scholarly journals Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach

2008 ◽  
Vol 74 (8) ◽  
pp. 2518-2525 ◽  
Author(s):  
Poornima Gourabathini ◽  
Maria T. Brandl ◽  
Katherine S. Redding ◽  
John H. Gunderson ◽  
Sharon G. Berk
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Pyriformis ◽  
Enteric Bacteria ◽  
Enteric Pathogens ◽  
Leafy Vegetables ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.

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.

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 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 Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens

2005 ◽  
Vol 71 (7) ◽  
pp. 4052-4056 ◽  
Author(s):  
Shlomo Sela ◽  
David Nestel ◽  
Riky Pinto ◽  
Esther Nemny-Lavy ◽  
Moshe Bar-Joseph
Keyword(s):  
Fluorescent Protein ◽  
Tap Water ◽  
Bacterial Load ◽  
Fruit Fly ◽  
Mediterranean Fruit Fly ◽  
Human Pathogens ◽  
Potential Vector ◽  
E Coli ◽  
Wild Fruits ◽  
Green Fluorescent

ABSTRACT The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.

scholarly journals Persistence of Human Pathogens in Manure-Amended Australian Soils Used for Production of Leafy Vegetables

Agriculture ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 14
Author(s):  
Jennifer Ekman ◽  
Adam Goldwater ◽  
Mark Bradbury ◽  
Jim Matthews ◽  
Gordon Rogers
Keyword(s):  
Production Systems ◽  
Limit Of Detection ◽  
Probability Of Detection ◽  
Enteric Pathogens ◽  
Leafy Vegetables ◽  
Vegetable Production ◽  
Human Pathogens ◽  
Salmonella Spp ◽  
E Coli ◽  
Vegetable Farm

Incorporation of manures into soil can add nutrients, improve soil structure and enhance biodiversity, thereby improving the sustainability of vegetable production systems. Unfortunately, manures can also potentially contain human enteric pathogens. To reduce the risk of contamination, Australian guidelines recommend a withholding period of 90 days between manure application and harvest for high-risk products such as leafy salad greens. Our study examined the appropriateness of these guidelines under conditions replicating those on a commercial vegetable farm. Cow manure and poultry litter with/without addition of non-pathogenic strains of Escherichia coli (E. coli), Listeriainnocua and Salmonella sv. sofia were added to sandy and clay loam soils typical of those used to grow vegetables. Bacterial populations were monitored in the soil and on crops of cos lettuce during spring (A), summer (B) and autumn (C) trials, with testing conducted by a commercial laboratory. Significant declines in E. coli occurred within 6 to 16 days in all trials. Modelling indicated that E. coli populations would be at or close to the limit of detection within 50 days for all of the combinations tested. A 2–3 log die-off of Salmonella spp. occurred within three weeks. However, occasional detections continued throughout trial A. As a result, the probability of detection after 50 days fell from 1.0 to 0.1 and 0.02 in trials B and C, respectively, but remained at 0.44 in trial A. Listeria spp. was the most persistent in soil but was not detected on lettuce at commercial maturity. While this study was limited in scope, the results suggest that a 90 day withholding period between application of manure and harvest significantly reduces risk from enteric pathogens under Australian field conditions.

Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report

2005 ◽  
Vol 14 (5) ◽  
pp. 323-330 ◽  
Author(s):  
L. H. Granicka ◽  
M. Wdowiak ◽  
A. Kosek ◽  
S. Świezewski ◽  
D. Wasilewska ◽  
...  
Keyword(s):  
Hollow Fiber ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Hollow Fiber Membrane ◽  
In Vitro Experiments ◽  
Bacteria Growth ◽  
E Coli ◽  
Green Fluorescent

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.

scholarly journals The N Terminus of MinD Contains Determinants Which Affect Its Dynamic Localization and Enzymatic Activity

2004 ◽  
Vol 186 (21) ◽  
pp. 7175-7185 ◽  
Author(s):  
Jason Szeto ◽  
Sudeep Acharya ◽  
Nelson F. Eng ◽  
Jo-Anne R. Dillon
Keyword(s):  
Enzymatic Activity ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Conserved Residues ◽  
Dynamic Localization ◽  
Mutant Proteins ◽  
E Coli ◽  
N Terminus ◽  
Green Fluorescent

ABSTRACT MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinDNg) on protein dynamism, localization, and interactions with MinDNg and with MinENg. Deletions or mutations were generated in the first five residues of MinDNg, and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinDNg interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinDNg mutants could still oscillate from pole to pole in E. coli, the GFP-MinDNg oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinDNg proteins lacking the first three residues or with an I5E substitution possessed higher MinENg-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinDNg are implicated in regulating the enzymatic activity and dynamic localization of the protein.

scholarly journals The Tubulin-Like RepX Protein Encoded by the pXO1 Plasmid Forms Polymers In Vivo in Bacillus anthracis

2009 ◽  
Vol 191 (8) ◽  
pp. 2493-2500 ◽  
Author(s):  
Parvez Akhtar ◽  
Syam P. Anand ◽  
Simon C. Watkins ◽  
Saleem A. Khan
Keyword(s):  
Bacillus Anthracis ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Host Cells ◽  
E Coli ◽  
Green Fluorescent ◽  
Related Proteins ◽  
Polymeric Structures

ABSTRACT Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.

scholarly journals Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 74 ◽  
Author(s):  
Jaime H. Amorim ◽  
Monica R. Jesus ◽  
Wilson B. Luiz ◽  
Bruna F.M.M. Porchia ◽  
Rita C.C. Ferreira ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Lytic Replication ◽  
Biologically Active ◽  
Gene Encoding ◽  
E Coli ◽  
Green Fluorescent

Shiga toxin (Stx) is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC) infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter. Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli K12 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP). Tracking GFP expression using an In Vivo Imaging System (IVIS), we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of D-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated in vitro and in vivo. These findings bring promising perspectives for the prevention and treatment of hemolytic uremic syndrome (HUS) cases.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

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