scholarly journals Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

2006 ◽  
Vol 72 (9) ◽  
pp. 6405-6410 ◽  
Author(s):  
Raul R. Raya ◽  
Peter Varey ◽  
Rebecca A. Oot ◽  
Michael R. Dyen ◽  
Todd R. Callaway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Dose ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Isolation And Characterization ◽  
Unit Reduction

ABSTRACT Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals Use of In Vivo-Induced Antigen Technology for Identification of Escherichia coli O157:H7 Proteins Expressed during Human Infection

2005 ◽  
Vol 73 (5) ◽  
pp. 2665-2679 ◽  
Author(s):  
Manohar John ◽  
Indira T. Kudva ◽  
Robert W. Griffin ◽  
Allen W. Dodson ◽  
Bethany McManus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Human Infection ◽  
Escherichia Coli O157 ◽  
Standard Laboratory ◽  
E Coli ◽  
Phase Culture ◽  
Stool Specimens

ABSTRACT Using in vivo-induced antigen technology (IVIAT), a modified immunoscreening technique that circumvents the need for animal models, we directly identified immunogenic Escherichia coli O157:H7 (O157) proteins expressed either specifically during human infection but not during growth under standard laboratory conditions or at significantly higher levels in vivo than in vitro. IVIAT identified 223 O157 proteins expressed during human infection, several of which were unique to this study. These in vivo-induced (ivi) proteins, encoded by ivi genes, mapped to the backbone, O islands (OIs), and pO157. Lack of in vitro expression of O157-specific ivi proteins was confirmed by proteomic analysis of a mid-exponential-phase culture of E. coli O157 grown in LB broth. Because ivi proteins are expressed in response to specific cues during infection and might help pathogens adapt to and counter hostile in vivo environments, those identified in this study are potential targets for drug and vaccine development. Also, such proteins may be exploited as markers of O157 infection in stool specimens.

scholarly journals Isolation and characterization of a novel temperate Escherichia coli bacteriophage, Kapi1, which modifies the O-antigen and contributes to the competitiveness of its host during colonization of the murine gastrointestinal tract.

2021 ◽  
Author(s):  
Kat Pick ◽  
Tingting Ju ◽  
Benjamin P. Willing ◽  
Tracy Lyn Raivio
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Molecular Mechanisms ◽  
Shigella Flexneri ◽  
Simulated Intestinal Fluid ◽  
O Antigen ◽  
Isolation And Characterization

In this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri, and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways.  Kapi1 displays unstable lysogeny, and we find that lysogeny is favored during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their non-lysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be.

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

scholarly journals Isolation and characterization of Escherichia coli mutants with altered rates of deletion formation.

Genetics ◽  
1991 ◽  
Vol 127 (1) ◽  
pp. 21-30 ◽  
Author(s):  
S K Whoriskey ◽  
M A Schofield ◽  
J H Miller
Keyword(s):  
Escherichia Coli ◽  
Genetic System ◽  
Repeated Sequences ◽  
E Coli ◽  
Template Strand ◽  
Deletion Formation ◽  
Precise Excision ◽  
Isolation And Characterization

Abstract Using site-specific mutagenesis in vitro we constructed a genetic system to detect mutants with altered rates of deletion formation between short repeated sequences in Escherichia coli. After in vivo mutagenesis with chemical mutagens and transposons, the system allowed the identification of mutants with either increased or decreased deletion frequencies. One mutational locus, termed mutR, that results in an increase in deletion formation, was studied in detail. The mutR gene maps at 38.5 min on the E. coli genetic map. Since the precise excision of many transposable elements is also mediated at short repeated sequences, we investigated the effects of the mutant alleles, as well as recA, on precise excision of the transposon Tn9. Neither mutR nor recA affect precise excision of the transposon Tn9, from three different insertions in lacI, whereas these alleles do affect other spontaneous deletions in the same system. These results indicate that deletion events leading to precise excision occur principally via a different pathway than other random spontaneous deletions. It is suggested that, whereas precise excision occurs predominantly via a pathway involving replication enzymes (for instance template strand slippage), deletions on an F'factor are stimulated by recombination enzymes.

scholarly journals ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1041-1059
Author(s):  
Joan M Henson ◽  
Herman Chu ◽  
Carleen A Irwin ◽  
James R Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
E Coli ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX  + and Y  +; the shorter F210 carries dnaY  +, but not X  +. Lambda transducing phages that carry dnaX  + or Y  + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

Isolation and characterization of Escherichia coli O157:H7 and shiga toxin-converting bacteriophages from strains of human, bovine and porcine origin

2002 ◽  
Vol 2 (3) ◽  
pp. 29-38 ◽  
Author(s):  
E.E. Müller ◽  
M.B. Taylor ◽  
W.O.K. Grabow ◽  
M.M. Ehlers
Keyword(s):  
Escherichia Coli ◽  
Pcr Analysis ◽  
Sewage Sample ◽  
Natural Environments ◽  
Escherichia Coli O157 ◽  
Electron Microscopic ◽  
E Coli ◽  
Isolation And Characterization ◽  
Faecal Samples

Toxin-converting bacteriophages encoding the Stx2 gene were induced from strains of Escherichia coli O157:H7 isolated from sewage, bovine and porcine faeces. Toxin synthesis can be stimulated by the induction of integrated toxin-converting phages from the host E. coli O157:H7 organism by ultra-violet (UV) exposure. The UV-mediated DNA damage of E. coli O157:H7 triggers a bacterial SOS response resulting in phage release. Free ranging phages outside their E. coli O157:H7 hosts were detected but could not be isolated directly from environmental samples such as sewage and river water. E. coli O157:H7 colonies carrying the genes coding for Stx2 were isolated from 1 sewage sample (0.76% of positive samples), 8 cattle faecal samples (16.67% of positive samples) and 2 pig faecal samples (14.28% of positive samples). Characterization of E. coli O157:H7 was done by repetitive sequence analysis using ERIC-PCR to determine the relationships between the individual E. coli O157:H7 strains. The ERIC-PCR analysis revealed distinct patterns for all E. coli O157:H7 strains with some small differences between the strains. DNA sequencing of some of the E. coli O157:H7 positive isolates carrying the Stx2 genes were performed confirming the amplified DNA nucleotide sequences of Stx2. Electron microscopic analysis revealed, for the first time in South Africa, that Stx2-converting phages induced from E. coli O157:H7 have different morphologies to that of phage lambda which was previously described. The role of the induced integrated Stx2 phages in natural environments such as river and dam water remains unclear. With the induction of Stx2-converting phages from environmental E. coli O157:H7 isolates, it is now possible to determine the potential of these phages to convert non-pathogenic E. coli strains and other enterobacteriaciae into pathogenic strains.

scholarly journals Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

2004 ◽  
Vol 70 (6) ◽  
pp. 3417-3424 ◽  
Author(s):  
G. O'Flynn ◽  
R. P. Ross ◽  
G. F. Fitzgerald ◽  
A. Coffey
Keyword(s):  
Escherichia Coli ◽  
Biocontrol Agents ◽  
Lytic Phage ◽  
O157 Strain ◽  
E Coli ◽  
Phage Cocktail ◽  
Phage Sensitivity ◽  
Unit Reduction

ABSTRACT Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37�C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10−6 CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10−4 CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10−6 CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.

Detection, Isolation, and Characterization of Shiga Toxin–Producing Escherichia coli in Flour

2019 ◽  
Vol 82 (1) ◽  
pp. 164-167 ◽  
Author(s):  
PATRICK KINDLE ◽  
MAGDALENA NÜESCH-INDERBINEN ◽  
NICOLE CERNELA ◽  
ROGER STEPHAN
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Pcr Amplification ◽  
The United States ◽  
The European Union ◽  
Conventional Pcr ◽  
E Coli ◽  
Isolation And Characterization

ABSTRACT Wheat flour has recently been described as a novel vehicle for transmission of Shiga toxin–producing Escherichia coli (STEC). Very recently, an outbreak of STEC O121 and STEC O26 infections was linked to flour in the United States. The aim of the present study was to generate baseline data for the occurrence of STEC in flour samples from different retailers in Switzerland. In total, 70 flour samples were analyzed. After enrichment, the samples were screened for stx1 and stx2 by the Assurance GDS MPX ID assay. STEC strains were isolated and serotyped by the E. coli SeroGenoTyping AS-1 kit. The determination of stx subtypes was performed with conventional PCR amplification. Screening for eae, aggR, elt, and estIa/Ib was performed by real-time PCR. Nine (12.9%) of the flour samples tested positive for stx by PCR. STEC was recovered from eight (88.9%) of the positive samples. Two isolates were STEC O11:H48 harboring stx1c/stx1d, two were O146:H28 containing stx2b, one was O103:H2 containing stx1a and eae, and three were O nontypeable: Ont:H12 (stx2a), Ont:H14 (stx2a/stx2g), and Ont:H31 (stx1c/stx1d). STEC O103 belongs to the “top five” serogroups of human pathogenic STEC in the European Union, and STEC O146 is frequently isolated from diseased humans in Switzerland. Our results show that flour may be contaminated with a variety of STEC serogroups. Consumption of raw or undercooked flour may constitute a risk for STEC infection.

scholarly journals Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

2009 ◽  
Vol 75 (15) ◽  
pp. 4975-4983 ◽  
Author(s):  
Xianhua Yin ◽  
James R. Chambers ◽  
Roger Wheatcroft ◽  
Roger P. Johnson ◽  
Jing Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Cultured Cells ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
Wild Type Level ◽  
E Coli

ABSTRACT There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA 15 (gene from OI-15) and aidA 48 (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA 48 had no effect on adherence, whereas deletion of aidA 15 resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.

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