniques

1982 ◽  
Vol 15 (4) ◽  
pp. 227-231 ◽  
Author(s):  
Brigitte Dauce-Le Reverend ◽  
Mich�le Boitel ◽  
Alain M. Deschamps ◽  
Jean-Michel Lebeault ◽  
Konosuke Sano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Dna ◽  
Recombinant Dna Techniques

Detection of enterotoxigenic Escherichia coli in water by polymerase chain reaction amplification and hybridization

1994 ◽  
Vol 40 (4) ◽  
pp. 243-249 ◽  
Author(s):  
Z. Tamanai-Shacoori ◽  
A. Jolivet-Gougeon ◽  
M. Pommepuy ◽  
M. Cormier ◽  
R. R. Colwell
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Pcr Amplification ◽  
Environmental Water ◽  
Latex Agglutination ◽  
Enterotoxigenic Escherichia Coli ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Enterotoxigenic Escherichia coli was studied in waste water, river water, and seawater from six locations along the west coast of Normandy by using the polymerase chain reaction (PCR) to amplify the heat labile (LT) gene. Cellular DNA was extracted from centrifugation pellets and amplified using PCR. The PCR products were detected by gel electrophoresis and confirmed by hybridization assay, using an 850 base pair HindIII DNA fragment probe from pEWD299 conjugated to digoxigenin and specific for the LT gene. Results of the PCR amplification were compared with those of GM1 enzyme-linked immunosorbent assay, latex agglutination, and colony hybridization. The PCR method was found to be more precise and less time consuming, especially when compared with methods requiring culture of isolates for enumeration of enterotoxigenic E. coli in water.Key words: enterotoxigenic Escherichia coli, PCR, environmental water, digoxigenin.

scholarly journals In Vitro Evaluation of Dietary Fiber Anti-Infectious Properties against Food-Borne Enterotoxigenic Escherichia coli

Nutrients ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 3188
Author(s):  
Thomas Sauvaitre ◽  
Claude Durif ◽  
Adeline Sivignon ◽  
Sandrine Chalancon ◽  
Tom Van de Wiele ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture Media ◽  
Toxin Production ◽  
Enterotoxigenic Escherichia Coli ◽  
In Vitro Assays ◽  
Dietary Fibers ◽  
Mucus Cell ◽  
Etec Strain ◽  
Beneficial Effects

Dietary fibers have well-known beneficial effects on human health, but their anti-infectious properties against human enteric pathogens have been poorly investigated. Enterotoxigenic Escherichia coli (ETEC) is the main agent of travelers’ diarrhea, against which targeted preventive strategies are currently lacking. ETEC pathogenesis relies on multiple virulence factors allowing interactions with the intestinal mucosal layer and toxins triggering the onset of diarrheal symptoms. Here, we used complementary in vitro assays to study the antagonistic properties of eight fiber-containing products from cereals, legumes or microbes against the prototypical human ETEC strain H10407. Inhibitory effects of these products on the pathogen were tested through growth, toxin production and mucus/cell adhesion inhibition assays. None of the tested compounds inhibited ETEC strain H10407 growth, while lentil extract was able to decrease heat labile toxin (LT) concentration in culture media. Lentil extract and specific yeast cell walls also interfered with ETEC strain H10407 adhesion to mucin beads and human intestinal cells. These results constitute a first step in the use of dietary fibers as a nutritional strategy to prevent ETEC infection. Further work will be dedicated to the study of fiber/ETEC interactions within a complex gut microbial background.

scholarly journals Detection of Verotoxigenic Escherichia coli by Magnetic Capture-Hybridization PCR

1998 ◽  
Vol 64 (1) ◽  
pp. 147-152 ◽  
Author(s):  
Jinru Chen ◽  
Roger Johnson ◽  
Mansel Griffiths
Keyword(s):  
Escherichia Coli ◽  
Magnetic Beads ◽  
Detection System ◽  
Toxin Production ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Pcr ◽  
Magnetic Capture ◽  
Pcr Products ◽  
Pure Cultures ◽  
Particle Separator

ABSTRACT Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin [VT]-producing)Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures. The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization. The hybrids formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR. The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products. With 5, 7, or 10 h of enrichment, the limits of detection were 103, 102, and 100 CFU/ml, respectively, by agarose gel electrophoresis. Southern hybridization did not seem to improve the limit of the detection. When applied to food, MCH-PCR was capable of detecting 100 CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The results of MCH-PCR for pure cultures of VT1- and/or VT2-producing E. coli cells were in total agreement with toxin production as measured by a VT enzyme-linked immunosorbent assay.

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