scholarly journals Differences in Colonization and Shedding Patterns after Oral Challenge of Cattle with Three Escherichia coli O157:H7 Strains

2012 ◽  
Vol 78 (22) ◽  
pp. 8045-8055 ◽  
Author(s):  
Megan J. Kulow ◽  
Tina K. Gonzales ◽  
Kelly M. Pertzborn ◽  
James Dahm ◽  
Bret A. Miller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Oral Challenge ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli ◽  
Group 4 ◽  
Group 3

ABSTRACTExperimental oral challenge studies with three different genotypes ofEscherichia coliO157:H7 were conducted in cattle to determine the genotype-specific variability in shedding frequencies and concentrations and the frequency and extent of contamination of the environment. The results indicated that theE. coliO157:H7 genotype and ecological origin maybe important factors for the occurrence and concentration in the cattle host. Four groups of six young Holstein steers each were orally challenged with 106CFU of one of threeE. coliO157:H7 strains: FRIK 47 (groups 1 and 2), FRIK 1641 (group 3), and FRIK 2533 (group 4). Recto-anal mucosal swabs (RAMS) and environmental samples were taken on alternate days over 30 days. The numbers ofE. coliO157:H7 cells and genericE. colicells per sample were determined. Also, the presence and absence of 28 gene targets were determined for 2,411 isolates using high-throughput real-time PCR. Over the study period, strains FRIK 47, FRIK 1641, and FRIK 2533 were detected in 52%, 42%, and 2% of RAMS, respectively. Environmental detection of the challenge strains was found mainly in samples of the hides and pen floors, with strains FRIK 47, FRIK 1641, and FRIK 2533 detected in 22%, 27%, and 0% of environmental samples, respectively. Based on the panel of 28 gene targets, genotypes of enterohemorrhagicE. coli(EHEC) and genericE. colifrom the experimental samples were clustered into three subgroups. In conclusion, the results suggested that the type and intensity of measures to control this pathogen at the preharvest level may need to be strain specific.

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals Fecal Metagenome Sequences from Lactating Dairy Cows Shedding Escherichia coli O157:H7

2018 ◽  
Vol 7 (18) ◽  
Author(s):  
Serajus Salaheen ◽  
Seon Woo Kim ◽  
Jeffrey S. Karns ◽  
Bradd J. Haley ◽  
Jo Ann S. Van Kessel
Keyword(s):  
Escherichia Coli ◽  
Dairy Cows ◽  
Causative Agent ◽  
Public Domain ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
The Public ◽  
E Coli ◽  
Lactating Dairy Cows ◽  
Human Infections

Cattle are primary reservoirs of Escherichia coli O157:H7, a causative agent of severe human infections. To facilitate analyses of the communities in which this pathogen is found, we sequenced the fecal metagenomes of 10 dairy cows shedding E. coli O157:H7 and added them to the public domain.

Comparative Performance Evaluation of Real-Time PCR and Dual-Labeled Fluorescence Resonance Energy Transfer Probe-Based Melt Peak Analysis for the Detection of Escherichia coli O157:H7 in Beef Products

2019 ◽  
Vol 82 (3) ◽  
pp. 507-512
Author(s):  
JOSEPH M. BOSILEVAC ◽  
HARI P. DWIVEDI ◽  
PATRICE CHABLAIN ◽  
MICHAEL ULLERY ◽  
JOSEPH S. BAILEY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Performance Evaluation ◽  
Energy Transfer ◽  
Real Time Pcr ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Escherichia Coli O157 ◽  
Comparative Performance ◽  
E Coli ◽  
Beef Products

ABSTRACT Contaminated beef and beef products remain a frequent vehicle for the transmission of Escherichia coli O157:H7. The current U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) regulatory testing for E. coli O157:H7 uses the method described in the USDA-FSIS Microbiology Laboratory Guidebook (MLG), chapter 5. At times, described presumptive test results are nonconfirmable, suggesting that recent PCR technological advancements and presumed enhanced sensitivity and specificity may offer beneficial changes. Here, we have evaluated the precision and sensitivity of a fluorescence resonance energy transfer–based real-time PCR assay called ECO for the detection of E. coli O157:H7. ECO detects the gene target specific to both E. coli O157:H7 and E. coli O157:non-H7 but distinguishes the two by using a melt curve analysis. A total of 3,113 O157:H7 and O157:non-H7 isolates were used to define this melting temperature–based criteria. The simulated comparative performance evaluation in the spiked beef samples indicated detection of 3 of 3 samples by ECO at &lt;3.3 log CFU/mL, whereas MLG only detected 1 of 3 (&lt;3.3 log CFU/mL). Using modified tryptic soy broth–enriched natural beef and veal product samples (n = 452), the comparative sensitivity, specificity, false-positive rate, and false-negative rate against culture between MLG and ECO were 75 versus 92%, 91 versus 99%, 8.9 versus 0.77%, and 25 versus 8.3%, respectively. Positive predictive value, negative predictive value, and the overall accuracy were found to be 56 versus 94%, 96 versus 98%, and 88 versus 98%, for MLG and ECO, respectively. These data demonstrate that the ECO assay is comparable to MLG detection of E. coli O157:H7 and offers improved sensitivity.

scholarly journals Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms

2012 ◽  
Vol 81 (3) ◽  
pp. 838-849 ◽  
Author(s):  
Francis Dziva ◽  
Heidi Hauser ◽  
Thomas R. Connor ◽  
Pauline M. van Diemen ◽  
Graham Prescott ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Core Genome ◽  
Systemic Disease ◽  
Genomic Islands ◽  
Content Type ◽  
E Coli ◽  
Group 4 ◽  
Pathogenic Escherichia Coli ◽  
Transposon Mutants ◽  
Avian Disease

ABSTRACTAvian pathogenicEscherichia coli(APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resemblesE. colicausing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenicE. coli(ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity ofE. colistrains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution ofE. colistrains adapted to cause avian or human disease via acquisition of distinct virulence genes.

scholarly journals Salmonella and Escherichia coli O157:H7 Survival in Soil and Translocation into Leeks (Allium porrum) as Influenced by an Arbuscular Mycorrhizal Fungus (Glomus intraradices)

2013 ◽  
Vol 79 (6) ◽  
pp. 1813-1820 ◽  
Author(s):  
Joshua B. Gurtler ◽  
David D. Douds ◽  
Brian P. Dirks ◽  
Jennifer J. Quinlan ◽  
April M. Nicholson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glomus Intraradices ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Allium Porrum ◽  
Escherichia Coli O157 ◽  
Content Type ◽  
E Coli ◽  
The Mean

ABSTRACTA study was conducted to determine the influence of arbuscular mycorrhizal (AM) fungi onSalmonellaand enterohemorrhagicEscherichia coliO157:H7 (EHEC) in autoclaved soil and translocation into leek plants. Six-week-old leek plants (with [Myc+] or without [Myc−] AM fungi) were inoculated with composite suspensions ofSalmonellaor EHEC at ca. 8.2 log CFU/plant into soil. Soil, root, and shoot samples were analyzed for pathogens on days 1, 8, 15, and 22 postinoculation. Initial populations (day 1) were ca. 3.1 and 2.1 log CFU/root, ca. 2.0 and 1.5 log CFU/shoot, and ca. 5.5 and 5.1 CFU/g of soil forSalmonellaand EHEC, respectively. Enrichments indicated that at days 8 and 22, only 31% of root samples were positive for EHEC, versus 73% positive forSalmonella. The meanSalmonellalevel in soil was 3.4 log CFU/g at day 22, while EHEC populations dropped to ≤0.75 log CFU/g by day 15. Overall,Salmonellasurvived in a greater number of shoot, root, and soil samples, compared with the survival of EHEC. EHEC was not present in Myc− shoots after day 8 (0/16 samples positive); however, EHEC persisted in higher numbers (P= 0.05) in Myc+ shoots (4/16 positive) at days 15 and 22.Salmonella, likewise, survived in statistically higher numbers of Myc+ shoot samples (8/8) at day 8, compared with survival in Myc− shoots (i.e., only 4/8). These results suggest that AM fungi may potentially enhance the survival ofE. coliO157:H7 andSalmonellain the stems of growing leek plants.

scholarly journals Physical Covering for Control of Escherichia coli O157:H7 and Salmonella spp. in Static and Windrow Composting Processes

2015 ◽  
Vol 81 (6) ◽  
pp. 2063-2074 ◽  
Author(s):  
Jitendra R. Patel ◽  
Irene Yossa ◽  
Dumitru Macarisin ◽  
Patricia Millner
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Bacterial Reduction ◽  
Salmonella Spp ◽  
Rapid Reduction ◽  
Content Type ◽  
E Coli ◽  
Windrow Composting ◽  
Composting Systems ◽  
Feedstock Material

ABSTRACTThis study investigated the effect of a 30-cm covering of finished compost (FC) on survival ofEscherichia coliO157:H7 andSalmonellaspp. in active static and windrow composting systems. Feedstocks inoculated withE. coliO157:H7 (7.41 log CFU/g) andSalmonella(6.46 log CFU/g) were placed in biosentry tubes (7.5-cm diameter, 30-cm height) at three locations: (i and ii) two opposing sides at the interface between the FC cover layer (where present) and the feedstock material (each positioned approximately 10 cm below the pile's surface) and (iii) an internal location (top) (approximately 30 cm below the surface). On specific sampling days, surviving populations of inoculatedE. coliO157:H7 andSalmonella, genericE. coli, and coliforms in compost samples were determined.Salmonellaspp. were reduced significantly within 24 h in windrow piles and were below the detection limit after 3 and 7 days at internal locations of windrow and static piles containing FC covering, respectively. Likewise,E. coliO157:H7 was undetectable after 1 day in windrow piles covered with finished compost. Use of FC as a covering layer significantly increased the number of days that temperatures in the windrows remained ≥55°C at all locations and in static piles at internal locations. These time-temperature exposures resulted in rapid reduction of inoculated pathogens, and the rate of bacterial reduction was rapid in windrow piles. The sample location significantly influenced the survival of these pathogens at internal locations compared to that at interface locations of piles. Finished compost covering of compost piles aids in the reduction of pathogens during the composting process.

Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

2007 ◽  
Vol 70 (12) ◽  
pp. 2717-2724 ◽  
Author(s):  
SUNEE HIMATHONGKHAM ◽  
MARY LEE DODD ◽  
JENNY K. YEE ◽  
DAVID K. LAU ◽  
RAYMOND G. BRYANT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Simple Method ◽  
E Coli ◽  
Low Levels ◽  
Spinach Leaves

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

scholarly journals Analysis of the Resistome of a Multidrug-Resistant NDM-1-Producing Escherichia coli Strain by High-Throughput Genome Sequencing

2011 ◽  
Vol 55 (9) ◽  
pp. 4224-4229 ◽  
Author(s):  
Laurent Poirel ◽  
Rémy A. Bonnin ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Genome Sequencing ◽  
High Throughput ◽  
Multidrug Resistant ◽  
Replicase Gene ◽  
Content Type ◽  
E Coli ◽  
Resistance Determinants ◽  
16S Rna ◽  
Carbapenemase Gene

ABSTRACTThe resistome of the multidrug-resistantEscherichia colistrain 271 carrying the plasmid-mediatedblaNDM-1carbapenemase gene was analyzed by high-throughput genome sequencing. The p271A plasmid carrying theblaNDM-1gene was 35.9 kb in size and possessed an IncN-type backbone that harbored a novel replicase gene. Acquisition of theblaNDM-1gene on plasmid p271A had been likely the result of a cointegration event involving the transposase of Tn5403. The expression ofblaNDM-1was associated with the insertion sequence ISAba125likely originating fromAcinetobacter baumannii. E. coli271 accumulated multiple resistance determinants, including five β-lactamase genes (comprising the extended-spectrum β-lactamase CTX-M-15), two 16S RNA methylase ArmA- and RmtB-encoding genes, and theqepAgene encoding an efflux pump involved in resistance to fluoroquinolones. These resistance genes were located on three additional plasmids, of 160 kb (IncA/C), 130 kb (IncF), and 110 kb (IncI1). In addition, several chromosomally encoded resistance determinants were identified, such as topoisomerase mutations, porin modifications and truncations, and the intrinsicampCgene ofE. colithat was weakly expressed. The multidrug resistance pattern observed forE. coli271 was therefore the result of combined chromosome- and plasmid-encoded mechanisms.

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