Microscopic Observation and Processing Validation of Fruit Sanitizing Treatments for the Enhanced Microbiological Safety of Fresh Orange Juice†

2001 ◽  
Vol 64 (3) ◽  
pp. 310-314 ◽  
Author(s):  
STEVEN PAO ◽  
CRAIG L. DAVIS ◽  
MICKEY E. PARISH
Keyword(s):  
Fluorescent Protein ◽  
Citrus Fruit ◽  
Microbial Reduction ◽  
Hot Water ◽  
Near Surface ◽  
E Coli ◽  
Surface Areas ◽  
Orange Fruit ◽  
Green Fluorescent ◽  
The Impact

Studies were conducted to evaluate the infiltration of dye and bacteria into the interior of orange fruit and the impact of possible infiltration on achieving a 5-log microbial reduction during fresh juice processing. Fresh orange fruit were treated at the stem end area with dye and either Salmonella Rubislaw or Escherichia coli strains expressing green fluorescent protein. Microscopic images showed that bacterial contaminants localized at the surface or near surface areas that may be sanitized by surface treatments. Dye infiltration was not a reliable indicator of bacterial penetration in citrus fruit. To quantify the reduction of bacterial contamination, orange fruit were inoculated with E. coli and processed with and without hot water treatments. Greater than 5-log reductions were achieved in juice extracted from fruit immersed in hot water for 1 or 2 min at 80°C, in comparison to the E. coli level detected in the control juice obtained by homogenization of inoculated fruit.

Efficacy of Antimicrobial Agents in Lettuce Leaf Processing Water for Control of Escherichia coli O157:H7

2009 ◽  
Vol 72 (7) ◽  
pp. 1392-1397 ◽  
Author(s):  
GUODONG ZHANG ◽  
LI MA ◽  
VANESSA H. PHELAN ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Organic Load ◽  
Escherichia Coli O157 ◽  
Peroxyacetic Acid ◽  
E Coli ◽  
Processing Water ◽  
Green Fluorescent ◽  
The Impact

The objectives of this research were to study transfer and control of Escherichia coli O157:H7 during simultaneous washing of inoculated and uninoculated lettuce pieces and to determine the efficacy of antimicrobial agents (peroxyacetic acid, mixed peracid, and sodium hypochlorite) on reducing the transfer of E. coli O157:H7 through processing water with or without organic load. Lettuce leaf pieces (5 by 5 cm) were inoculated with a five-strain mixture of green fluorescent protein–labeled E. coli O157:H7 at 5.6 log CFU per piece. One inoculated lettuce piece was added to five uninoculated leaves during washing. Peroxyacetic acid and mixed peracid were tested at 10, 20, and 30 ppm, and chlorine was tested at 30 and 50 ppm. No organic load (liquefied lettuce leaves) and 10% organic load in processing water were compared. Without organic load, peroxyacetic acid at 30 ppm, mixed peracid at 10, 20, and 30 ppm, and chlorine at 30 and 50 ppm all significantly reduced E. coli O157: H7 in processing water by 1.83, 1.73, 1.50, 1.83, 1.34, and 1.83 log CFU/ml, respectively, compared with washing with water alone. These antimicrobials at all concentrations tested also significantly reduced transfer of the bacteria from an inoculated leaf to uninoculated leaves in the processing water by 0.96 to 2.57 log CFU per piece. A 10% organic load in the processing water reduced efficacy of antimicrobial agents. In this contaminated water, peroxyacetic acid at 10 and 20 ppm and chlorine at 30 ppm produced effects not significantly different from those of water alone. Therefore, it is important to understand the impact of organic load when validating the effectiveness of antimicrobial treatments.

Soil wetting state and preferential transport of Escherichia coli in clay soils

2007 ◽  
Vol 87 (1) ◽  
pp. 61-72 ◽  
Author(s):  
L K Tallon ◽  
B C Si ◽  
D. Korber ◽  
X. Guo
Keyword(s):  
Escherichia Coli ◽  
Preferential Flow ◽  
Fluorescent Protein ◽  
Transport Pathways ◽  
Near Surface ◽  
E Coli ◽  
Significant Difference ◽  
Preferential Transport ◽  
Soil Wetting ◽  
Green Fluorescent

Transport of Escherichia coli (E. coli) through soil to drinking and recreational water may pose a serious health risk. The objective of this study was to determine how initial preferred soil wetting state influences the preferential transport of E. coli in a clay soil. A strain of E. coli marked with green fluorescent protein (gfp) was gravity-fed-sprinkler-applied as simulated rainfall to three replicates in different wetting states, along with Cl- and adsorptive dye near Plenty, SK. Canada. After 48 h, a 50 × 50 × 50 cm3 block was excavated to determine the transport pathways. Digital image analysis of horizontal sections provided estimates of dye coverage. Escherichia coli were significantly filtered in the top 10 cm of soil with concentration profiles similar to that of Cl-. Ratios of E. coli to Cl- did not show significant differences among treatments (P < 0.05) and indicated that below 10 cm depth, E. coli and Cl- were preferentially transported along the same pathways with no significant difference between plots. Results show that the majority of E. coli and Cl- were filtered when transported through the discontinuous pores of the near-surface matrix and suggest a saturated layer that controlled infiltration into organized root channels, resulting in preferential flow. Key words: Preferential flow, Escherichia coli, wetting state, Vertisol, conducting areas

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.

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 Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Shireen M. Kotay ◽  
Rodney M. Donlan ◽  
Christine Ganim ◽  
Katie Barry ◽  
Bryan E. Christensen ◽  
...  
Keyword(s):  
Patient Care ◽  
Sampling Methods ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Air Sampling ◽  
Multidrug Resistant ◽  
Hand Washing ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

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 &lt; 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.

Designing Authentic Undergraduate Research Experiences in a Single-Semester Lab Course

2015 ◽  
Vol 77 (7) ◽  
pp. 526-531 ◽  
Author(s):  
Thomas A. Mennella
Keyword(s):  
Fluorescent Protein ◽  
Undergraduate Research ◽  
Research Experience ◽  
Research Project ◽  
Research Experiences ◽  
Laboratory Course ◽  
E Coli ◽  
Undergraduate Research Experiences ◽  
Green Fluorescent ◽  
Scheduled Time

The importance of a robust undergraduate research experience has been demonstrated time and again. However, too few undergraduates engage in genuine research and leverage this opportunity. Here, I present a laboratory course in cell and molecular biology that is designed to mimic a true research project. Students work through a 10-step experimental design culminating in the construction, expression, and visualization of microtubules fused to green fluorescent protein in baker's yeast. The steps of this project include the isolation of the tubulin gene from yeast genomic DNA, the cloning of that gene into an expression vector, the amplification of this plasmid in E. coli, and the expression of fluorescent tubulin in yeast. Controls and validation steps are embedded throughout the project, as they would be in a genuine research project. This laboratory course more closely resembles a one-semester undergraduate research experience than a typical lab course. However, because this course reaches a much larger number of students compared with undergraduate research opportunities, it provides students with a valuable research experience that remains confined to the scheduled time block of a typical lab course. In this way, many of the benefits of research are experienced by a large number of undergraduates.

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.

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