scholarly journals Engineered Microbes to Sense and Respond to Enterotoxigenic Escherichia coli

2018 ◽  
Author(s):  
A. Bete ◽  
J. Carter ◽  
C. Davis ◽  
J. Dong ◽  
M. Herrmann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Genetically Engineered ◽  
Surface Proteins ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Autoinducer 2 ◽  
Binding Domains ◽  
Sense And Respond ◽  
Green Fluorescent

AbstractEvery year, Enterotoxigenic Escherichia coli (ETEC), the most common form of traveler’s diarrhea, affects thousands of military personnel deployed overseas. The goal of this research was to engineer non-pathogenic E. coli to sense ETEC, respond to its presence, and package the non-pathogenic E. coli in a cellulose matrix to enable environmental detection of ETEC. Two plasmids were created: ‘sense-respond’; and ‘packaging’. The sense-respond plasmid detected autoinducer 2 (AI-2), a quorum sensing molecule created by most ETEC strains, by expressing LsrR which switches on the Lsr promoter. Activation of the Lsr promoter expresses superfolder green fluorescent protein (sfGFP), indicating the presence of ETEC. The packaging plasmid expresses a fusion protein consisting of curli fibers and cellulose binding domains. These modified surface proteins permit the bacteria to bind to cellulose, encapsulating the sense-response module. This genetically engineered machine could be deployed in both the internal and external environment to detect ETEC.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

scholarly journals Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic

2020 ◽  
Vol 8 (7) ◽  
pp. 1051 ◽  
Author(s):  
Aleksandar Božić ◽  
Robin C. Anderson ◽  
Tawni L. Crippen ◽  
Christina L. Swaggerty ◽  
Michael E. Hume ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Saccharomyces Boulardii ◽  
Binding Activity ◽  
Intestinal Cells ◽  
E Coli ◽  
Enteric Infections ◽  
Green Fluorescent

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

scholarly journals An improved molecular tool for screening bacterial colonies using GFP expression enhanced by a Dictyostelium sequence

BioTechniques ◽  
2020 ◽  
Vol 68 (2) ◽  
pp. 91-95 ◽  
Author(s):  
Tomo Kondo ◽  
Shigehiko Yumura
Keyword(s):  
Escherichia Coli ◽  
Gene Sequence ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Magnetospirillum Gryphiswaldense ◽  
E Coli ◽  
Molecular Tool ◽  
Visual Confirmation ◽  
Green Fluorescent ◽  
User Friendly

During molecular cloning, screening bacterial transformants is a time-consuming and labor-intensive process; however, tractable tools that can be applied to various vectors for visual confirmation of desired colonies are limited. Recently, we reported that translational enhancement by a Dictyostelium gene sequence (TED) boosted protein expression even without an expression inducer in Escherichia coli. Here, we demonstrate a generally applicable molecular tool using the expression of green fluorescent protein enhanced by TED. By inserting a module related to TED into the cloning site in advance, we effectively screened E. coli colonies harboring the desired plasmid functions in a prokaryote ( Magnetospirillum gryphiswaldense) or eukaryote ( Dictyostelium discoideum). Thus, our system represents a user-friendly technique for cloning.

scholarly journals Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

2002 ◽  
Vol 68 (6) ◽  
pp. 3114-3120 ◽  
Author(s):  
A. O. Charkowski ◽  
J. D. Barak ◽  
C. Z. Sarreal ◽  
R. E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Irrigation Water ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Human Pathogens ◽  
E Coli ◽  
Inoculum Dose ◽  
Alfalfa Sprouts ◽  
Green Fluorescent ◽  
Alfalfa Seeds

ABSTRACT Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log10 on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log10. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.

scholarly journals Fate of Escherichia coli during Ensiling of Wheat and Corn

2005 ◽  
Vol 71 (9) ◽  
pp. 5163-5170 ◽  
Author(s):  
Y. Chen ◽  
S. Sela ◽  
M. Gamburg ◽  
R. Pinto ◽  
Z. G. Weinberg
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Dry Matter ◽  
Fluorescent Protein ◽  
Rapid Decrease ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
E Coli ◽  
Aerobic Stability ◽  
Green Fluorescent

ABSTRACT A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 × 108 CFU/g into direct-cut wheat (348 g of dry matter kg−1), wilted wheat (450 g of dry matter kg−1), and corn (375 g of dry matter kg−1). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.

scholarly journals Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

2008 ◽  
Vol 191 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Michael C. Konopka ◽  
Kem A. Sochacki ◽  
Benjamin P. Bratton ◽  
Irina A. Shkel ◽  
M. Thomas Record ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
Fluorescent Protein ◽  
Volume Fraction ◽  
E Coli ◽  
Surprising Effect ◽  
Wide Range ◽  
Underlying Causes ◽  
The Mean ◽  
Green Fluorescent

ABSTRACT Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction (〈φ〉) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar 〈φ〉 range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D GFP) in the cytoplasm of E. coli cells as a function of 〈φ〉 for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D GFP ( \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(D_{GFP}^{m}\) \end{document} ) decreases by a factor of 70 as 〈φ〉 increases from 0.16 to 0.33. In sharp contrast, for adapted cells, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(D_{GFP}^{m}\) \end{document} decreases only by a factor of 2.1 as 〈φ〉 increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by 〈φ〉 alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

scholarly journals Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Shireen Kotay ◽  
Weidong Chai ◽  
William Guilford ◽  
Katie Barry ◽  
Amy J. Mathers
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hand Washing ◽  
Resistant Bacteria ◽  
Droplet Dispersion ◽  
Content Type ◽  
E Coli ◽  
Mode Of Transmission ◽  
Green Fluorescent

ABSTRACT There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

Efficacy of Commercial Produce Sanitizers against Nontoxigenic Escherichia coli O157:H7 during Processing of Iceberg Lettuce in a Pilot-Scale Leafy Green Processing Line

2013 ◽  
Vol 76 (11) ◽  
pp. 1838-1845 ◽  
Author(s):  
GORDON R. DAVIDSON ◽  
ANNEMARIE L. BUCHHOLZ ◽  
ELLIOT T. RYSER
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Pilot Scale ◽  
Wash Water ◽  
Escherichia Coli O157 ◽  
Cross Contamination ◽  
E Coli ◽  
Iceberg Lettuce ◽  
Processing Line ◽  
Green Fluorescent

Chemical sanitizers are routinely used during commercial flume washing of fresh-cut leafy greens to minimize cross-contamination from the water. This study assessed the efficacy of five commercial sanitizer treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line. Iceberg lettuce (5.4 kg) was inoculated to contain 106 CFU/g of a four-strain cocktail of nontoxigenic, green fluorescent protein–labeled, ampicillin-resistant E. coli O157:H7 and processed after 1 h of draining at ~22°C. Lettuce was shredded using a commercial slicer, step-conveyed to a flume tank, washed for 90 s using six different treatments (water alone, 50 ppm of peroxyacetic acid, 50 ppm of mixed peracid, or 50 ppm of available chlorine either alone or acidified to pH 6.5 with citric acid [CA] or T-128), and then dried using a shaker table and centrifugal dryer. Various product (25-g) and water (50-ml) samples collected during processing along with equipment surface samples (100 cm2) from the flume tank, shaker table, and centrifugal dryer were homogenized in neutralizing buffer and plated on tryptic soy agar. During and after iceberg lettuce processing, none of the sanitizers were significantly more effective (P ≤ 0.05) than water alone at reducing E. coli O157:H7 populations on lettuce, with reductions ranging from 0.75 to 1.4 log CFU/g. Regardless of the sanitizer treatment used, the centrifugal dryer surfaces yielded E. coli O157:H7 populations of 3.49 to 4.98 log CFU/100 cm2. Chlorine, chlorine plus CA, and chlorine plus T-128 were generally more effective (P ≤ 0.05) than the other treatments, with reductions of 3.79, 5.47, and 5.37 log CFU/ml after 90 s of processing, respectively. This indicates that chlorine-based sanitizers will likely prevent wash water containing low organic loads from becoming a vehicle for cross-contamination.

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