Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7.

ABSTRACT: In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 10(4) cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 10(2) and 10(3) cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coli O157:H7 in broth cultures.

SUBMITTER: Goodridge L

PROVIDER: S-EPMC91198 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Development and characterization of a fluorescent-bacteriophage assay for detection of Escherichia coli O157:H7.

Goodridge L L Chen J J Griffiths M M

Applied and environmental microbiology 19990401 4

In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coli O157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 10(4) cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated tha ...[more]

PMID: 10103228

Similar Datasets

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage.

Project description:A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.

| S-EPMC321238 | biostudies-literature

Multiparametric Magneto-fluorescent Nanosensors for the Ultrasensitive Detection of Escherichia coli O157:H7.

Project description:Enterohemorrhagic Escherichia coli O157:H7 presents a serious threat to human health and sanitation and is a leading cause in many food- and waterborne ailments. While conventional bacterial detection methods such as PCR, fluorescent immunoassays and ELISA exhibit high sensitivity and specificity, they are relatively laborious and require sophisticated instruments. In addition, these methods often demand extensive sample preparation and have lengthy readout times. We propose a simpler and more sensitive diagnostic technique featuring multiparametric magneto-fluorescent nanosensors (MFnS). Through a combination of magnetic relaxation and fluorescence measurements, our nanosensors are able to detect bacterial contamination with concentrations as little as 1 colony-forming unit (CFU). The magnetic relaxation property of our MFnS allow for sensitive screening at low target CFU, which is complemented by fluorescence measurements of higher CFU samples. Together, these qualities allow for the detection and quantification of broad-spectrum contaminations in samples ranging from aquatic reservoirs to commercially produced food.

| S-EPMC6736527 | biostudies-literature

Rapid Detection of Escherichia coli O157: H7 by Fluorescent Amplification-Based Specific Hybridization (FLASH) PCR.

Project description:BackgroundEscherichia coli O157:H7 is an enteric pathogen which can be frequently found asymptomatically in ruminant mammals, but can cause diseases from mild diarrhea to hemolytic uremic syndrome in humans.MethodsWe developed fluorescent amplification-based specific hybridization (FLASH-PCR) assay to detect the Stx-encoding gene Stx-1 of E. coli O157:H7.ResultPCR product of 336 bp was successfully amplified in a FLASH-PCR.ConclusionAs rapid detection and confirmation of the presence of E. coli O157:H7 are of importance for the medical, food, and water industries, FLASH-PCR is one of selective methods for detection of E. coli O157:H7.

| S-EPMC3482334 | biostudies-literature

Immunological characterization of Escherichia coli O157:H7 intimin gamma1.

Project description:Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+. Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E. coli strains such as E. coli O157:H7.

| S-EPMC119882 | biostudies-literature

Characterization of novel bacteriophage phiC119 capable of lysing multidrug-resistant Shiga toxin-producing Escherichia coli O157:H7.

Project description:BackgroundShiga toxin-producing Escherichia coli (STEC) is one of the most common and widely distributed foodborne pathogens that has been frequently implicated in gastrointestinal and urinary tract infections. Moreover, high rates of multiple antibiotic-resistant E. coli strains have been reported worldwide. Due to the emergence of antibiotic-resistant strains, bacteriophages are considered an attractive alternative to biocontrol pathogenic bacteria. Characterization is a preliminary step towards designing a phage for biocontrol.MethodsIn this study, we describe the characterization of a bacteriophage designated phiC119, which can infect and lyse several multidrug-resistant STEC strains and some Salmonella strains. The phage genome was screened to detect the stx-genes using PCR, morphological analysis, host range was determined, and genome sequencing were carried out, as well as an analysis of the cohesive ends and identification of the type of genetic material through enzymatic digestion of the genome.ResultsAnalysis of the bacteriophage particles by transmission electron microscopy showed that it had an icosahedral head and a long tail, characteristic of the family Siphoviridae. The phage exhibits broad host range against multidrug-resistant and highly virulent E. coli isolates. One-step growth experiments revealed that the phiC119 phage presented a large burst size (210 PFU/cell) and a latent period of 20 min. Based on genomic analysis, the phage contains a linear double-stranded DNA genome with a size of 47,319 bp. The phage encodes 75 putative proteins, but lysogeny and virulence genes were not found in the phiC119 genome.ConclusionThese results suggest that phage phiC119 may be a good biological control agent. However, further studies are required to ensure its control of STEC and to confirm the safety of phage use.

| S-EPMC5028729 | biostudies-literature

Complete Genome Sequence of Bacteriophage EC6, Capable of Lysing Escherichia coli O157:H7.

Project description:A bacteriophage, EC6, capable of lysing Escherichia coli serotype O157:H7 was isolated and found to be a member of the family Myoviridae. The genomic sequence of phage EC6 was composed of 86,231 bp with a G+C content of 38.9% and is very similar to the sequences of Escherichia phage wV8 and Salmonella phage Felix O1.

| S-EPMC3569278 | biostudies-literature

The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5.

Project description:BackgroundBacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs.MethodsWe examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set.ResultsPhage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.

| S-EPMC3606486 | biostudies-literature

Complete Genome Sequence of Escherichia coli O157:H7 Bacteriophage phiJLA23 Isolated in Mexico.

Project description:The bacteriophage phiJLA23 was isolated from an animal feces sample and lytic activity was demonstrated against the Escherichia coli O157:H7 strain. We report the complete nucleotide sequence of bacteriophage phiJLA23, information which may be useful for determining whether this phage is a candidate for biocontrol or another biotechnological application.

| S-EPMC3587941 | biostudies-literature

Characterization of the Escherichia coli O157:H7 Sakai GadE regulon.

Project description:Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB, and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the DeltagadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the DeltagadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.

| S-EPMC2648353 | biostudies-literature

Bacteriophage cocktail for biocontrol of Escherichia coli O157:H7: Stability and potential allergenicity study.

Project description:Escherichia coli O157:H7 has become a global public health and a food safety problem. Despite the implementation of control strategies that guarantee the safety in various products, outbreaks persist and new alternatives are necessary to reduce this pathogen along the food chain. Recently, our group isolated and characterised lytic bacteriophages against E. coli O157:H7 with potential to be used as biocontrol agents in food. To this end, phages need certain requirements to allow their manufacture and application. The aim of this study was to determine the physical stability and allergenic potential of free and microencapsulated (ME) bacteriophage cocktails against E. coli O157:H7. In vitro and in vivo studies were performed to determine phage survival under different pH, gastrointestinal conditions, temperature and UV light intensities. Results showed that the stability of ME phages was significantly (P<0.05) higher than free phages after ultraviolet irradiation, pH conditions between 3 to 7, and exposure to temperatures between at -80°C and 70°C. Both formulations were highly sensitive to very low pH in simulated gastric fluid, but stable in bile salts. In vivo studies in mice confirmed these phages passed through the gastrointestinal tract and were excreted in faeces. In silico, full-length alignment analysis showed that all phage proteins were negative for allergenic potential, but different predicting criteria classified seven phage proteins with a very low probability to be an allergen. In conclusion, these data demonstrated that microencapsulation provided a greater stability to phage formulation under stress conditions and assure a more suitable commercial formulation for the biological control of E. coli O157:H7.

| S-EPMC5953568 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data