Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

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Reham Wasfi

Full Text Available The purpose of this study was to: (i evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram against Escherichia coli; (ii investigate the effects of these honeys on bacterial ultrastructure; and (iii assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival in the test organism. The minimum inhibitory concentration (MIC of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM and quantitative real-time polymerase chain reaction (qPCR analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli

DEFF Research Database (Denmark)

Koehler, JF; Birkelund, Svend; Stephens, RS

1992-01-01

The Chlamydia trachomatis major outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP in Escherichia coli. The MOMP is surface exposed in C. trachomatis....... The induction of MOMP expression had a rapidly lethal effect on the L2rMOMP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli clone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including...

Identification of Characteristic Macromolecules of Escherichia coli Genotypes by Atomic Force Microscope Nanoscale Mechanical Mapping

Science.gov (United States)

Chang, Alice Chinghsuan; Liu, Bernard Haochih

2018-02-01

The categorization of microbial strains is conventionally based on the molecular method, and seldom are the morphological characteristics in the bacterial strains studied. In this research, we revealed the macromolecular structures of the bacterial surface via AFM mechanical mapping, whose resolution was not only determined by the nanoscale tip size but also the mechanical properties of the specimen. This technique enabled the nanoscale study of membranous structures of microbial strains with simple specimen preparation and flexible working environments, which overcame the multiple restrictions in electron microscopy and label-enable biochemical analytical methods. The characteristic macromolecules located among cellular surface were considered as surface layer proteins and were found to be specific to the Escherichia coli genotypes, from which the averaged molecular sizes were characterized with diameters ranging from 38 to 66 nm, and the molecular shapes were kidney-like or round. In conclusion, the surface macromolecular structures have unique characteristics that link to the E. coli genotype, which suggests that the genomic effects on cellular morphologies can be rapidly identified using AFM mechanical mapping. [Figure not available: see fulltext.

Nanoparticle Surface Functionality Dictates Cellular and Systemic Toxicity

DEFF Research Database (Denmark)

Saei, Amir Ata; Yazdani, Mahdieh; Lohse, Samuel E.

2017-01-01

can greatly enhance subsequent therapeutic effects of NPs while diminishing their adverse side effects. In this review, we will focus on the effect of surface functionality on the cellular uptake and the transport of NPs by various subcellular processes.......Engineered nanoparticles (NPs) have opened new frontiers in therapeutics and diagnostics in recent years. The surface functionality of these nanoparticles often predominates their interactions with various biological components of human body, and proper selection or control of surface functionality...

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli

Science.gov (United States)

Bhat, Supriya V.; Kamencic, Belma; Körnig, André; Shahina, Zinnat; Dahms, Tanya E. S.

2018-01-01

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage. PMID:29472899

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

International Nuclear Information System (INIS)

Ferreira, L.C.S.; Almeida, D.F. de

1987-01-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and 131 I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42 0 C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12. (author) [pt

Iodo-gen-catalysed iodination for identification of surface-exposed outer membrane proteins of Escherichia coli K12

Energy Technology Data Exchange (ETDEWEB)

Ferreira, L C.S.; Almeida, D.F. de

1987-12-01

Surface proteins of Escherichia coli K12 were identified by radiolabelling using 1,3,4,6 - tatrachloro, 3-alpha, 6-alpha - diphenylgycoluryl (Iodo-Gen) and /sup 131/I. Labelled proteins were localized in the outer membrane of the cells. Using this technique it has been possible to observe technique it has been possible to observe that the eletrophoretic pattern of surface proteins changes according to the growth phases in culture. Radiolabelling of E.coli cells inculbated at 42/sup 0/C showed that the syntheses of two surface proteins were temperature-inducible. At least one such protein may be involved in the process of cell division in E.coli K12.

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

OpenAIRE

Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

2011-01-01

NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. co...

Escherichia coli in the surface waters and in oysters of two cultivations of Guaratuba Bay - Paraná - Brazil

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Helenita Catharina Dalla-Lana Forcelini

2013-04-01

Full Text Available The present work aimed to evaluate the contamination of Escherichia coli in the surface waters and oysters from two cultivations of Guaratuba Bay and to analyze the correlation patterns among the concentrations of E. coli in the waters and in the oysters with the local physical-chemical parameters. Samples were collected in the spring of 2007 and summer, autumn and winter of 2008 from two points of the bay (internal point and external point. From each cultivation and sampling period, 18 oysters were collected. The samples of surface water were collected for the measurement of physical-chemical parameters (pH, salinity, temperature, dissolved oxygen, seston, particulate organic matter and quantification of E. coli. The surface water analyzed in the summer presented the largest most probable number of E. coli, (1,659.22 MPN.100 ml-1 and 958,55 MPN.100 ml-1 at external and internal points, respectively. The oysters from the internal point presented more E. coli, except in the winter sampling. The largest contamination was observed in the spring, at the internal point (979,78 MPN.g-1. The Principal Components Analysis showed direct correlation among the amount of E. coli in the oysters and in the surface water.

Surface ligand controls silver ion release of nanosilver and its antibacterial activity against Escherichia coli

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Long Y

2017-04-01

Full Text Available Yan-Min Long,1,2 Li-Gang Hu,1,3 Xue-Ting Yan,1,3 Xing-Chen Zhao,1,3 Qun-Fang Zhou,1,3 Yong Cai,2,4 Gui-Bin Jiang1,3 1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Beijing, China; 2Institute of Environment and Health, Jianghan University, Wuhan, Hubei, China; 3College of Resources and Environment, University of Chinese Academy of Sciences, Beijing, China; 4Department of Chemistry and Biochemistry, Southeast Environmental Research Center, Florida International University, Miami, FL, USA Abstract: Understanding the mechanism of nanosilver-dependent antibacterial activity against microorganisms helps optimize the design and usage of the related nanomaterials. In this study, we prepared four kinds of 10 nm-sized silver nanoparticles (AgNPs with dictated surface chemistry by capping different ligands, including citrate, mercaptopropionic acid, mercaptohexanoic acid, and mercaptopropionic sulfonic acid. Their surface-dependent chemistry and antibacterial activities were investigated. Owing to the weak bond to surface Ag, short carbon chain, and low silver ion attraction, citrate-coated AgNPs caused the highest silver ion release and the strongest antibacterial activity against Escherichia coli, when compared to the other tested AgNPs. The study on the underlying antibacterial mechanisms indicated that cellular membrane uptake of Ag, NAD+/NADH ratio increase, and intracellular reactive oxygen species (ROS generation were significantly induced in both AgNP and silver ion exposure groups. The released silver ions from AgNPs inside cells through a Trojan-horse-type mechanism were suggested to interact with respiratory chain proteins on the membrane, interrupt intracellular O2 reduction, and induce ROS production. The further oxidative damages of lipid peroxidation and membrane breakdown caused the lethal effect on E. coli. Altogether, this study demonstrated that AgNPs exerted

Pathogenic Escherichia coli producing Extended-Spectrum β-Lactamases isolated from surface water and wastewater.

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Franz, Eelco; Veenman, Christiaan; van Hoek, Angela H A M; de Roda Husman, Ana; Blaak, Hetty

2015-09-24

To assess public health risks from environmental exposure to Extended-Spectrum β-Lactamases (ESBL)-producing bacteria, it is necessary to have insight in the proportion of relative harmless commensal variants and potentially pathogenic ones (which may directly cause disease). In the current study, 170 ESBL-producing E. coli from Dutch wastewater (n = 82) and surface water (n = 88) were characterized with respect to ESBL-genotype, phylogenetic group, resistance phenotype and virulence markers associated with enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), extraintesinal E. coli (ExPEC), and Shiga toxin-producing E. coli (STEC). Overall, 17.1% of all ESBL-producing E. coli were suspected pathogenic variants. Suspected ExPECs constituted 8.8% of all ESBL-producing variants and 8.3% were potential gastrointestinal pathogens (4.1% EAEC, 1.8% EPEC, 1.2% EIEC, 1.2% ETEC, no STEC). Suspected pathogens were significantly associated with ESBL-genotype CTX-M-15 (X(2) = 14.7, P antibiotics. In conclusion, this study demonstrates that the aquatic environment is a potential reservoir of E. coli variants that combine ESBL-genes, a high level of multi-drug resistance and virulence factors, and therewith pose a health risk to humans upon exposure.

Electrolyte effects on the surface chemistry and cellular response of anodized titanium

International Nuclear Information System (INIS)

Ohtsu, Naofumi; Kozuka, Taro; Hirano, Mitsuhiro; Arai, Hirofumi

2015-01-01

Highlights: • Ti samples were anodized using various electrolytes. • Anodization decreased carbon adsorption, improving hydrophilicity. • Improved hydrophilicity led to improved cellular attachment. • Only one electrolyte showed any heteroatom incorporation into the TiO 2 layer. • Choice of electrolyte played no role on the effects of anodization. - Abstract: Anodic oxidation of titanium (Ti) material is used to enhance biocompatibility, yet the effects of various electrolytes on surface characteristics and cellular behavior have not been completely elucidated. To investigate this topic, oxide layers were produced on Ti substrates by anodizing them in aqueous electrolytes of (NH 4 ) 2 O·5B 2 O 3 , (NH 4 ) 2 SO 4 , or (NH 4 ) 3 PO 4 , after which their surface characteristics and cellular responses were examined. Overall, no surface differences between the electrolytes were visually observed. X-ray photoelectron spectroscopy (XPS) revealed that the anodized surfaces are composed of titanium dioxide (TiO 2 ), while incorporation from electrolyte was only observed for (NH 4 ) 3 PO 4 . Surface adsorption of carbon contaminants during sterilization was suppressed by anodization, leading to lower water contact angles. The attachment of MC3T3-E1 osteoblast-like cells was also improved by anodization, as evidenced by visibly enlarged pseudopods. This improved attachment performance is likely due to TiO 2 formation. Overall, electrolyte selection showed no effect on either surface chemistry or cellular response of Ti materials

Surface topography and chemistry shape cellular behavior on wide band-gap semiconductors.

Science.gov (United States)

Bain, Lauren E; Collazo, Ramon; Hsu, Shu-Han; Latham, Nicole Pfiester; Manfra, Michael J; Ivanisevic, Albena

2014-06-01

The chemical stability and electrical properties of gallium nitride make it a promising material for the development of biocompatible electronics, a range of devices including biosensors as well as interfaces for probing and controlling cellular growth and signaling. To improve the interface formed between the probe material and the cell or biosystem, surface topography and chemistry can be applied to modify the ways in which the device interacts with its environment. PC12 cells are cultured on as-grown planar, unidirectionally polished, etched nanoporous and nanowire GaN surfaces with and without a physisorbed peptide sequence that promotes cell adhesion. While cells demonstrate preferential adhesion to roughened surfaces over as-grown flat surfaces, the topography of that roughness also influences the morphology of cellular adhesion and differentiation in neurotypic cells. Addition of the peptide sequence generally contributes further to cellular adhesion and promotes development of stereotypic long, thin neurite outgrowths over alternate morphologies. The dependence of cell behavior on both the topographic morphology and surface chemistry is thus demonstrated, providing further evidence for the importance of surface modification for modulating bio-inorganic interfaces. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Surface modification of PLGA nanoparticles to deliver nitric oxide to inhibit Escherichia coli growth

Energy Technology Data Exchange (ETDEWEB)

Reger, Nina A. [Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 (United States); Meng, Wilson S. [Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282 (United States); Gawalt, Ellen S., E-mail: gawalte@duq.edu [Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219 (United States)

2017-04-15

Highlights: • Thin film functionalized PLGA nanoparticles were modified to release nitric oxide from an s-nitrosothiol donor. • The nitric oxide modified nanoparticles were bacteriostatic against Escherichia coli. • The nitric oxide modified nanoparticles increased the effectiveness of tetracycline against Escherichia coli. • The modified nitric oxide nanoparticles did not exhibit cytotoxic effects against fibroblasts. - Abstract: Polymer nanoparticles consisting of poly (DL-lactic-co-glycolic acid) were surface functionalized to deliver nitric oxide. These biodegradable and biocompatible nanoparticles were modified with an S-nitrosothiol molecule, S-nitrosocysteamine, as the nitric oxide delivery molecule. S-nitrosocysteamine was covalently immobilized on the nanoparticle surface using small organic molecule linkers and carbodiimide coupling. Nanoparticle size, zeta potential, and morphology were determined using dynamic light scattering and scanning electron microscopy, respectively. Subsequent attachment of the S-nitrosothiol resulted in a nitric oxide release of 37.1 ± 1.1 nmol per milligram of nanoparticles under physiological conditions. This low concentration of nitric oxide reduced Escherichia coli culture growth by 31.8%, indicating that the nitric oxide donor was effective at releasing nitric oxide even after attachment to the nanoparticle surface. Combining the nitric oxide modified nanoparticles with tetracycline, a commonly prescribed antibiotic for E. coli infections, increased the effectiveness of the antibiotic by 87.8%, which allows for lower doses of antibiotics to be used in order to achieve the same effect. The functionalized nanoparticles were not cytotoxic to mouse fibroblasts.

Surface modification of PLGA nanoparticles to deliver nitric oxide to inhibit Escherichia coli growth

International Nuclear Information System (INIS)

Reger, Nina A.; Meng, Wilson S.; Gawalt, Ellen S.

2017-01-01

Highlights: • Thin film functionalized PLGA nanoparticles were modified to release nitric oxide from an s-nitrosothiol donor. • The nitric oxide modified nanoparticles were bacteriostatic against Escherichia coli. • The nitric oxide modified nanoparticles increased the effectiveness of tetracycline against Escherichia coli. • The modified nitric oxide nanoparticles did not exhibit cytotoxic effects against fibroblasts. - Abstract: Polymer nanoparticles consisting of poly (DL-lactic-co-glycolic acid) were surface functionalized to deliver nitric oxide. These biodegradable and biocompatible nanoparticles were modified with an S-nitrosothiol molecule, S-nitrosocysteamine, as the nitric oxide delivery molecule. S-nitrosocysteamine was covalently immobilized on the nanoparticle surface using small organic molecule linkers and carbodiimide coupling. Nanoparticle size, zeta potential, and morphology were determined using dynamic light scattering and scanning electron microscopy, respectively. Subsequent attachment of the S-nitrosothiol resulted in a nitric oxide release of 37.1 ± 1.1 nmol per milligram of nanoparticles under physiological conditions. This low concentration of nitric oxide reduced Escherichia coli culture growth by 31.8%, indicating that the nitric oxide donor was effective at releasing nitric oxide even after attachment to the nanoparticle surface. Combining the nitric oxide modified nanoparticles with tetracycline, a commonly prescribed antibiotic for E. coli infections, increased the effectiveness of the antibiotic by 87.8%, which allows for lower doses of antibiotics to be used in order to achieve the same effect. The functionalized nanoparticles were not cytotoxic to mouse fibroblasts.

Escherichia coli lipoprotein binds human plasminogen via an intramolecular domain

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Tammy eGonzalez

2015-10-01

Full Text Available Escherichia coli lipoprotein (Lpp is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysines in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen, a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to plasminogen, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp-plasminogen interactions were examined. Additionally, the ability of Lpp-bound plasminogen to be converted to active plasmin was analyzed. We determined that Lpp binds plasminogen via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that plasminogen bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding plasminogen are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria.

Inactivation of Escherichia coli O157:H7 in biofilm on food-contact surfaces by sequential treatments of aqueous chlorine dioxide and drying.

Science.gov (United States)

Bang, Jihyun; Hong, Ayoung; Kim, Hoikyung; Beuchat, Larry R; Rhee, Min Suk; Kim, Younghoon; Ryu, Jee-Hoon

2014-11-17

We investigated the efficacy of sequential treatments of aqueous chlorine and chlorine dioxide and drying in killing Escherichia coli O157:H7 in biofilms formed on stainless steel, glass, plastic, and wooden surfaces. Cells attached to and formed a biofilm on wooden surfaces at significantly (P ≤ 0.05) higher levels compared with other surface types. The lethal activities of sodium hypochlorite (NaOCl) and aqueous chlorine dioxide (ClO₂) against E. coli O157:H7 in a biofilm on various food-contact surfaces were compared. Chlorine dioxide generally showed greater lethal activity than NaOCl against E. coli O157:H7 in a biofilm on the same type of surface. The resistance of E. coli O157:H7 to both sanitizers increased in the order of wood>plastic>glass>stainless steel. The synergistic lethal effects of sequential ClO₂ and drying treatments on E. coli O157:H7 in a biofilm on wooden surfaces were evaluated. When wooden surfaces harboring E. coli O157:H7 biofilm were treated with ClO₂ (200 μg/ml, 10 min), rinsed with water, and subsequently dried at 43% relative humidity and 22 °C, the number of E. coli O157:H7 on the surface decreased by an additional 6.4 CFU/coupon within 6 h of drying. However, when the wooden surface was treated with water or NaOCl and dried under the same conditions, the pathogen decreased by only 0.4 or 1.0 log CFU/coupon, respectively, after 12 h of drying. This indicates that ClO₂ treatment of food-contact surfaces results in residual lethality to E. coli O157:H7 during the drying process. These observations will be useful when selecting an appropriate type of food-contact surfaces, determining a proper sanitizer for decontamination, and designing an effective sanitization program to eliminate E. coli O157:H7 on food-contact surfaces in food processing, distribution, and preparation environments. Copyright © 2014 Elsevier B.V. All rights reserved.

Specificity for field enumeration of Escherichia coli in tropical surface waters

DEFF Research Database (Denmark)

Jensen, Peter Kjær Mackie; Aalbaek, B; Aslam, R

2001-01-01

In remote rural areas in developing countries, bacteriological monitoring often depends on the use of commercial field media. This paper evaluates a commercial field medium used for the enumeration of Escherichia coli in different surface waters under primitive field conditions in rural Pakistan....... In order to verify the field kit, 117 presumptive E. coli isolates have been tested, finding a specificity of only 40%. By excluding some strains based on colony colours, the calculated specificity could be increased to 65%. Thus, it is suggested that prior to use in a tropical environment, the specificity...... of any commercial medium used should be tested with representative tropical isolates, in order to increase the specificity....

Escherichia Coli

Science.gov (United States)

Goodsell, David S.

2009-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

Visualizing Escherichia coli sub-cellular structure using sparse deconvolution Spatial Light Interference Tomography.

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Mustafa Mir

Full Text Available Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM. In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT, to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.

Subtractive Inhibition Assay for the Detection of E. coli O157:H7 Using Surface Plasmon Resonance

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Chengyan Si

2011-03-01

Full Text Available A surface plasmon resonance (SPR immunosensor was developed for the detection of E. coli O157:H7 by means of a new subtractive inhibition assay. In the subtractive inhibition assay, E. coli O157:H7 cells and goat polyclonal antibodies for E. coli O157:H7 were incubated for a short of time, and then the E. coli O157:H7 cells which bound antibodies were removed by a stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using a BIAcore 3000 biosensor. The results showed that the signal was inversely correlated with the concentration of E. coli O157:H7 cells in a range from 3.0 × 104 to 3.0 × 108 cfu/mL with a detection limit of 3.0 × 104 cfu/mL. Compared with direct SPR by immobilizing antibodies on the chip surface to capture the bacterial cells and ELISA for E. coli O157:H7 (detection limit: both 3.0 × 105 cfu/mL in this paper, the detection limit of subtractive inhibition assay method was reduced by one order of magnitude. The method simplifies bacterial cell detection to protein-protein interaction, which has the potential for providing a practical alternative for the monitoring of E. coli O157:H7 and other pathogens.

Translational errors in expression of Shiga toxin from pathogenic Escherichia coli as measured by MALDI-TOF-TOF and Orbitrap mass spectrometry

Science.gov (United States)

Introduction: Shiga toxin (Stx) is an AB5 toxin expressed by Shiga toxin-producing E. coli (STEC) and Shigella dysenteriae. The Stx holotoxin attaches to surface receptors of eukaryotic cells. After cellular envelopment, the toxin disrupts ribosomal protein synthesis causing cell death. Variations i...

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

Science.gov (United States)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering

Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents.

Science.gov (United States)

Byerly, Douglas W; McElroy, Craig A; Foster, Mark P

2002-07-01

Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded (15)N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens.

Genetic diversity of Escherichia coli isolates from surface water and groundwater in a rural environment.

Science.gov (United States)

Gambero, Maria Laura; Blarasin, Monica; Bettera, Susana; Giuliano Albo, Jesica

2017-10-01

The genetic characteristics among Escherichia coli strains can be grouped by origin of isolation. Then, it is possible to use the genotypes as a tool to determine the source of water contamination. The aim of this study was to define water aptitude for human consumption in a rural basin and to assess the diversity of E. coli water populations. Thus, it was possible to identify the main sources of fecal contamination and to explore linkages with the hydrogeological environment and land uses. The bacteriological analysis showed that more than 50% of samples were unfit for human consumption. DNA fingerprinting analysis by BOX-PCR indicated low genotypic diversity of E. coli isolates taken from surface water and groundwater. The results suggested the presence of a dominant source of fecal contamination. The relationship between low genotypic diversity and land use would prove that water contamination comes from livestock. The genetic diversity of E. coli isolated from surface water was less than that identified in groundwater because of the different hydraulic features of both environments. Furthermore, each one of the two big strain groups identified in this basin is located in different sub-basins, showing that hydrological dynamics exerts selective pressure on bacteria DNA.

RECOGNITION DYNAMICS OF ESCHERICHIA COLI THIOREDOXIN PROBED USING MOLECULAR DYNAMICS AND BINDING FREE ENERGY CALCULATIONS

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M. S. Shahul Hameed

2016-03-01

Full Text Available E. coli thioredoxin has been regarded as a hub protein as it interacts with, and regulates, numerous target proteins involved in a wide variety of cellular processes. Thioredoxin can form complexes with a variety of target proteins with a wide range of affinity, using a consensus binding surface. In this study an attempt to deduce the molecular basis for the observed multispecificity of E. coli thioredoxin has been made. In this manuscript it has been shown that structural plasticity, adaptable and exposed hydrophobic binding surface, surface electrostatics, closely clustered multiple hot spot residues and conformational changes brought about by the redox status of the protein have been shown to account for the observed multispecificity and molecular recognition of thioredoxin. Dynamical differences between the two redox forms of the enzyme have also been studied to account for their differing interactions with some target proteins.

The upper surface of an Escherichia coli swarm is stationary.

Science.gov (United States)

Zhang, Rongjing; Turner, Linda; Berg, Howard C

2010-01-05

When grown in a rich medium on agar, many bacteria elongate, produce more flagella, and swim in a thin film of fluid over the agar surface in swirling packs. Cells that spread in this way are said to swarm. The agar is a solid gel, with pores smaller than the bacteria, so the swarm/agar interface is fixed. Here we show, in experiments with Escherichia coli, that the swarm/air interface also is fixed. We deposited MgO smoke particles on the top surface of an E. coli swarm near its advancing edge, where cells move in a single layer, and then followed the motion of the particles by dark-field microscopy and the motion of the underlying cells by phase-contrast microscopy. Remarkably, the smoke particles remained fixed (diffusing only a few micrometers) while the swarming cells streamed past underneath. The diffusion coefficients of the smoke particles were smaller over the virgin agar ahead of the swarm than over the swarm itself. Changes between these two modes of behavior were evident within 10-20 microm of the swarm edge, indicating an increase in depth of the fluid in advance of the swarm. The only plausible way that the swarm/air interface can be fixed is that it is covered by a surfactant monolayer pinned at its edges. When a swarm is exposed to air, such a monolayer can markedly reduce water loss. When cells invade tissue, the ability to move rapidly between closely opposed fixed surfaces is a useful trait.

Efficacy of a Blend of Sulfuric Acid and Sodium Sulfate against Shiga Toxin-Producing Escherichia coli, Salmonella, and Nonpathogenic Escherichia coli Biotype I on Inoculated Prerigor Beef Surface Tissue.

Science.gov (United States)

Scott-Bullard, Britteny R; Geornaras, Ifigenia; Delmore, Robert J; Woerner, Dale R; Reagan, James O; Morgan, J Bred; Belk, Keith E

2017-12-01

A study was conducted to investigate the efficacy of a sulfuric acid-sodium sulfate blend (SSS) against Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella, and nonpathogenic E. coli biotype I on prerigor beef surface tissue. The suitability of using the nonpathogenic E. coli as a surrogate for in-plant validation studies was also determined by comparing the data obtained for the nonpathogenic inoculum with those for the pathogenic inocula. Prerigor beef tissue samples (10 by 10 cm) were inoculated (ca. 6 log CFU/cm 2 ) on the adipose side in a laboratory-scale spray cabinet with multistrain mixtures of E. coli O157:H7 (5 strains), non-O157 STEC (12 strains), Salmonella (6 strains), or E. coli biotype I (5 strains). Treatment parameters evaluated were two SSS pH values (1.5 and 1.0) and two spray application pressures (13 and 22 lb/in 2 ). Untreated inoculated beef tissue samples served as controls for initial bacterial populations. Overall, the SSS treatments lowered inoculated (6.1 to 6.4 log CFU/cm 2 ) bacterial populations by 0.6 to 1.5 log CFU/cm 2 (P SSS was applied to samples inoculated with any of the tested E. coli inocula; however, solution pH did have a significant effect (P SSS was applied to samples inoculated with Salmonella. Results indicated that the response of the nonpathogenic E. coli inoculum to the SSS treatments was similar (P ≥ 0.05) to that of the pathogenic inocula tested, making the E. coli biotype I strains viable surrogate organisms for in-plant validation of SSS efficacy on beef. The application of SSS at the tested parameters to prerigor beef surface tissue may be an effective intervention for controlling pathogens in a commercial beef harvest process.

Campylobacter jejuni CsrA complements an Escherichia coli csrA mutation for the regulation of biofilm formation, motility and cellular morphology but not glycogen accumulation

Science.gov (United States)

2012-01-01

Background Although Campylobacter jejuni is consistently ranked as one of the leading causes of bacterial diarrhea worldwide, the mechanisms by which C. jejuni causes disease and how they are regulated have yet to be clearly defined. The global regulator, CsrA, has been well characterized in several bacterial genera and is known to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus Campylobacter. Previously, we reported data illustrating the requirement for CsrA in several virulence related phenotypes of C. jejuni strain 81–176, indicating that the Csr pathway is important for Campylobacter pathogenesis. Results We compared the Escherichia coli and C. jejuni orthologs of CsrA and characterized the ability of the C. jejuni CsrA protein to functionally complement an E. coli csrA mutant. Phylogenetic comparison of E. coli CsrA to orthologs from several pathogenic bacteria demonstrated variability in C. jejuni CsrA relative to the known RNA binding domains of E. coli CsrA and in several amino acids reported to be involved in E. coli CsrA-mediated gene regulation. When expressed in an E. coli csrA mutant, C. jejuni CsrA succeeded in recovering defects in motility, biofilm formation, and cellular morphology; however, it failed to return excess glycogen accumulation to wild type levels. Conclusions These findings suggest that C. jejuni CsrA is capable of efficiently binding some E. coli CsrA binding sites, but not others, and provide insight into the biochemistry of C. jejuni CsrA. PMID:23051923

Impact of enumeration method on diversity of Escherichia coli genotypes isolated from surface water.

Science.gov (United States)

Martin, E C; Gentry, T J

2016-11-01

There are numerous regulatory-approved Escherichia coli enumeration methods, but it is not known whether differences in media composition and incubation conditions impact the diversity of E. coli populations detected by these methods. A study was conducted to determine if three standard water quality assessments, Colilert ® , USEPA Method 1603, (modified mTEC) and USEPA Method 1604 (MI), detect different populations of E. coli. Samples were collected from six watersheds and analysed using the three enumeration approaches followed by E. coli isolation and genotyping. Results indicated that the three methods generally produced similar enumeration data across the sites, although there were some differences on a site-by-site basis. The Colilert ® method consistently generated the least diverse collection of E. coli genotypes as compared to modified mTEC and MI, with those two methods being roughly equal to each other. Although the three media assessed in this study were designed to enumerate E. coli, the differences in the media composition, incubation temperature, and growth platform appear to have a strong selective influence on the populations of E. coli isolated. This study suggests that standardized methods of enumeration and isolation may be warranted if researchers intend to obtain individual E. coli isolates for further characterization. This study characterized the impact of three USEPA-approved Escherichia coli enumeration methods on observed E. coli population diversity in surface water samples. Results indicated that these methods produced similar E. coli enumeration data but were more variable in the diversity of E. coli genotypes observed. Although the three methods enumerate the same species, differences in media composition, growth platform, and incubation temperature likely contribute to the selection of different cultivable populations of E. coli, and thus caution should be used when implementing these methods interchangeably for

Mycoplasma hyopneumoniae and Mycoplasma flocculare differential domains from orthologous surface proteins induce distinct cellular immune responses in mice.

Science.gov (United States)

Leal, Fernanda Munhoz Dos Anjos; Virginio, Veridiana Gomes; Martello, Carolina Lumertz; Paes, Jéssica Andrade; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Ferreira, Henrique Bunselmeyer

2016-07-15

Mycoplasma hyopneumoniae and Mycoplasma flocculare are two genetically close species found in the swine respiratory tract. Despite their similarities, while M. hyopneumoniae is the causative agent of porcine enzootic pneumonia, M. flocculare is a commensal bacterium. Genomic and transcriptional comparative analyses so far failed to explain the difference in pathogenicity between these two species. We then hypothesized that such difference might be, at least in part, explained by amino acid sequence and immunological or functional differences between ortholog surface proteins. In line with that, it was verified that approximately 85% of the ortholog surface proteins from M. hyopneumoniae 7448 and M. flocculare present one or more differential domains. To experimentally assess possible immunological implications of this kind of difference, the extracellular differential domains from one pair of orthologous surface proteins (MHP7448_0612, from M. hyopneumoniae, and MF_00357, from M. flocculare) were expressed in E. coli and used to immunize mice. The recombinant polypeptides (rMHP61267-169 and rMF35767-196, respectively) induced distinct cellular immune responses. While, rMHP61267-169 induced both Th1 and Th2 responses, rMF35767-196 induced just an early pro-inflammatory response. These results indicate that immunological properties determined by differential domains in orthologous surface protein might play a role in pathogenicity, contributing to elicit specific and differential immune responses against each species. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacteria hold their breath upon surface contact as shown in a strain of Escherichia coli, using dispersed surfaces and flow cytometry analysis.

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Jing Geng

Full Text Available Bacteria are ubiquitously distributed throughout our planet, mainly in the form of adherent communities in which cells exhibit specific traits. The mechanisms underpinning the physiological shift in surface-attached bacteria are complex, multifactorial and still partially unclear. Here we address the question of the existence of early surface sensing through implementation of a functional response to initial surface contact. For this purpose, we developed a new experimental approach enabling simultaneous monitoring of free-floating, aggregated and adherent cells via the use of dispersed surfaces as adhesive substrates and flow cytometry analysis. With this system, we analyzed, in parallel, the constitutively expressed GFP content of the cells and production of a respiration probe--a fluorescent reduced tetrazolium ion. In an Escherichia coli strain constitutively expressing curli, a major E. coli adhesin, we found that single cell surface contact induced a decrease in the cell respiration level compared to free-floating single cells present in the same sample. Moreover, we show here that cell surface contact with an artificial surface and with another cell caused reduction in respiration. We confirm the existence of a bacterial cell "sense of touch" ensuring early signalling of surface contact formation through respiration down modulation.

Transfer coefficient models for escherichia coli O157:H7 on contacts between beef tissue and high-density polyethylene surfaces.

Science.gov (United States)

Flores, Rolando A; Tamplin, Mark L; Marmer, Benne S; Phillips, John G; Cooke, Peter H

2006-06-01

Risk studies have identified cross-contamination during beef fabrication as a knowledge gap, particularly as to how and at what levels Escherichia coli O157:H7 transfers among meat and cutting board (or equipment) surfaces. The objectives of this study were to determine and model transfer coefficients (TCs) between E. coli O157:H7 on beef tissue and high-density polyethylene (HDPE) cutting board surfaces. Four different transfer scenarios were evaluated: (i) HDPE board to agar, (ii) beef tissue to agar, (iii) HDPE board to beef tissue to agar, and (iv) beef tissue to HDPE board to agar. Also, the following factors were studied for each transfer scenario: two HDPE surface roughness levels (rough and smooth), two beef tissues (fat and fascia), and two conditions of the initial beef tissue inoculation with E. coli O157:H7 (wet and dry surfaces), for a total of 24 treatments. The TCs were calculated as a function of the plated inoculum and of the cells recovered from the first contact. When the treatments were compared, all of the variables evaluated interacted significantly in determining the TC. An overall TC-per-treatment model did not adequately represent the reduction of the cells on the original surface after each contact and the interaction of the factors studied. However, an exponential model was developed that explained the experimental data for all treatments and represented the recontamination of the surfaces with E. coli O157:H7. The parameters for the exponential model for cross-contamination with E. coli O157:H7 between beef tissue and HDPE surfaces were determined, allowing for the use of the resulting model in quantitative microbial risk assessment.

Roles of ionic strength and biofilm roughness on adhesion kinetics of Escherichia coli onto groundwater biofilm grown on PVC surfaces

Science.gov (United States)

Janjaroen, Dao; Ling, Fangqiong; Monroy, Guillermo; Derlon, Nicolas; Mogenroth, Eberhard; Boppart, Stephen A.; Liu, Wen-Tso; Nguyen, Thanh H.

2013-01-01

Mechanisms of Escherichia coli attachment on biofilms grown on PVC coupons were investigated. Biofilms were grown in CDC reactors using groundwater as feed solution over a period up to 27 weeks. Biofilm physical structure was characterized at the micro- and meso-scales using Scanning Electron Microscopy (SEM) and Optical Coherence Tomography (OCT), respectively. Microbial community diversity was analyzed with Terminal Restricted Fragment Length Polymorphism (T-RFLP). Both physical structure and microbial community diversity of the biofilms were shown to be changing from 2 weeks to 14 weeks, and became relatively stable after 16 weeks. A parallel plate flow chamber coupled with an inverted fluorescent microscope was also used to monitor the attachment of fluorescent microspheres and E. coli on clean PVC surfaces and biofilms grown on PVC surfaces for different ages. Two mechanisms of E. coli attachment were identified. The adhesion rate coefficients (kd) of E. coli on nascent PVC surfaces and 2-week biofilms increased with ionic strength. However, after biofilms grew for 8 weeks, the adhesion was found to be independent of solution chemistry. Instead, a positive correlation between kd and biofilm roughness as determined by OCT was obtained, indicating that the physical structure of biofilms could play an important role in facilitating the adhesion of E. coli cells. PMID:23497979

Surface Enhanced Raman Scattering for Quantification of p-Coumaric Acid Produced by Escherichia coli

DEFF Research Database (Denmark)

Morelli, Lidia; Zor, Kinga; Jendresen, Christian Bille

2017-01-01

The number of newly developed genetic variants of microbial cell factories for production of biochemicals has been rapidly growing in recent years, leading to an increased need for new screening techniques. We developed a method based on surface-enhanced Raman scattering (SERS) coupled with liquid......-liquid extraction (LLE) for quantification of p-coumaric acid (pHCA) in the supernatant of genetically engineered Escherichia coli (E. coli) cultures. pHCA was measured in a dynamic range from 1 μM up to 50 μM on highly uniform SERS substrates based on leaning gold-capped nanopillars, which showed an in...

Antibiotic resistance patterns of Escherichia coli strains isolated from surface water and groundwater samples in a pig production area

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Roger Neto Schneider

2009-09-01

Full Text Available The use of antibiotics, so excessive and indiscriminate in intensive animal production, has triggered an increase in the number of resistant microorganisms which can be transported to aquatic environments. The aim of this study was to determine the profile of the antimicrobial resistance of samples of Escherichia coli isolated from groundwater and surface water in a region of pig breeding. Through the test of antimicrobial susceptibility, we analyzed 205 strains of E. coli. A high rate of resistance to cefaclor was observed, both in surface water (51.9% and groundwater (62.9%, while all samples were sensitive to amikacin. The percentages of multi-resistant samples were 25.96% and 26.73% in surface water and groundwater, respectively, while 19.23% and 13.86% were sensitive to all antibiotics tested. It was determined that the rate of multiple antibiotic resistance (MAR was 0.164 for surface water and 0.184 for groundwater. No significant differences were found in the profile of the antimicrobial resistance in strains of E. coli isolated in surface water and groundwater, but the index MAR calculated in certain points of groundwater may offer a potential risk of transmission of resistant genes.

Inhibition of Escherichia coli respiratory enzymes by short visible femtosecond laser irradiation

International Nuclear Information System (INIS)

Lu, Chieh-Han; Hsu, Yung-Yuan; Lin, Kung-Hsuan; Tsen, Kong-Thon; Kuan, Yung-Shu

2014-01-01

A visible femtosecond laser is shown to be capable of selectively inactivating a wide spectrum of microorganisms in a wavelength and pulse width dependent manner. However, the mechanism of how a visible femtosecond laser affects the viability of different microorganisms is still elusive. In this paper, the cellular surface properties, membrane integrity and metabolic rate of Escherichia coli (E. coli) irradiated by a visible femtosecond laser (λ = 415 nm, pulse width = 100 fs) with different exposure times were investigated. Our results showed that femtosecond laser treatment for 60 min led to cytoplasmic leakage, protein aggregation and alternation of the physical properties of the E. coli cell membrane. In comparison, a 10 min exposure of bacteria to femtosecond laser irradiation induced an immediate reduction of 75% in the glucose-dependent respiratory rate, while the cytoplasmic leakage was not detected. Results from enzymatic assays showed that oxidases and dehydrogenases involved in the E. coli respiratory chain exhibited divergent susceptibility after laser irradiation. This early commencement of respiratory inhibition after a short irradiation is presumed to have a dominant effect on the early stage of bacteria inactivation. (paper)

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters.

Science.gov (United States)

Jenkins, M B; Endale, D M; Fisher, D S; Gay, P A

2009-02-01

To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method. The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0.1 MPN per litre, with a 95% confidence level of 0.01-0.7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0.9 MPN per litre for pond outflow and from not detectable to 8.3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from 10(4) MPN per hour. This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife. This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.

Transfer, attachment, and formation of biofilms by Escherichia coli O157:H7 on meat-contact surface materials.

Science.gov (United States)

Simpson Beauchamp, Catherine; Dourou, Dimitra; Geornaras, Ifigenia; Yoon, Yohan; Scanga, John A; Belk, Keith E; Smith, Gary C; Nychas, George-John E; Sofos, John N

2012-06-01

Studies examined the effects of meat-contact material types, inoculation substrate, presence of air at the liquid-solid surface interface during incubation, and incubation substrate on the attachment/transfer and subsequent biofilm formation by Escherichia coli O157:H7 on beef carcass fabrication surface materials. Materials studied as 2 × 5 cm coupons included stainless steel, acetal, polypropylene, and high-density polyethylene. A 6-strain rifampicin-resistant E. coli O157:H7 composite was used to inoculate (6 log CFU/mL, g, or cm²) tryptic soy broth (TSB), beef fat/lean tissue homogenate (FLH), conveyor belt-runoff fluids, ground beef, or beef fat. Coupons of each material were submerged (4 °C, 30 min) in the inoculated fluids or ground beef, or placed between 2 pieces of inoculated beef fat with pressure (20 kg) applied. Attachment/transfer of the pathogen was surface material and substrate dependent, although beef fat appeared to negate differences among surface materials. Beef fat was the most effective (P transfer and subsequent biofilm formation by E. coli O157:H7. The results highlight the importance of thoroughly cleaning soiled surfaces to remove all remnants of beef fat or other organic material that may harbor or protect microbial contaminants during otherwise lethal antimicrobial interventions. © 2012 Institute of Food Technologists®

Simulation of Escherichia coli Dynamics in Biofilms and Submerged Colonies with an Individual-Based Model Including Metabolic Network Information.

Science.gov (United States)

Tack, Ignace L M M; Nimmegeers, Philippe; Akkermans, Simen; Hashem, Ihab; Van Impe, Jan F M

2017-01-01

Clustered microbial communities are omnipresent in the food industry, e.g., as colonies of microbial pathogens in/on food media or as biofilms on food processing surfaces. These clustered communities are often characterized by metabolic differentiation among their constituting cells as a result of heterogeneous environmental conditions in the cellular surroundings. This paper focuses on the role of metabolic differentiation due to oxygen gradients in the development of Escherichia coli cell communities, whereby low local oxygen concentrations lead to cellular secretion of weak acid products. For this reason, a metabolic model has been developed for the facultative anaerobe E. coli covering the range of aerobic, microaerobic, and anaerobic environmental conditions. This metabolic model is expressed as a multiparametric programming problem, in which the influence of low extracellular pH values and the presence of undissociated acid cell products in the environment has been taken into account. Furthermore, the developed metabolic model is incorporated in MICRODIMS, an in-house developed individual-based modeling framework to simulate microbial colony and biofilm dynamics. Two case studies have been elaborated using the MICRODIMS simulator: (i) biofilm growth on a substratum surface and (ii) submerged colony growth in a semi-solid mixed food product. In the first case study, the acidification of the biofilm environment and the emergence of typical biofilm morphologies have been observed, such as the mushroom-shaped structure of mature biofilms and the formation of cellular chains at the exterior surface of the biofilm. The simulations show that these morphological phenomena are respectively dependent on the initial affinity of pioneer cells for the substratum surface and the cell detachment process at the outer surface of the biofilm. In the second case study, a no-growth zone emerges in the colony center due to a local decline of the environmental pH. As a result

Simulation of Escherichia coli Dynamics in Biofilms and Submerged Colonies with an Individual-Based Model Including Metabolic Network Information

Directory of Open Access Journals (Sweden)

Ignace L. M. M. Tack

2017-12-01

Full Text Available Clustered microbial communities are omnipresent in the food industry, e.g., as colonies of microbial pathogens in/on food media or as biofilms on food processing surfaces. These clustered communities are often characterized by metabolic differentiation among their constituting cells as a result of heterogeneous environmental conditions in the cellular surroundings. This paper focuses on the role of metabolic differentiation due to oxygen gradients in the development of Escherichia coli cell communities, whereby low local oxygen concentrations lead to cellular secretion of weak acid products. For this reason, a metabolic model has been developed for the facultative anaerobe E. coli covering the range of aerobic, microaerobic, and anaerobic environmental conditions. This metabolic model is expressed as a multiparametric programming problem, in which the influence of low extracellular pH values and the presence of undissociated acid cell products in the environment has been taken into account. Furthermore, the developed metabolic model is incorporated in MICRODIMS, an in-house developed individual-based modeling framework to simulate microbial colony and biofilm dynamics. Two case studies have been elaborated using the MICRODIMS simulator: (i biofilm growth on a substratum surface and (ii submerged colony growth in a semi-solid mixed food product. In the first case study, the acidification of the biofilm environment and the emergence of typical biofilm morphologies have been observed, such as the mushroom-shaped structure of mature biofilms and the formation of cellular chains at the exterior surface of the biofilm. The simulations show that these morphological phenomena are respectively dependent on the initial affinity of pioneer cells for the substratum surface and the cell detachment process at the outer surface of the biofilm. In the second case study, a no-growth zone emerges in the colony center due to a local decline of the environmental p

Ultrasensitive detection and quantification of E. coli O157:H7 using a giant magneto impedance sensor in an open-surface micro fluidic cavity covered with an antibody-modified gold surface

International Nuclear Information System (INIS)

Yang, Zhen; Liu, Yan; Lei, Chong; Sun, Xue-cheng; Zhou, Yong

2016-01-01

We report on a method for ultrasensitive detection and quantification of the pathogen Escherichia coli (E. coli), type O157:H7. It is using a tortuous-shaped giant magneto impedance (GMI) sensor in combination with an open-surface micro fluidic system coated with a gold film for performing the sandwich immuno binding on its surface. Streptavidin-coated super magnetic Dynabeads were loaded with biotinylated polyclonal antibody to capture E. coli O157:H7. The E. coli-loaded Dynabeads are then injected into the microfluidics system where it comes into contact with the surface of gold nanofilm carrying the monoclonal antibody to form the immuno complex. As a result, the GMI ratio is strongly reduced at high frequencies if E. coli O157:H7 is present. The sensor has a linear response in the 50 to 500 cfu·mL"−"1 concentration range, and the detection limit is 50 cfu·mL"−"1 at a working frequency of 2.2 MHz. In our perception, this method provides a valuable tool for developing GMI-based micro fluidic sensors systems for ultrasensitive and quantitative analysis of pathogenic bacteria. The method may also be extended to other sensing applications by employing respective immuno reagents. (author)

Surface Sampling Collection and Culture Methods for Escherichia coli in Household Environments with High Fecal Contamination.

Science.gov (United States)

Exum, Natalie G; Kosek, Margaret N; Davis, Meghan F; Schwab, Kellogg J

2017-08-22

Empiric quantification of environmental fecal contamination is an important step toward understanding the impact that water, sanitation, and hygiene interventions have on reducing enteric infections. There is a need to standardize the methods used for surface sampling in field studies that examine fecal contamination in low-income settings. The dry cloth method presented in this manuscript improves upon the more commonly used swabbing technique that has been shown in the literature to have a low sampling efficiency. The recovery efficiency of a dry electrostatic cloth sampling method was evaluated using Escherichia coli and then applied to household surfaces in Iquitos, Peru, where there is high fecal contamination and enteric infection. Side-by-side measurements were taken from various floor locations within a household at the same time over a three-month period to compare for consistency of quantification of E. coli bacteria. The dry cloth sampling method in the laboratory setting showed 105% (95% Confidence Interval: 98%, 113%) E. coli recovery efficiency off of the cloths. The field application demonstrated strong agreement of side-by-side results (Pearson correlation coefficient for dirt surfaces was 0.83 ( p samples (Pearson (0.53, p method can be utilized in households with high bacterial loads using either continuous (quantitative) or categorical (semi-quantitative) data. The standardization of this low-cost, dry electrostatic cloth sampling method can be used to measure differences between households in intervention and non-intervention arms of randomized trials.

Regulation of the E. coli SOS response by the lexA gene product

International Nuclear Information System (INIS)

Brent, R.

1983-01-01

In an Escherichia coli that is growing normally, transcription of many genes is repressed by the product of the lexA gene. If cellular DNA is damaged, proteolytically competent recA protein (recA protease) inactivates lexA protein and these genes are induced. Many of the cellular phenomena observed during the cellular response to DNA damage (the SOS response) are the consequence of the expression of these lexA-prepressed genes. Since the SOS response of E. coli has recently been the subject of a comprehensive review, in this paper I would like to concentrate on some modifications to the picture based on new data. 12 references, 2 figures

Maximized Autotransporter-Mediated Expression (MATE for Surface Display and Secretion of Recombinant Proteins in Escherichia coli

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Shanna Sichwart

2015-01-01

Full Text Available A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression. It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the β-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 μg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. coli.

TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli

NARCIS (Netherlands)

Carre, Gaelle; Hamon, Erwann; Ennahar, Said; Estner, Maxime; Lett, Marie-Claire; Horvatovich, Peter; Gies, Jean-Pierre; Keller, Valerie; Keller, Nicolas; Andre, Philippe

This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by

Cytotoxicity and cellular uptake of tri-block copolymer nanoparticles with different size and surface characteristics

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Bhattacharjee Sourav

2012-04-01

Full Text Available Abstract Background Polymer nanoparticles (PNP are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. Results Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm and surface charges (positive and negative were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm showed a higher cytotoxicity compared to the positive bigger PNP90 (90 nm particles including reduction in mitochondrial membrane potential (ΔΨm, induction of reactive oxygen species (ROS production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm, uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM. Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP, caveolin (for negative PNP and mannose receptors (for hydroxylated PNP were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP90. Conclusions The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose leading to cellular internalization were observed to depend on

Action of peracetic acid on Escherichia coli and Staphylococcus aureus in suspension or settled on stainless steel surfaces

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Kunigk Leo

2001-01-01

Full Text Available The efficiency of a commercial peracetic acid sanitizer on destruction of Staphylococcus aureus and Escherichia coli was evaluated using two distinct methods. The first method is the AOAC suspension test and the second is a method proposed by one of the authors in which the microbial cells are settled on a stainless steel surface and then treated with the sanitizer. The results showed that when in suspension S. aureus was more resistant to the sanitizer than E. coli. When S. aureus was settled on the stainless steel surface, the contact time between the sanitizer and the microorganisms to attain a 6.5 log reduction in the number of viable cells was three times greater than when the cells were in suspension.

Escherichia coli in the surface waters and in oysters of two cultivations of Guaratuba Bay - Paraná - Brazil

OpenAIRE

Forcelini,Helenita Catharina Dalla-Lana; Kolm,Hedda Elisabeth; Absher,Theresinha Monteiro

2013-01-01

The present work aimed to evaluate the contamination of Escherichia coli in the surface waters and oysters from two cultivations of Guaratuba Bay and to analyze the correlation patterns among the concentrations of E. coli in the waters and in the oysters with the local physical-chemical parameters. Samples were collected in the spring of 2007 and summer, autumn and winter of 2008 from two points of the bay (internal point and external point). From each cultivation and sampling period, 18 oyst...

Inhibition of P-fimbriated Escherichia coli adhesion by multivalent galabiose derivatives studied by a live-bacteria application of surface plasmon resonance.

Science.gov (United States)

Salminen, Annika; Loimaranta, Vuokko; Joosten, John A F; Khan, A Salam; Hacker, Jörg; Pieters, Roland J; Finne, Jukka

2007-09-01

Uropathogenic P-fimbriated Escherichia coli adheres to host cells by specific adhesins recognizing galabiose (Galalpha1-4Gal)-containing structures on cell surfaces. In search of agents inhibiting this first step of infection, the inhibition potency of a set of synthetic mono- and multivalent galabiose compounds was evaluated. In order to mimic the flow conditions of natural infections, a live-bacteria application of surface plasmon resonance (SPR) was established. For the measurement of the binding of E. coli to a surface containing galabiose, live bacteria were injected over the flow cell, and the inhibition of adhesion caused by the galabiose inhibitors was recorded. Quantitative binding data were recorded in real-time for each inhibitor. The results were compared with those of conventional static haemagglutination and ELISA-based cell adhesion assays. Compared with the Gram-positive Streptococcus suis bacteria, which also bind to galabiose and whose binding inhibition is strongly dependent on the multivalency of the inhibitor, E. coli inhibition was only moderately affected by the valency. However, a novel octavalent compound was found to be the most effective inhibitor of E. coli PapG(J96) adhesion, with an IC50 value of 2 microM. Measurement of bacterial adhesion by SPR is an efficient way to characterize the adhesion of whole bacterial cells and allows the characterization of the inhibitory potency of adhesion inhibitors under dynamic flow conditions. Under these conditions, multivalency increases the anti-adhesion potency of galabiose-based inhibitors of P-fimbriated E. coli adhesion and provides a promising approach for the design of high-affinity anti-adhesion agents.

UV laser-ablated surface textures as potential regulator of cellular response.

Science.gov (United States)

Chandra, Prafulla; Lai, Karen; Sung, Hak-Joon; Murthy, N Sanjeeva; Kohn, Joachim

2010-06-01

Textured surfaces obtained by UV laser ablation of poly(ethylene terephthalate) films were used to study the effect of shape and spacing of surface features on cellular response. Two distinct patterns, cones and ripples with spacing from 2 to 25 μm, were produced. Surface features with different shapes and spacings were produced by varying pulse repetition rate, laser fluence, and exposure time. The effects of the surface texture parameters, i.e., shape and spacing, on cell attachment, proliferation, and morphology of neonatal human dermal fibroblasts and mouse fibroblasts were studied. Cell attachment was the highest in the regions with cones at ∼4 μm spacing. As feature spacing increased, cell spreading decreased, and the fibroblasts became more circular, indicating a stress-mediated cell shrinkage. This study shows that UV laser ablation is a useful alternative to lithographic techniques to produce surface patterns for controlling cell attachment and growth on biomaterial surfaces.

Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli

International Nuclear Information System (INIS)

Possan, A.L.; Menti, C.; Beltrami, M.; Santos, A.D.; Roesch-Ely, M.; Missell, F.P.

2016-01-01

Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembled monolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift. - Highlights: • Magnetoelastic biosensors to capture Escherichia coli were produced. • Surface roughness of biosensors was varied in the range R a = 0.3–0.52 μm. • Rough surfaces show faster response, polished surfaces have larger frequency shift.

Effect of surface roughness on performance of magnetoelastic biosensors for the detection of Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Possan, A.L. [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Menti, C. [Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Beltrami, M. [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Santos, A.D. [Instituto de Física, Universidade de São Paulo, São Paulo, SP (Brazil); Roesch-Ely, M. [Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil); Missell, F.P., E-mail: fmissell@yahoo.com [Centro de Ciências Exatas e Tecnologia, Universidade de Caxias do Sul, Caxias do Sul, RS (Brazil)

2016-01-01

Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembled monolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift. - Highlights: • Magnetoelastic biosensors to capture Escherichia coli were produced. • Surface roughness of biosensors was varied in the range R{sub a} = 0.3–0.52 μm. • Rough surfaces show faster response, polished surfaces have larger frequency shift.

Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli

DEFF Research Database (Denmark)

Holm, Anders Koefoed; Blank, L.M.; Oldiges, M.

2010-01-01

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli...... of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli....

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

Science.gov (United States)

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-05-02

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding

Multiple loci affecting photoreactivation in Escherichia coli

International Nuclear Information System (INIS)

Sutherland, B.M.; Hausrath, S.G.

1979-01-01

Sutherland et al. mapped a phr gene in Escherichia coli at 17 min and found that induction of an E. coli stain lysogenic for a lambda phage carrying this gene increased photoreactivating enzyme levels 2,000-fold. Recently, Smith and Youngs and Sancar and Rupert located a phr gene at 15.9 min. We have therefore investigated the properties of photoreactivating enzyme and cellular photoreactivation in cells containing deletions of the gene at 17 min. Cells with this deletion photoreactivated ultraviolet-induced killing at a rate 20% of normal; they also contained approximately 20% of the normal photoreactivating enzyme level. The residual enzyme in these cells was characterized to determine whether the reduced cellular photoreactivation rate and photoreactivating enzyme levels resulted from reduced numbers of normal enzymes or from an altered enzyme. Photoreactivating enzymes from strains carrying a deletion of the region at 17 min has an apparent K/sub m/ about two- to threefold higher than normal enzyme and showed markedly increased heat lability. The gene at 17 min thus contains information determining the function of the E. coli photoreactivating enzyme rather than the quantity of the enzyme. It is proposed that the gene at 17 min be termed phrA and that located at 15.9 min be termed phrB

Antibiotic resistance patterns of Escherichia coli strains isolated from surface water and groundwater samples in a pig production area

OpenAIRE

Roger Neto Schneider; André Nadvorny; Verônica Schmidt

2009-01-01

The use of antibiotics, so excessive and indiscriminate in intensive animal production, has triggered an increase in the number of resistant microorganisms which can be transported to aquatic environments. The aim of this study was to determine the profile of the antimicrobial resistance of samples of Escherichia coli isolated from groundwater and surface water in a region of pig breeding. Through the test of antimicrobial susceptibility, we analyzed 205 strains of E. coli. A high rate of res...

Interaction of Escherichia coli with growing salad spinach plants.

Science.gov (United States)

Warriner, Keith; Ibrahim, Faozia; Dickinson, Matthew; Wright, Charles; Waites, William M

2003-10-01

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this study's findings with regard to the microbiological safety of minimally processed vegetables are discussed.

Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

Science.gov (United States)

Schrand, Amanda M; Lin, Jonathan B; Hens, Suzanne Ciftan; Hussain, Saber M

2011-02-01

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

Regulation of gene expression in Escherichia coli and its bacteriophage

International Nuclear Information System (INIS)

Higgins, C.F.

1986-01-01

This chapter reviews the study of prokaryotic gene expression beginning with a look at the regulation of the lactose operon and the mechanism of attenuation in the tryptophan operon to the more recent development of recombinant DNA technology. The chapter deals almost entirely with escherichia coli and its bacteriophage. The only experimental technique which the authors explore in some detail is the construction and use of gene and operon fusions which have revolutionized the study of gene expression. Various mechanisms by which E. Coli regulate the cellular levels of individual messenger-RNA species are described. Translational regulation of the cellular levels of messenger-RNA include signals encoded within the messenger-RNA molecule itself and regulatory molecules which interact with the messenger-RNA and alter it translational efficiency

Cellular localization of the Escherichia coli SpoT protein.

OpenAIRE

Gentry, D R; Cashel, M

1995-01-01

The SpoT protein of Escherichia coli serves as a source of degradation as well as an apparent source of synthesis of (p)ppGpp. Since the subcellular localization of SpoT might be a clue to its function, we have used SpoT-specific antisera to analyze cell extracts fractionated on sucrose gradients. We find that the SpoT protein is not bound to ribosomes or to either inner or outer membrane fractions. Although the SpoT protein is found in large aggregates, its localization is probably cytosolic.

Effect of chlorine treatment on inhibition of E. coli serogroup O2 incorporation into 7-day-old biofilm on polyvinylchloride surface.

Science.gov (United States)

Maharjan, P; Dey, S; Huff, G; Zhang, W; Phillips, G K; Watkins, S

2017-08-01

Poultry waterlines are constructed using polyvinylchloride (PVC) material on which bacterial biofilm can easily form. Biofilm can harbor pathogens including avian pathogenic E. coli (APEC) strains. An in vitro evaluation was performed to determine if E. coli sero group O2 (avian pathogenic) could attach on a PVC surface that had pre-formed biofilm and if this phenomenon could be affected when water was treated with chlorine. Initially, biofilm growth was induced in PVC test coupons (15.16 cm2) for a 7-day period mimicking the waterline scenario in the first wk of poultry brooding; and then this biofilm was challenged with E. coli O2 seeded water in presence/absence of chlorine treatment. After rinsing, test coupons were sampled for bacterial (APC) and E. coli O2 enumeration at various occasions post seeding the pathogen and chlorine treatment. Day 7 APC recovered from coupons was 4.35 log10 cfu/cm2 in trial 1 and 3.66 log10 cfu/cm2 in trial 2. E. coli O2 was not recovered from chlorine treated test coupons (P  3 log10 cfu/cm2 in trial 1 and > 2 log10 cfu/cm2 in trial 2). This study suggests that E. coli O2 can incorporate into pre-formed biofilm on a PVC surface within 24 h if water sanitation is not present, and the attachment time of the pathogen can prolong in the absence of already formed biofilm. © 2017 Poultry Science Association Inc.

Action of sodium deoxycholate on Escherichia coli

International Nuclear Information System (INIS)

D'Mello, A.; Yotis, W.W.

1987-01-01

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant total cell yield of this microorganisms. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of [U- 14 C]glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order

Action of sodium deoxycholate on Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

D' Mello, A.; Yotis, W.W.

1987-08-01

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant total cell yield of this microorganisms. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of (U-/sup 14/C)glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.

A flow chamber assay for quantitative evaluation of bacterial surface colonization used to investigate the influence of temperature and surface hydrophilicity on the biofilm forming capacity of uropathogenic Escherichia coli

DEFF Research Database (Denmark)

Andersen, Thomas Emil; Kingshott, Peter; Palarasah, Yaseelan

2010-01-01

to those found on an implanted device. We have used the method to evaluate the biofilm forming capacity of clinically isolated Escherichia coli on silicone rubber and on silicone rubber containing a hydrophilic coating. It was found that the surface chemistry influenced the colonization of the isolates...... very differently. In addition, the temperature was found to have a considerable influence upon the adhesion and biofilm forming capacity of some of the isolates, and that the influence of surface chemistry depended on temperature. Our results suggest that the step from using E. coli laboratory strains...

An aptamer-based biosensor for colorimetric detection of Escherichia coli O157:H7.

Directory of Open Access Journals (Sweden)

Wenhe Wu

Full Text Available BACKGROUND: An aptamer based biosensor (aptasensor was developed and evaluated for rapid colorimetric detection of Escherichia coli (E. coli O157:H7. METHODOLOGY/PRINCIPAL FINDINGS: The aptasensor was assembled by modifying the truncated lipopolysaccharides (LPS-binding aptamer on the surface of nanoscale polydiacetylene (PDA vesicle using peptide bonding between the carboxyl group of the vesicle and the amine group of the aptamer. Molecular recognition between E. coli O157:H7 and aptamer at the interface of the vesicle lead to blue-red transition of PDA which was readily visible to the naked eyes and could be quantified by colorimetric responses (CR. Confocal laser scanning microscope (CLSM and transmission electron microscopy (TEM was used to confirm the specific interactions between the truncated aptamer and E. coli O157:H7. The aptasensor could detect cellular concentrations in a range of 10(4~ 10(8 colony-forming units (CFU/ml within 2 hours and its specificity was 100% for detection of E. coli O157:H7. Compared with the standard culture method, the correspondent rate was 98.5% for the detection of E. coli O157:H7 on 203 clinical fecal specimens with our aptasensor. CONCLUSIONS: The new aptasensor represents a significant advancement in detection capabilities based on the combination of nucleic acid aptamer with PDA vesicle, and offers a specific and convenient screening method for the detection of pathogenic bacteria. This technic could also be applied in areas from clinical analysis to biological terrorism defense, especially in low-resource settings.

Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Science.gov (United States)

Laverty, Garry; Gorman, Sean P.; Gilmore, Brendan F.

2014-01-01

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation. PMID:25438014

Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Directory of Open Access Journals (Sweden)

Garry Laverty

2014-07-01

Full Text Available Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl, pellicle Formation (Pel and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Effect of durable γ-radiation on E.Coli

International Nuclear Information System (INIS)

Koudela, K.; Drashil, V.

1990-01-01

Effect of prolonged low intensity γ-radiation on changes of frequency of reversion mutations was studied in Escherichia Coli. Frequency of His + revertants was shown to depend on growth phase. Cellular DNA absorbed more energy in stationary than DNA in growth phase. K-12 AB2497 strain of Escherichia Coli D-37 comprised about 60 Gy. This dose wasn't absorbed under continuous irradiation at dose rate of 0.21 R/min in 5 hours. The dose rate was considered to be sufficient to induce SOS-system and thus to increase mutations number. 2 refs

Attachment behaviour of Escherichia coli K12 and Salmonella Typhimurium P6on food contact surfaces for food transportation

DEFF Research Database (Denmark)

Abban, Stephen; Jakobsen, Mogens; Jespersen, Lene

2012-01-01

The role of cargo container lining materials aluminium, a fibre reinforced plastic (FRP) and stainless steel in bacterial cross contamination during transport was assessed. For this, attachment and detachment of Escherichia coli K12 and Salmonella Typhimurium P6 on the three surfaces in the absence....... Typhimurium P6 respectively. Correlation with roughness average was poor; r = -0.425 and -0.413 respectively for E. coli K12 and S. Typhimurium P6. Presence of residue caused significant reduction (p ... material sections of the same surfaces. We report these observations for the first time for aluminium and the FRP material and in part for stainless steel. The S. Typhimurium P6 strain also had significantly higher level of attachment than the E. coli K12 strain. Our findings show that food residue...

Can E. coli fly?

DEFF Research Database (Denmark)

Lindeberg, Yrja Lisa; Egedal, Karen; Hossain, Zenat Zebin

2018-01-01

, and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of E. coli and genes of diarrheagenic E. coli and Shigella strains. RESULTS: Rice was at greater risk (p ... with E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (p ...-landings, the average CFU per fly-landing was > 0·6 x 103 CFU. Genes of diarrheagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified; the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala). CONCLUSION: Flies may...

Free water surface constructed wetlands limit the dissemination of extended-spectrum beta-lactamase producing Escherichia coli in the natural environment.

Science.gov (United States)

Vivant, Anne-Laure; Boutin, Catherine; Prost-Boucle, Stéphanie; Papias, Sandrine; Hartmann, Alain; Depret, Géraldine; Ziebal, Christine; Le Roux, Sophie; Pourcher, Anne-Marie

2016-11-01

The fates of Escherichia coli and extended-spectrum beta-lactamase-producing E. coli (ESBL E. coli) were studied over a period of one year in a free water surface constructed wetland (FWS CW) with a succession of open water zones and vegetation ponds (Typha or Phragmites), that received the effluent from a wastewater treatment plant. ESBL E. coli were detected and isolated from all sampling areas of the FWS CW throughout the study period. They represented 1‰ of the total E. coli population regardless of the origin of samples. Two main factors affected the log removal of E. coli and of ESBL E. coli: the season and the presence of vegetation. Between the inlet and the outlet of the FWS CW, the log removal of E. coli ranged from 1.5 in the warmer season (summer and fall) to 3.0 in the colder season (winter and spring). The concentrations of E. coli decreased significantly in the vegetated areas during the colder season, but increased in the warmer season, suggesting an effect of the plant growth stage on the survival of E. coli. Among the 369 ESBL E. coli isolates collected during our study, 84% harbored the CTX-M-ESBL type and 55.3% carried bla genes on plasmid DNA. Furthermore, 93% of the ESBL E. coli isolates were multidrug resistant but the proportion of resistant strains did not change significantly along the FWS CW. ESBL E. coli were characterized by MLST analysis using the 7 genes based Achtman Scheme. ESBL E. coli isolated from water, sediments, roots and feces of myocastors collected in the FWS CW and in the recipient river were genotypically related, suggesting persistence and circulation of the ESBL producing E. coli throughout the FWS CW and in the receiving river. Overall, these observations show that FWS CW could be an efficient treatment for ESBL E. coli disinfection of wastewater and could limit their dissemination in the aquatic environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phylogenetic Backgrounds and Virulence-Associated Traits of Escherichia coli Isolates from Surface Waters and Diverse Animals in Minnesota and Wisconsin.

Science.gov (United States)

Johnson, James R; Johnston, Brian D; Delavari, Parissa; Thuras, Paul; Clabots, Connie; Sadowsky, Michael J

2017-12-15

Possible external reservoirs for extraintestinal pathogenic Escherichia coli (ExPEC) strains that cause infections in humans are poorly defined. Because of the tremendous human health importance of ExPEC infections, we assessed surface waters and domesticated and wild animals in Minnesota and Wisconsin as potential reservoirs of ExPEC of human health relevance. We characterized 595 E. coli isolates (obtained from 1999 to 2002; 280 from seven surface water sites, 315 from feces of 13 wild and domesticated animal species) for phylogroup and virulence genotype, including inferred ExPEC status, by using multiplex PCR-based methods. We also compared the pulsed-field gel electrophoresis (PFGE) profiles of the isolates with a large private PFGE profile library. We found a predominance of non-ExPEC strains (95% and 93% among water and animal isolates, respectively), which were mainly from phylogroups A and B1, plus a minority of ExPEC strains (5% and 7% among water isolates and animal isolates, respectively), predominantly from phylogroup B2. The ExPEC strains, although significantly associated with cats, dogs, and turkeys, occurred in several additional animal species (goat, horse, chicken, pig) and were distributed broadly across all surface water sites. Virulence gene content among the animal source ExPEC isolates segregated significantly in relation to host species, following established patterns. PFGE analysis indicated that 11 study isolates closely matched (94% to 100% profile similarity) reference human clinical and fecal isolates. These findings imply what probably is a low but non-zero risk to humans from environmental and animal source E. coli isolates, especially those from specific human-associated animal species. IMPORTANCE Our detection of potentially pathogenic strains that may pose a health threat to humans among E. coli isolates from surface waters and wild and domesticated animals suggests a need for heightened attention to these reservoirs as possible

Increased acellular and cellular surface mineralization induced by nanogrooves in combination with a calcium-phosphate coating.

NARCIS (Netherlands)

Klymov, A.; Song, J.; Cai, X; Riet, J. te; Leeuwenburgh, S.C.; Jansen, J.A.; Walboomers, X.F.

2016-01-01

The current work evaluated the influence of nanoscale surface-topographies in combination with a calcium phosphate (CaP) coating on acellular and cellular surface mineralization. Four groups of substrates were produced, including smooth, grooved (940nm pitch, 430nm groove width, 185nm depth), smooth

Surface conditioning with Escherichia coli cell wall components can reduce biofilm formation by decreasing initial adhesion

Directory of Open Access Journals (Sweden)

Luciana C. Gomes

2017-07-01

Full Text Available Bacterial adhesion and biofilm formation on food processing surfaces pose major risks to human health. Non-efficient cleaning of equipment surfaces and piping can act as a conditioning layer that affects the development of a new biofilm post-disinfection. We have previously shown that surface conditioning with cell extracts could reduce biofilm formation. In the present work, we hypothesized that E. coli cell wall components could be implicated in this phenomena and therefore mannose, myristic acid and palmitic acid were tested as conditioning agents. To evaluate the effect of surface conditioning and flow topology on biofilm formation, assays were performed in agitated 96-well microtiter plates and in a parallel plate flow chamber (PPFC, both operated at the same average wall shear stress (0.07 Pa as determined by computational fluid dynamics (CFD. It was observed that when the 96-well microtiter plate and the PPFC were used to form biofilms at the same shear stress, similar results were obtained. This shows that the referred hydrodynamic feature may be a good scale-up parameter from high-throughput platforms to larger scale flow cell systems as the PPFC used in this study. Mannose did not have any effect on E. coli biofilm formation, but myristic and palmitic acid inhibited biofilm development by decreasing cell adhesion (in about 50%. These results support the idea that in food processing equipment where biofilm formation is not critical below a certain threshold, bacterial lysis and adsorption of cell components to the surface may reduce biofilm buildup and extend the operational time.

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

Science.gov (United States)

Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

2011-01-01

NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD+ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD+ biosynthesis in cells harboring the ntt4 gene. The minimal NAD+ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. PMID:21742902

Fimbrial adhesins from extraintestinal Escherichia coli

DEFF Research Database (Denmark)

Klemm, Per; Hancock, Viktoria; Schembri, Mark A.

2010-01-01

Extraintestinal pathogenic Escherichia coli (ExPEC) represent an important subclass of E. coli that cause a wide spectrum of diseases in human and animal hosts. Fimbriae are key virulence factors of ExPEC strains. These long surface located rod-shaped organelles mediate receptor-specific attachment...

Escherichia coli producing colibactin triggers premature and transmissible senescence in mammalian cells.

Directory of Open Access Journals (Sweden)

Thomas Secher

Full Text Available Cellular senescence is an irreversible state of proliferation arrest evoked by a myriad of stresses including oncogene activation, telomere shortening/dysfunction and genotoxic insults. It has been associated with tumor activation, immune suppression and aging, owing to the secretion of proinflammatory mediators. The bacterial genotoxin colibactin, encoded by the pks genomic island is frequently harboured by Escherichia coli strains of the B2 phylogenetic group. Mammalian cells exposed to live pks+ bacteria exhibit DNA-double strand breaks (DSB and undergo cell-cycle arrest and death. Here we show that cells that survive the acute bacterial infection with pks+ E. coli display hallmarks of cellular senescence: chronic DSB, prolonged cell-cycle arrest, enhanced senescence-associated β-galactosidase (SA-β-Gal activity, expansion of promyelocytic leukemia nuclear foci and senescence-associated heterochromatin foci. This was accompanied by reactive oxygen species production and pro-inflammatory cytokines, chemokines and proteases secretion. These mediators were able to trigger DSB and enhanced SA-β-Gal activity in bystander recipient cells treated with conditioned medium from senescent cells. Furthermore, these senescent cells promoted the growth of human tumor cells. In conclusion, the present data demonstrated that the E. coli genotoxin colibactin induces cellular senescence and subsequently propel bystander genotoxic and oncogenic effects.

The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles

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Fröhlich E

2012-11-01

Full Text Available Eleonore FröhlichCenter for Medical Research, Medical University of Graz, Graz, AustriaAbstract: Many types of nanoparticles (NPs are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging.Keywords: endocytosis, plasma membrane, lysosomes, polystyrene particles, quantum dots, dendrimers

Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

Science.gov (United States)

Tan, Guoqiang; Yang, Jing; Li, Tang; Zhao, Jin; Sun, Shujuan; Li, Xiaokang; Lin, Chuxian; Li, Jianghui; Zhou, Huaibin; Lyu, Jianxin; Ding, Huangen

2017-08-15

While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they

Quantitative risk analysis for potentially resistant E. coli in surface waters caused by antibiotic use in agricultural systems.

Science.gov (United States)

Limayem, Alya; Martin, Elizabeth M

2014-01-01

Antibiotics are frequently used in agricultural systems to promote livestock health and to control bacterial contaminants. Given the upsurge of the resistant fecal indicator bacteria (FIB) in the surface waters, a novel statistical method namely, microbial risk assessment (MRA) was performed, to evaluate the probability of infection by resistant FIB on populations exposed to recreational waters. Diarrheagenic Escherichia coli, except E. coli O157:H7, were selected for their prevalence in aquatic ecosystem. A comparative study between a typical E. coli pathway and a case scenario aggravated by antibiotic use has been performed via Crystal Ball® software in an effort to analyze a set of available inputs provided by the US institutions including E. coli concentrations in US Great Lakes through using random sampling and probability distributions. Results from forecasting a possible worst-case scenario dose-response, accounted for an approximate 50% chance for 20% of the exposed human populations to be infected by recreational water in the U.S. However, in a typical scenario, there is a 50% chance of infection for only 1% of the exposed human populations. The uncertain variable, E. coli concentration accounted for approximately 92.1% in a typical scenario as the major contributing factor of the dose-response model. Resistant FIB in recreational waters that are exacerbated by a low dose of antibiotic pollutants would increase the adverse health effects in exposed human populations by 10 fold.

Inactivation and Gene Expression of a Virulent WastewaterEscherichia coliStrain and the Nonvirulent CommensalEscherichia coliDSM1103 Strain upon Solar Irradiation

KAUST Repository

Aljassim, Nada I.; Mantilla-Calderon, David; Wang, Tiannyu; Hong, Pei-Ying

2017-01-01

This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

Inactivation and Gene Expression of a Virulent WastewaterEscherichia coliStrain and the Nonvirulent CommensalEscherichia coliDSM1103 Strain upon Solar Irradiation

KAUST Repository

Aljassim, Nada I.

2017-03-06

This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

[E75, R78 and D82 of Escherichia coli FtsZ are key residues for FtsZ cellular self-assembly and FtsZ-MreB interaction].

Science.gov (United States)

Huo, Yujia; Lu, Qiaonan; Zheng, Xiaowei; Ma, Yuanfang; Lu, Feng

2016-02-04

To explore effects of FtsZ mutants FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) on FtsZ self-assembly and interaction of FtsZ with MreB in Escherichia coli strains. METHODS) We constructed FtsZ and its mutant's plasmids by molecular clone and site-directed mutagenesis methods, and purified targeted proteins by affinity chromatography. QN6(ftsZ::yfp-cat), QN7(tsZ::yfp-cat), QN8(ftsZ(R78G)::yfp-cat) and QN9 (ftsZ(D82A):.:yfp-cat) strains were constructed by linear DNA homologous recombination. We observed cellular localization pattern of FtsZ and its mutants in E. coli by living cell imaging experiments, examined interaction of FtsZ/FtsZ*-FtsZ* and FtsZ/FtsZ*-MreB by Coimmunoprecipitation and bacteria two hybrid, and analyzed assembly characteristics of FtsZ mutants by Light scattering. RESULTS) The Yfp-labeled FtsZ(E75A), FtsZ(R78G) and FtsZ(D82A) mutant proteins failed to assemble into functional Z-ring structure and localize correctly in E. coli strains. Interaction of FtsZ with its mutants, or FtsZ*-FtsZ* and FtsZ*-MreB interaction were weakened or completely disappeared. In addition, in vitro experiments show that E75A, R78G and D82A mutations decreased the polymerization efficiency of FtsZ monomer. FtsZ E75, R78 and D82 are critical amino acids in the assembly, function of FtsZ protein and FtsZ-MreB interaction in E. coli strains.

Cellular automaton model for hydrogen transport dynamics through metallic surface

International Nuclear Information System (INIS)

Shimura, K.; Yamaguchi, K.; Terai, T.; Yamawaki, M.

2002-01-01

Hydrogen re-emission and re-combination at the surface of first wall materials are a crucial issue for the understanding of the fuel recycling and for the tritium inventory in plasma facing materials. It is know to be difficult to model the transient behaviour of those processes due to their complex time-transient nature. However, cellular automata (CA) are powerful tools to model such complex systems because of their nature of discreteness in both dependent and independent variables. Then the system can be represented by the fully local interactions between cells. For that reason, complex physical and chemical systems can be described by fairly simple manner. In this study, the kinetics of desorption of adsorbed hydrogen from an ideal metallic surface is modelled in CA. Thermal desorption is simulated with this model and the comparison with the theory of rate processes is performed to identify the validity of this model. The overall results show that this model is reasonable to express the desorption kinetics

The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

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Min Wang

Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

Cellular Homeostasis and Antioxidant Response in Epithelial HT29 Cells on Titania Nanotube Arrays Surface

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Rabiatul Basria SMN Mydin

2017-01-01

Full Text Available Cell growth and proliferative activities on titania nanotube arrays (TNA have raised alerts on genotoxicity risk. Present toxicogenomic approach focused on epithelial HT29 cells with TNA surface. Fledgling cell-TNA interaction has triggered G0/G1 cell cycle arrests and initiates DNA damage surveillance checkpoint, which possibly indicated the cellular stress stimuli. A profound gene regulation was observed to be involved in cellular growth and survival signals such as p53 and AKT expressions. Interestingly, the activation of redox regulator pathways (antioxidant defense was observed through the cascade interactions of GADD45, MYC, CHECK1, and ATR genes. These mechanisms furnish to protect DNA during cellular division from an oxidative challenge, set in motion with XRRC5 and RAD50 genes for DNA damage and repair activities. The cell fate decision on TNA-nanoenvironment has been reported to possibly regulate proliferative activities via expression of p27 and BCL2 tumor suppressor proteins, cogent with SKP2 and BCL2 oncogenic proteins suppression. Findings suggested that epithelial HT29 cells on the surface of TNA may have a positive regulation via cell-homeostasis mechanisms: a careful circadian orchestration between cell proliferation, survival, and death. This nanomolecular knowledge could be beneficial for advanced medical applications such as in nanomedicine and nanotherapeutics.

Injury and recovery of salmonella, Escherichia coli 0157:H7 and Listeria Monocytogenes on cantaloupe rind surfaces after hyrdogren peroxide and minimal thermal treatment

Science.gov (United States)

Introduction: Produce surface structures vary and complicate decontamination treatments for reducing attached bacteria. Purpose: The objective of this study on survival and recovery of injured population of Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes on cantaloupe rind surfaces...

Changes in antibiotic sensitivity and cell surface hydrophobicity in Escherichia coli injured by heating, freezing, drying or gamma radiation

International Nuclear Information System (INIS)

Mackey, B.M.

1983-01-01

Escherichia coli cells exposed to mild heating, freezing and thawing, drying or γ-radiation were sensitised to hydrophobic antibiotics and sodium deoxycholate but not to small hydrophilic antibiotics. These stress treatments also caused increases in cell surface hydrophobicity broadly reflecting the degree of sensitivity to hydrophobic antibiotics. (Auth.)

Correlation between E. coli levels and the presence of foodborne pathogens in surface irrigation water: Establishment of a sampling program.

Science.gov (United States)

Truchado, Pilar; Hernandez, Natalia; Gil, Maria I; Ivanek, Renata; Allende, Ana

2018-01-01

To establish the association between microbial indicators and the presence of foodborne pathogens in irrigation water, Escherichia coli was enumerated using two quantification methods (plate counts and PMA-qPCR) and presence/absence of pathogenic microorganisms, including five strains from the Shiga toxigenic E. coli (O157:H7, O26, O103, O111 and O145) and Salmonella spp. were evaluated. The results confirmed that surface water can be considered a microbial hazard when used for irrigation. The levels of viable E. coli were very similar to those of cultivable E. coli, except for irrigation water obtained from water reservoirs. Comparison between the E. coli counts in samples positive and negative for the presence of pathogenic bacteria for the evaluated water sources identified E. coli level of 2.35 log cfu/100 mL as a cut-off able to correctly predict positive and negative samples with 93% sensitivity and 66% specificity, respectively. Thus, for the samples with levels of E. coli under 2.35 log cfu/100 mL (e.g., 2.24 log cfu/100 mL) there was a 90% probability that the samples were not contaminated with pathogenic microorganism in locations with similar prevalence. E. coli levels in irrigation water were affected by the ambient temperature confirming that water source and climate conditions should be taken into account by growers when designing a sampling program and the frequency of the monitoring to make a better and more efficient use of their resources. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular automaton for surface reactions

Energy Technology Data Exchange (ETDEWEB)

Pechatnikov, E L [AN SSSR, Chernogolovka (Russian Federation). Otdelenie Inst. Khimicheskoj Fiziki; Frankowicz, A; Danielak, R [Uniwersytet Jagiellonski, Cracow (Poland)

1994-06-01

A new algorithm which overcomes some specific difficulties arising in modeling of heterogeneous catalytic processes by cellular automata (CA) technique is proposed. The algorithm was tested with scheme introduced by Ziff, Gulari and Barshad and showed a good agreement with their results. The problem of the physical adequacy and interpretation of the algorithm was discussed. (author). 10 refs, 4 figs.

Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire.

Science.gov (United States)

Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H

2013-10-03

Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method, we analyzed over 1,500 single cells throughout the mouse hematopoietic system and illustrate its utility for revealing important biological insights. The comprehensive single cell data set permits mapping of the mouse hematopoietic stem cell differentiation hierarchy by computational lineage progression analysis. Further profiling of 180 intracellular regulators enabled construction of a genetic network to assign the earliest differentiation event during hematopoietic lineage specification. Analysis of acute myeloid leukemia elicited by MLL-AF9 uncovered a distinct cellular hierarchy containing two independent self-renewing lineages with different clonal activities. The strategy has broad applicability in other cellular systems. Copyright © 2013 Elsevier Inc. All rights reserved.

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

International Nuclear Information System (INIS)

Xu, H. Howard; Real, Lilian; Bailey, Melissa Wu

2006-01-01

With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli

NARCIS (Netherlands)

Schulze, Egbert; Westphal, Adrie H.; Veenhuis, Marten; Kok, Arie de

1992-01-01

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been

FimH-mediated autoaggregation of Escherichia coli

DEFF Research Database (Denmark)

Schembri, Mark; Christiansen, G.; Klemm, Per

2001-01-01

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate D-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component...... FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within...

Secretion of clostridium cellulase by E. coli

Science.gov (United States)

Yu, Ida Kuo

1998-01-01

A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

International Nuclear Information System (INIS)

Wang, Lin-Xu; Mellon, Michael; Bowder, Dane; Quinn, Meghan; Shea, Danielle; Wood, Charles; Xiang, Shi-Hua

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture

Antimicrobial activity of Bacillus amyloliquefaciens LBM 5006 is enhanced in the presence of Escherichia coli.

Science.gov (United States)

Benitez, Lisianne; Correa, AnaPaula; Daroit, Daniel; Brandelli, Adriano

2011-03-01

Increased antimicrobial activity was observed when Bacillus amyloliquefaciens LBM 5006 strain was cultivated in the presence of thermally inactivated cells of Escherichia coli, but not with Staphylococcus aureus, Listeria monocytogenes, or Bacillus cereus. E. coli also enhanced the antimicrobial activity when it was added to the medium in the form of living cells or as cell debris after cellular fractionation. No inducing activity was observed with addition of cell-free supernatant of E. coli cultures, suggesting that inducing factor is associated to the cells. Polyacrylamide gel electrophoresis revealed that additional peptide bands are secreted when B. amyloliquefaciens was cultivated in the presence of cell debris of E. coli. These results suggest that the presence of intact or inactivated E. coli enhanced the synthesis of antimicrobial peptides by B. amyloliquefaciens LBM 5006.

Adherence to abiotic surface induces SOS response in Escherichia coli K-12 strains under aerobic and anaerobic conditions.

Science.gov (United States)

Costa, Suelen B; Campos, Ana Carolina C; Pereira, Ana Claudia M; de Mattos-Guaraldi, Ana Luiza; Júnior, Raphael Hirata; Rosa, Ana Cláudia P; Asad, Lídia M B O

2014-09-01

During the colonization of surfaces, Escherichia coli bacteria often encounter DNA-damaging agents and these agents can induce several defence mechanisms. Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species (ROS) generated by chemical and physical agents or by metabolism. In this work, we have evaluated whether the interaction with an abiotic surface by mutants derived from E. coli K-12 deficient in some enzymes that are part of BER causes DNA damage and associated filamentation. Moreover, we studied the role of endonuclease V (nfi gene; 1506 mutant strain) in biofilm formation. Endonuclease V is an enzyme that is involved in DNA repair of nitrosative lesions. We verified that endonuclease V is involved in biofilm formation. Our results showed more filamentation in the xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) than in the wild-type strain (AB1157). By contrast, the mutant nfi did not present filamentation in biofilm, although its wild-type strain (1466) showed rare filaments in biofilm. The filamentation of bacterial cells attaching to a surface was a consequence of SOS induction measured by the SOS chromotest. However, biofilm formation depended on the ability of the bacteria to induce the SOS response since the mutant lexA Ind(-) did not induce the SOS response and did not form any biofilm. Oxygen tension was an important factor for the interaction of the BER mutants, since these mutants exhibited decreased quantitative adherence under anaerobic conditions. However, our results showed that the presence or absence of oxygen did not affect the viability of BW9091 and BW535 strains. The nfi mutant and its wild-type did not exhibit decreased biofilm formation under anaerobic conditions. Scanning electron microscopy was also performed on the E. coli K-12 strains that had adhered to the glass, and we observed the presence of a structure similar to an extracellular matrix that depended on the

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology.

Science.gov (United States)

Shafiee, Fatemeh; Rabbani, Mohammad; Jahanian-Najafabadi, Ali

2017-01-01

The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli ( E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3), followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM). Finally, the best culture medium was selected. Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml). Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Optimization of the Expression of DT386-BR2 Fusion Protein in Escherichia coli using Response Surface Methodology

Directory of Open Access Journals (Sweden)

Fatemeh Shafiee

2017-01-01

Full Text Available Background: The aim of this study was to determine the best condition for the production of DT386-BR2 fusion protein, an immunotoxin consisting of catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, for targeted eradication of cancer cells, in terms of the host, cultivation condition, and culture medium. Materials and Methods: Recombinant pET28a vector containing the codons optimized for the expression of the DT386-BR2 gene was transformed to different strains of Escherichia coli (E. coli BL21 DE3, E. coli Rosetta DE3 and E. coli Rosetta-gami 2 DE3, followed by the induction of expression using 1 mM IPTG. Then, the strain with the highest ability to produce recombinant protein was selected and used to determine the best expression condition using response surface methodology (RSM. Finally, the best culture medium was selected. Results: Densitometry analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expressed fusion protein showed that E. coli Rosetta DE3 produced the highest amounts of the recombinant fusion protein when quantified by 1 mg/ml bovine serum albumin (178.07 μg/ml. Results of RSM also showed the best condition for the production of the recombinant fusion protein was induction with 1 mM IPTG for 2 h at 37°C. Finally, it was established that terrific broth could produce higher amounts of the fusion protein when compared to other culture media. Conclusion: In this study, we expressed the recombinant DT386-BR2 fusion protein in large amounts by optimizing the expression host, cultivation condition, and culture medium. This fusion protein will be subjected to purification and evaluation of its cytotoxic effects in future studies.

Transfer of Escherichia coli O157:H7 from equipment surfaces to fresh-cut leafy greens during processing in a model pilot-plant production line with sanitizer-free water.

Science.gov (United States)

Buchholz, Annemarie L; Davidson, Gordon R; Marks, Bradley P; Todd, Ewen C D; Ryser, Elliot T

2012-11-01

Escherichia coli O157:H7 contamination of fresh-cut leafy greens has become a public health concern as a result of several large outbreaks. The goal of this study was to generate baseline data for E. coli O157:H7 transfer from product-inoculated equipment surfaces to uninoculated lettuce during pilot-scale processing without a sanitizer. Uninoculated cored heads of iceberg and romaine lettuce (22.7 kg) were processed using a commercial shredder, step conveyor, 3.3-m flume tank with sanitizer-free tap water, shaker table, and centrifugal dryer, followed by 22.7 kg of product that had been dip inoculated to contain ∼10(6), 10(4), or 10(2) CFU/g of a four-strain avirulent, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 cocktail. After draining the flume tank and refilling the holding tank with tap water, 90.8 kg of uninoculated product was similarly processed and collected in ∼5-kg aliquots. After processing, 42 equipment surface samples and 46 iceberg or 36 romaine lettuce samples (25 g each) from the collection baskets were quantitatively examined for E. coli O157:H7 by direct plating or membrane filtration using tryptic soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Initially, the greatest E. coli O157:H7 transfer was seen from inoculated lettuce to the shredder and conveyor belt, with all equipment surface populations decreasing 90 to 99% after processing 90.8 kg of uncontaminated product. After processing lettuce containing 10(6) or 10(4) E. coli O157:H7 CFU/g followed by uninoculated lettuce, E. coli O157:H7 was quantifiable throughout the entire 90.8 kg of product. At an inoculation level of 10(2) CFU/g, E. coli O157:H7 was consistently detected in the first 21.2 kg of previously uninoculated lettuce at 2 to 3 log CFU/100 g and transferred to 78 kg of product. These baseline E. coli O157:H7 transfer results will help determine the degree of sanitizer efficacy required to better ensure the safety of fresh-cut leafy

Protein-protein association and cellular localization of four essential gene products encoded by tellurite resistance-conferring cluster "ter" from pathogenic Escherichia coli.

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Valkovicova, Lenka; Vavrova, Silvia Minarikova; Mravec, Jozef; Grones, Jozef; Turna, Jan

2013-12-01

Gene cluster "ter" conferring high tellurite resistance has been identified in various pathogenic bacteria including Escherichia coli O157:H7. However, the precise mechanism as well as the molecular function of the respective gene products is unclear. Here we describe protein-protein association and localization analyses of four essential Ter proteins encoded by minimal resistance-conferring fragment (terBCDE) by means of recombinant expression. By using a two-plasmid complementation system we show that the overproduced single Ter proteins are not able to mediate tellurite resistance, but all Ter members play an irreplaceable role within the cluster. We identified several types of homotypic and heterotypic protein-protein associations among the Ter proteins by in vitro and in vivo pull-down assays and determined their cellular localization by cytosol/membrane fractionation. Our results strongly suggest that Ter proteins function involves their mutual association, which probably happens at the interface of the inner plasma membrane and the cytosol.

Prevalence of veterinary antibiotics and antibiotic-resistant Escherichia coli in the surface water of a livestock production region in northern China.

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Xuelian Zhang

Full Text Available This study investigated the occurrence of 12 veterinary antibiotics (VAs and the susceptibility of Escherichia coli (E. coli in a rural water system that was affected by livestock production in northern China. Each of the surveyed sites was determined with at least eight antibiotics with maximum concentration of up to 450 ng L(-1. The use of VAs in livestock farming probably was a primary source of antibiotics in the rivers. Increasing total antibiotics were measured from up- to mid- and downstream in the two tributaries. Eighty-eight percent of the 218 E. coli isolates that were derived from the study area exhibited, in total, 48 resistance profiles against the eight examined drugs. Significant correlations were found among the resistance rates of sulfamethoxazole-trimethoprim, chloromycetin and ampicillin as well as between tetracycline and chlortetracycline, suggesting a possible cross-selection for resistance among these drugs. The E. coli resistance frequency also increased from up- to midstream in the three rivers. E. coli isolates from different water systems showed varying drug numbers of resistance. No clear relationship was observed in the antibiotic resistance frequency with corresponding antibiotic concentration, indicating that the antibiotic resistance for E. coli in the aquatic environment might be affected by factors besides antibiotics. High numbers of resistant E. coli were also isolated from the conserved reservoir. These results suggest that rural surface water may become a large pool of VAs and resistant bacteria. This study contributes to current information on VAs and resistant bacteria contamination in aquatic environments particularly in areas under intensive agriculture. Moreover, this study indicates an urgent need to monitor the use of VAs in animal production, and to control the release of animal-originated antibiotics into the environment.

Prevalence of veterinary antibiotics and antibiotic-resistant Escherichia coli in the surface water of a livestock production region in northern China.

Science.gov (United States)

Zhang, Xuelian; Li, Yanxia; Liu, Bei; Wang, Jing; Feng, Chenghong; Gao, Min; Wang, Lina

2014-01-01

This study investigated the occurrence of 12 veterinary antibiotics (VAs) and the susceptibility of Escherichia coli (E. coli) in a rural water system that was affected by livestock production in northern China. Each of the surveyed sites was determined with at least eight antibiotics with maximum concentration of up to 450 ng L(-1). The use of VAs in livestock farming probably was a primary source of antibiotics in the rivers. Increasing total antibiotics were measured from up- to mid- and downstream in the two tributaries. Eighty-eight percent of the 218 E. coli isolates that were derived from the study area exhibited, in total, 48 resistance profiles against the eight examined drugs. Significant correlations were found among the resistance rates of sulfamethoxazole-trimethoprim, chloromycetin and ampicillin as well as between tetracycline and chlortetracycline, suggesting a possible cross-selection for resistance among these drugs. The E. coli resistance frequency also increased from up- to midstream in the three rivers. E. coli isolates from different water systems showed varying drug numbers of resistance. No clear relationship was observed in the antibiotic resistance frequency with corresponding antibiotic concentration, indicating that the antibiotic resistance for E. coli in the aquatic environment might be affected by factors besides antibiotics. High numbers of resistant E. coli were also isolated from the conserved reservoir. These results suggest that rural surface water may become a large pool of VAs and resistant bacteria. This study contributes to current information on VAs and resistant bacteria contamination in aquatic environments particularly in areas under intensive agriculture. Moreover, this study indicates an urgent need to monitor the use of VAs in animal production, and to control the release of animal-originated antibiotics into the environment.

Prevalence of Veterinary Antibiotics and Antibiotic-Resistant Escherichia coli in the Surface Water of a Livestock Production Region in Northern China

Science.gov (United States)

Zhang, Xuelian; Li, Yanxia; Liu, Bei; Wang, Jing; Feng, Chenghong; Gao, Min; Wang, Lina

2014-01-01

This study investigated the occurrence of 12 veterinary antibiotics (VAs) and the susceptibility of Escherichia coli (E. coli) in a rural water system that was affected by livestock production in northern China. Each of the surveyed sites was determined with at least eight antibiotics with maximum concentration of up to 450 ng L−1. The use of VAs in livestock farming probably was a primary source of antibiotics in the rivers. Increasing total antibiotics were measured from up- to mid- and downstream in the two tributaries. Eighty-eight percent of the 218 E. coli isolates that were derived from the study area exhibited, in total, 48 resistance profiles against the eight examined drugs. Significant correlations were found among the resistance rates of sulfamethoxazole-trimethoprim, chloromycetin and ampicillin as well as between tetracycline and chlortetracycline, suggesting a possible cross-selection for resistance among these drugs. The E. coli resistance frequency also increased from up- to midstream in the three rivers. E. coli isolates from different water systems showed varying drug numbers of resistance. No clear relationship was observed in the antibiotic resistance frequency with corresponding antibiotic concentration, indicating that the antibiotic resistance for E. coli in the aquatic environment might be affected by factors besides antibiotics. High numbers of resistant E. coli were also isolated from the conserved reservoir. These results suggest that rural surface water may become a large pool of VAs and resistant bacteria. This study contributes to current information on VAs and resistant bacteria contamination in aquatic environments particularly in areas under intensive agriculture. Moreover, this study indicates an urgent need to monitor the use of VAs in animal production, and to control the release of animal-originated antibiotics into the environment. PMID:25372873

Role of bolA and rpoS genes in biofilm formation and adherence pattern by Escherichia coli K-12 MG1655 on polypropylene, stainless steel, and silicone surfaces.

Science.gov (United States)

Adnan, Mohd; Sousa, Ana Margarida; Machado, Idalina; Pereira, Maria Olivia; Khan, Saif; Morton, Glyn; Hadi, Sibte

2017-06-01

Escherichia coli has developed sophisticated means to sense, respond, and adapt in stressed environment. It has served as a model organism for studies in molecular genetics and physiology since the 1960s. Stress response genes are induced whenever a cell needs to adapt and survive under unfavorable growth conditions. Two of the possible important genes are rpoS and bolA. The rpoS gene has been known as the alternative sigma (σ) factor, which controls the expression of a large number of genes, which are involved in responses to various stress factors as well as transition to stationary phase from exponential form of growth. Morphogene bolA response to stressed environment leads to round morphology of E. coli cells, but little is known about its involvement in biofilms and its development or maintenance. This study has been undertaken to address the adherence pattern and formation of biofilms by E. coli on stainless steel, polypropylene, and silicone surfaces after 24 h of growth at 37 °C. Scanning electron microscopy was used for direct examination of the cell attachment and biofilm formation on various surfaces and it was found that, in the presence of bolA, E. coli cells were able to attach to the stainless steel and silicone very well. By contrast, polypropylene surface was not found to be attractive for E. coli cells. This indicates that bolA responded and can play a major role in the presence and absence of rpoS in cell attachment.

Is central dogma a global property of cellular information flow?

Science.gov (United States)

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

Effects of Genetic Variation on the E. coli Host-Circuit Interface

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Stefano Cardinale

2013-07-01

Full Text Available Predictable operation of engineered biological circuitry requires the knowledge of host factors that compete or interfere with designed function. Here, we perform a detailed analysis of the interaction between constitutive expression from a test circuit and cell-growth properties in a subset of genetic variants of the bacterium Escherichia coli. Differences in generic cellular parameters such as ribosome availability and growth rate are the main determinants (89% of strain-specific differences of circuit performance in laboratory-adapted strains but are responsible for only 35% of expression variation across 88 mutants of E. coli BW25113. In the latter strains, we identify specific cell functions, such as nitrogen metabolism, that directly modulate circuit behavior. Finally, we expose aspects of carbon metabolism that act in a strain- and sequence-specific manner. This method of dissecting interactions between host factors and heterologous circuits enables the discovery of mechanisms of interference necessary for the development of design principles for predictable cellular engineering.

Influence of type-I fimbriae and fluid shear stress on bacterial behavior and multicellular architecture of early Escherichia coli biofilms at single-cell resolution.

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Wang, Liyun; Keatch, Robert; Zhao, Qi; Wright, John A; Bryant, Clare E; Redmann, Anna L; Terentjev, Eugene M

2018-01-12

Biofilm formation on abiotic surfaces in food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine cellular architecture of early biofilms and bacterial behavior of the constituent cells remains largely unknown. In this study we examine the specific role of type-I fimbriae in nascent stages of biofilm formation and the response of micro-colonies to environmental flow shear at single-cell resolution. The results show that type-I fimbriae are not required for reversible adhesion from plankton, but critical for irreversible adhesion of Escherichia coli ( E.coli ) MG1655 forming biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing a firm cell-surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E.coli on the surface. After application of shear stress, bacterial retention is dominated by the 3D architecture of colonies independent of the population and the multi-layered structure could protect the embedded cells from being insulted by fluid shear, while cell membrane permeability mainly depends on the biofilm population and the duration time of the shear stress. Importance Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level, thus little is known about how individual bacterial behavior within biofilms and multicellular architecture are influenced by bacterial appendages (e.g. pili/fimbriae) and environmental factors during early biofilm formation. We apply Confocal Laser Scanning Microscopy (CLSM) to visualize E.coli micro-colonies at single-cell resolution. Our findings suggest that type-I fimbriae are vital to the initiation of bacterial proliferation on surfaces and that the responses of biofilm architecture and cell membrane permeability of constituent bacteria to fluid shear stress are different, which are

Cellular Reflectarray Antenna

Science.gov (United States)

Romanofsky, Robert R.

2010-01-01

The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

Biosynthesis of Enterobacterial Common Antigen: The ECA-Trace Phenotype of Salmonella Typhimurium and The Role of the rfe Gene in 08 Side-Chain Synthesis in Escherichia Coli

Science.gov (United States)

1993-03-18

hitherto unidentified class of compounds for procaryotes . There are several possible explanations for the occurrence of lipids IIa and lIb . The first...SalOJoneiia TypbiOJuriUOl. Cellular and Molecular Biology .. Vol. I. American Society For Microbiology. Washington. D. C. Galanos. C. 0 .. and E. T...ill: 3697-3702 Hirota,Y. , H. Suzuki, Y. Nishimura, S. Yasuda. 1977. On the process of cellular division in Escherichia coli: a mutant of £. coli

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

International Nuclear Information System (INIS)

Peters, J.

1977-01-01

Some experimental work is described showing that near-U.V. irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-U.V., and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex. (U.K.)

In vivo photoinactivation of Escherichia coli ribonucleoside reductase by near-ultraviolet light

Energy Technology Data Exchange (ETDEWEB)

Peters, J [California Univ., Irvine (USA)

1977-06-09

Some experimental work is described showing that near-uv irradiation of E.coli cells selectively destroys RDP-reductase (ribonucleoside diphosphate reductase) activity in vivo are providing evidence relating the loss of RDP-reductase to loss of cellular visibility and the inactivity of irrdiated cells to support the replication of DNA phages. The data are consistent with the interpretation that the principal cause in the killing of exponentially growing E.coli cells by near-uv, and the loss of ability of irradiated host cells to support the replication of DNA phages, is the photoinactivation of the RDP-reductase complex.

Statistical Optimization of the Induction of Phytase Production by Arabinose in a recombinant E. coli using Response Surface Methodology

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Abd-El Aziem Farouk

2017-11-01

Full Text Available The production of phytase in a recombinant E.coli using the pBAD expression  system was optimized using response surface methodology with full-factorial faced centered central composite design. The ampicilin and arabinose concentration in the cultivation media and the incubation temperature were optimized in order to maximize phytase production using 2 3  central composite experimental design. With this design the number of actual experiment performed could be reduced while allowing eludidation of possible interactions among these factors. The most significant parameter was shown to be the linear and quadratic effect of the incubation temperature.  Optimal conditions for phytase production were determined to be 100 µg/ml ampicilin, 0.2 % arabinose and an incubation temperature of 37ºC. The production of phytase in the recombinant E. coli was scaled up to 100 ml and 1000 ml.

Characterization of Silk Fibroin Modified Surface: A Proteomic View of Cellular Response Proteins Induced by Biomaterials

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Ming-Hui Yang

2014-01-01

Full Text Available The purpose of this study was to develop the pathway of silk fibroin (SF biopolymer surface induced cell membrane protein activation. Fibroblasts were used as an experimental model to evaluate the responses of cellular proteins induced by biopolymer material using a mass spectrometry-based profiling system. The surface was covered by multiwalled carbon nanotubes (CNTs and SF to increase the surface area, enhance the adhesion of biopolymer, and promote the rate of cell proliferation. The amount of adhered fibroblasts on CNTs/SF electrodes of quartz crystal microbalance (QCM greatly exceeded those on other surfaces. Moreover, analyzing differential protein expressions of adhered fibroblasts on the biopolymer surface by proteomic approaches indicated that CD44 may be a key protein. Through this study, utilization of mass spectrometry-based proteomics in evaluation of cell adhesion on biopolymer was proposed.

Interaction force measurement between E. coli cells and nanoparticles immobilized surfaces by using AFM.

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Zhang, Wen; Stack, Andrew G; Chen, Yongsheng

2011-02-01

To better understand environmental behaviors of nanoparticles (NPs), we used the atomic force microscopy (AFM) to measure interaction forces between E. coli cells and NPs immobilized on surfaces in an aqueous environment. The results showed that adhesion force strength was significantly influenced by particle size for both hematite (α-Fe(2)O(3)) and corundum (α-Al(2)O(3)) NPs whereas the effect on the repulsive force was not observed. The adhesion force decreased from 6.3±0.7nN to 0.8±0.4nN as hematite NPs increased from 26nm to 98nm in diameter. Corundum NPs exhibited a similar dependence of adhesion force on particle size. The Johnson-Kendall-Roberts (JKR) model was employed to estimate the contact area between E. coli cells and NPs, and based on the JKR model a new model that considers local effective contact area was developed. The prediction of the new model matched the size dependence of adhesion force in experimental results. Size effects on adhesion forces may originate from the difference in local effective contact areas as supported by our model. These findings provide fundamental information for interpreting the environmental behaviors and biological interactions of NPs, which barely have been addressed. Copyright © 2010 Elsevier B.V. All rights reserved.

Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

Science.gov (United States)

Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

2014-01-01

In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

Is central dogma a global property of cellular information flow?

Directory of Open Access Journals (Sweden)

Vincent ePiras

2012-11-01

Full Text Available The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcript to protein show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber.

Science.gov (United States)

McClaine, Jennifer W; Ford, Roseanne M

2002-04-20

A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities. The effect of flagella on adhesion was investigated by performing experiments with several E. coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella). We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system. At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain. At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria. Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed. The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains. This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear. There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development. Copyright 2002 Wiley Periodicals, Inc.

The relationship between cellular adhesion and surface roughness for polyurethane modified by microwave plasma radiation

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Heidari S

2011-04-01

Full Text Available Saeed Heidari Keshel1, S Neda Kh Azhdadi2, Azadeh Asefnezhad2, Mohammad Sadraeian3, Mohamad Montazeri4, Esmaeil Biazar51Stem Cell Preparation Unit, Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences; 2Department of Biomaterial Engineering, Faculty of Biomedical Engineering, Science and Research Branch - Islamic Azad University; 3Young Researchers Club, Islamic Azad University, North Tehran Branch, Tehran; 4Faculty of Medical Sciences, Babol University of Medical Sciences, Babol; 5Department of Chemistry, Islamic Azad University, Tonekabon, IranAbstract: Surface modification of medical polymers is carried out to improve biocompatibility. In this study, conventional polyurethane was exposed to microwave plasma treatment with oxygen and argon gases for 30 seconds and 60 seconds. Attenuated total reflection Fourier transform infrared spectra investigations of irradiated samples indicated the presence of functional groups. Atomic force microscope images of samples irradiated with inert and active gases indicated the nanometric topography of the sample surfaces. Samples irradiated by oxygen plasma indicated high roughness compared with those irradiated by inert plasma for the different lengths of time. In addition, surface roughness increased with time, which can be due to a reduction of contact angle of samples irradiated by oxygen plasma. Contact angle analysis indicated a reduction in samples irradiated with both types of plasma. However, samples irradiated with oxygen plasma indicated lower contact angle compared with those irradiated by argon plasma. Cellular investigations with unrestricted somatic stem cells showed better adhesion, cell growth, and proliferation among samples radiated by oxygen plasma for longer than for normal samples.Keywords: surface topography, polyurethane, plasma treatment, cellular investigation

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions.

Science.gov (United States)

Ditu, Lia-Mara; Chifiriuc, Mariana Carmen; Bezirtzoglou, Eugenia; Voltsi, Chrysa; Bleotu, Coralia; Pelinescu, Diana; Mihaescu, Grigore; Lazar, Veronica

2011-12-01

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains. The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tunable recombinant protein expression with E. coli in a mixed-feed environment.

Science.gov (United States)

Sagmeister, Patrick; Schimek, Clemens; Meitz, Andrea; Herwig, Christoph; Spadiut, Oliver

2014-04-01

Controlling the recombinant protein production rate in Escherichia coli is of utmost importance to ensure product quality and quantity. Up to now, only the genetic construct, introduced into E. coli, and the specific growth rate of the culture were used to influence and stir the productivity. However, bioprocess technological means to control or even tune the productivity of E. coli are scarce. Here, we present a novel method for the process-technological control over the recombinant protein expression rate in E. coli. A mixed-feed fed-batch bioprocess based on the araBAD promoter expression system using both D-glucose and L-arabinose as assimilable C-sources was designed. Using the model product green fluorescent protein, we show that the specific product formation rate can be efficiently tuned even on the cellular level only via the uptake rate of L-arabinose. This novel approach introduces an additional degree of freedom for the design of recombinant bioprocesses with E. coli. We anticipate that the presented method will result in significant quality and robustness improvement as well as cost and process time reduction for recombinant bacterial bioprocesses in the future.

Modelling for near-surface interaction of lithium ceramics and sweep-gas by use of cellular automation

International Nuclear Information System (INIS)

Shimura, K.; Terai, T.; Yamawaki, M.; Yamaguchi, K.

2003-01-01

Tritium release from the lithium ceramics as a fusion reactor breeder material is strongly affected by the composition of the sweep-gas as result of its influences with the material's surface. The typical surface processes which play important roles are adsorption, desorption and interaction between vacancy site and the constituents of the sweep-gas. Among a large number of studies and models, yet it seems to be difficult to model the overall behaviour of those processes due to its complex time-transient nature. In the present work the coarse grained atomic simulation based on the Cellular Automaton (CA) is used to model the dynamics of near-surface interaction between Li 2 O surface and sweep-gas that is consisting of a noble gas, hydrogen gas and water vapour. (author)

G-quadruplex recognition activities of E. Coli MutS

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Ehrat Edward A

2012-07-01

Full Text Available Abstract Background Guanine quadruplex (G4 DNA is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA. Results Here, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements. Conclusions Taken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.

ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli ANALISIS CEMARAN BAKTERI Escherichia coli

OpenAIRE

ANGGREINI, RAHAYU

2015-01-01

2015 RAHAYU ANGGREINI coli Penelitian ini bertujuan untuk melakukan identifikasi cemaran bakteri E. coli O157:H7 pada daging sapi di kota Makassar. Sampel pada penelitian ini sebanyak 72 sampel Kata Kunci : Daging sapi, pasar tradisional, E. coli, E. coli O157:H7, kontaminasi bakteri, identifikasi E. coli O157:H7.

Electrophoretically deposited multiwalled carbon nanotube based amperometric genosensor for E.coli detection

International Nuclear Information System (INIS)

Bhardwaj, Hema; Solanki, Shipra; Sumana, Gajjala

2016-01-01

This work reports on a sensitive and selective genosensor fabrication method for Escherichia coli ( E.coli) detection. The functionalized multiwalled carbon nanotubes (MWCNT) synthesized via chemical vapour deposition have been deposited electrophoretically onto indium tin oxide coated glass surface and have been utilized as matrices for the covalent immobilization of E.coli specific probe oligonucleotide that was identified from the 16s rRNA coding region of the E.coli genome. This fabricated functionalized MWCNT based platform sought to provide improved fundamental characteristics to electrode interface in terms of electro-active surface area and diffusion coefficient. Electrochemical cyclic voltammetry revealed that this genosensor exhibits a linear response to complementary DNA in the concentration range of 10 -7 to 10 -12 M with a detection limit of 1×10 -12 M. (paper)

UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in a tolC Mutant Escherichia coli Strain Leads to Cell Death

OpenAIRE

Humnabadkar, Vaishali; Prabhakar, K. R.; Narayan, Ashwini; Sharma, Sreevalli; Guptha, Supreeth; Manjrekar, Praveena; Chinnapattu, Murugan; Ramachandran, Vasanthi; Hameed, Shahul P.; Ravishankar, Sudha; Chatterji, Monalisa

2014-01-01

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tol...

Influence of surface polysaccharides of Escherichia coli O157:H7 on plant defense response and survival of the human enteric pathogen on Arabidopsis thaliana and lettuce (Lactuca sativa).

Science.gov (United States)

Jang, Hyein; Matthews, Karl R

2018-04-01

This study aimed to determine the influence of bacterial surface polysaccharides (cellulose, colanic acid, and lipopolysaccharide; LPS) on the colonization or survival of Escherichia coli O157:H7 on plants and the plant defense response. Survival of E. coli O157:H7 were evaluated on Arabidopsis thaliana and romaine lettuce as a model plant and an edible crop (leafy vegetable), respectively. The population of the wild-type strain of E. coli O157:H7 on Arabidopsis plants and lettuce was significantly (P lettuce regardless of day post-inoculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modification of radiation response of E. coli B/r cells by phenothiazines

International Nuclear Information System (INIS)

Maniar, H.S.; Singh, B.B.

1983-01-01

Promethazine and trimeprazine sensitized anoxic E. coli B/r cells to 60 Co gamma-rays, but both drugs showed a radioprotective effect under euoxic conditions. Their radiosensitizing effect was found to be due to the reaction of radiolytically induced hydroxyl radicals with the sensitizers. The radioprotective effect of these drugs is attributed to changes in the membrane structure conducive with chemical repair of the damaged sites in the gel region of the cellular membrane by intracellular sulphydryl compounds. Pre-irradiation depletion of sulphydryls from E. coli B/r by treatment with N-ethyl maleimide abolished the radioprotective effect of these drugs under euoxic conditions. (author)

Inhibition of Escherichia coli O157:H7 on stainless steel using Pseudomonas veronii biofilms.

Science.gov (United States)

Kim, Y; Kim, H; Beuchat, L R; Ryu, J-H

2018-05-01

We produced a Pseudomonas veronii biofilm on the surface of a stainless steel that is inhibitory to Escherichia coli O157:H7. Pseudomonas veronii strain KACC 81051BP, isolated from lettuce, readily formed biofilm on the surface of stainless steel coupons (SSCs) immersed in tryptic soy broth at 25°C. Cells showed significantly (P ≤ 0·05) enhanced tolerance to desiccation stress (43% relative humidity (RH)) and retained antimicrobial activity against E. coli O157:H7. The number of E. coli O157:H7 (control; 4·1 ± 0·1 log CFU per coupon) on sterile SSCs decreased to 2·7 ± 0·2 log CFU per coupon after exposure to 43% RH at 25°C for 48 h, while the population of E. coli O157:H7 (4·1 ± 0·0 log CFU per coupon) on SSCs containing P. veronii biofilm decreased to below the theoretical detection limit (1·5 log CFU per coupon) within 24 h. The antimicrobial biofilm produced on stainless steel may have application in preventing cross-contamination by E. coli O157:H7 on other abiotic surfaces in food-contact environments. The presence of Escherichia coli O157:H7 on environmental surfaces of food manufacturing, transportation and storage facilities is a significant food safety concern because it can result in cross-contamination of food products. In this study, we developed a Pseudomonas veronii biofilm on the surface of a stainless steel that inhibits the growth of E. coli O157:H7. Since P. veronii in biofilm resists desiccation, it provides persistent antimicrobial activity. Information presented here provides novel and practical insights to developing biological strategies to inactivate E. coli O157:H7 on diverse surfaces in food processing and handling environments. © 2018 The Society for Applied Microbiology.

76 FR 20542 - Escherichia coli

Science.gov (United States)

2011-04-13

... beef, Escherichia coli and coliphages were found in chicken, fresh pork, fresh oyster, fresh mushrooms, lettuce, chicken pot pie, biscuit dough, deli loaf, deli roasted turkey, and package roasted chicken... surfaces, and in foods such as ground beef, pork sausage, chicken, oysters, cheese, fresh mushrooms, and...

Plasma Electrolytic Oxidation of Titanium Implant Surfaces: Microgroove-Structures Improve Cellular Adhesion and Viability.

Science.gov (United States)

Hartjen, Philip; Hoffmann, Alexia; Henningsen, Anders; Barbeck, Mike; Kopp, Alexander; Kluwe, Lan; Precht, Clarissa; Quatela, Olivia; Gaudin, Robert; Heiland, Max; Friedrich, Reinhard E; Knipfer, Christian; Grubeanu, Daniel; Smeets, Ralf; Jung, Ole

2018-01-01

Plasma electrolytic oxidation (PEO) is an established electrochemical treatment technique that can be used for surface modifications of metal implants. In this study we to treated titanium implants with PEO, to examine the resulting microstructure and to characterize adhesion and viability of cells on the treated surfaces. Our aim was to identify an optimal surface-modification for titanium implants in order to improve soft-tissue integration. Three surface-variants were generated on titanium alloy Ti6Al4V by PEO-treatment. The elemental composition and the microstructures of the surfaces were characterized using energy dispersive X-ray spectroscopy, scanning electron microscopy and profilometry. In vitro cytocompatibility of the surfaces was assessed by seeding L929 fibroblasts onto them and measuring the adhesion, viability and cytotoxicity of cells by means of live/dead staining, XTT assay and LDH assay. Electron microscopy and profilometry revealed that the PEO-surface variants differed largely in microstructure/topography, porosity and roughness from the untreated control material as well as from one another. Roughness was generally increased after PEO-treatment. In vitro, PEO-treatment led to improved cellular adhesion and viability of cells accompanied by decreased cytotoxicity. PEO-treatment provides a promising strategy to improve the integration of titanium implants with surrounding tissues. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Fluorinated cellular polypropylene films with time-invariant excellent surface electret properties by post-treatments

International Nuclear Information System (INIS)

An Zhenlian; Mao Mingjun; Yao Junlan; Zhang Yewen; Xia Zhongfu

2010-01-01

In this work, to improve the electret properties of cellular polypropylene films, they were fluorinated and post-treated with nitrous oxide and by isothermal crystallization. Surface electret properties of the samples were investigated by thermally stimulated discharge current measurements, and their compositions and structures were analysed by attenuated total reflection infrared spectroscopy and wide angle x-ray diffraction, respectively. Time-dependent deterioration of surface electret properties was observed for the fluorinated samples without the nitrous oxide post-treatment. However, deterioration did not occur for the fluorinated samples post-treated with nitrous oxide, and time-invariant excellent surface electret properties or deep surface charge traps were obtained by the combined post-treatments of the fluorinated samples with nitrous oxide and by isothermal crystallization. Based on the analyses of composition and structure of the treated samples, the deterioration was clarified to be due to a trace of oxygen in the reactive mixture, which led to the formation of peroxy RO 2 . radicals in the fluorinated surface layer. The time invariability of surface electret properties was owing to the rapid termination of the peroxy RO 2 . radicals by nitrous oxide. And the deep surface charge traps resulted from the isothermal crystallization treatment which led to an increase in the efficient charging interface between the crystallite and amorphous region and its property change.

Induction of sos response in Escherichia Coli cells by gamma rays

International Nuclear Information System (INIS)

Fuentes Lorenzo, J.L.; Padron Soler, E.; Martin Hernandez, G.; Perez Tamayo, N.; del Sol Abascal, E.R.; Almeida Varela, E.

1996-01-01

The kinetics of sos response induction in Escherichia Coli cells was studied by means of the gene fusion SfiA:LacZ. In these cells, the specific beta galactosidase activity and the cellular growth rate showed an exponential behaviour. The sensitivity of the GC 2181 starin to gamma irradiation is equal to Do -1= 0.00088/Gy. The beta galactosidase activity

Novel Materials for Cellular Nanosensors

DEFF Research Database (Denmark)

Sasso, Luigi

The monitoring of cellular behavior is useful for the advancement of biomedical diagnostics, drug development and the understanding of a cell as the main unit of the human body. Micro- and nanotechnology allow for the creation of functional devices that enhance the study of cellular dynamics...... modifications for electrochemical nanosensors for the detection of analytes released from cells. Two type of materials were investigated, each pertaining to the two different aspects of such devices: peptide nanostructures were studied for the creation of cellular sensing substrates that mimic in vivo surfaces...... and that offer advantages of functionalization, and conducting polymers were used as electrochemical sensor surface modifications for increasing the sensitivity towards relevant analytes, with focus on the detection of dopamine released from cells via exocytosis. Vertical peptide nanowires were synthesized from...

Cellular response of Campylobacter jejuni to trisodium phosphate

DEFF Research Database (Denmark)

Riedel, Charlotte Tandrup; Cohn, M. T.; Stabler, R. A.

2012-01-01

The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal...... exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E....... coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters...

Alterations in ambipolar characteristic of graphene due to adsorption of Escherichia coli bacteria

Science.gov (United States)

Mulyana, Yana; Uenuma, Mutsunori; Okamoto, Naofumi; Ishikawa, Yasuaki; Yamashita, Ichiro; Uraoka, Yukiharu

2018-03-01

In order to evaluate the interaction between biomaterials and graphene from the perspective of its ambipolar characteristic, we have investigated the alteration in ambipolarity of graphene-based field effect transistors (G-FET) after the adsorption of Escherichia coli (E. coli) bacteria onto its graphene layer. We confirmed a positive shift in the ambipolar curve of the G-FETs after the adsorption of E. coli, presumably due to the negative charge of the adsorbed E. coli. However, we did not observe any decrease in the electron mobility or conductivity of the G-FETs, which implied that E. coli did not chemically react with the carbon atoms of graphene, nor introduce any damage on the graphene lattice, but were only physically adsorbed onto the graphene surface. These findings may extend the prominence of graphene as a stable yet sensitive material to be fully utilized in future biosensing applications. These results were then compared to those of ferritin adsorption, which is a protein shell and biomaterial like E. coli, and radical oxygen doping onto the graphene surface.

Transcriptional and Physiological Characterizations of Escherichia coli MG1655 that have been grown under Low Shear Stress Environment for 1000 Generations

Science.gov (United States)

Karouia, Fathi; Tirumalai, Madhan R.; Nelman-Gonzalez, Mayra A.; Sams, Clarence F.; Ott, Mark C.; Pierson, Duane L.; Fofanov, Yuriy; Willson, Richard C.; Fox, George E.

Human space travelers experience a unique environment that affects homeostasis and physio-logic adaptation. One of the important regulatory biology interactions affected by space flight is the alteration of the immune response. As such, the impairment of the immune system may lead to higher risk of bacterial and/or viral infection during human space flight missions. Mi-crobiological contaminants have been a source of concern over the years for NASA and there is evidence to suggest that microbes in space do not behave like they do on Earth. Previ-ous studies have examined the physiological response of bacteria when exposed to short-term microgravity either during spaceflight or in a Low Shear Modeled Microgravity (LSMMG) en-vironment. Exposure to these environments has been found to induce increased resistance to stresses and antibiotics, and in one case increase of virulence. As NASA increases the duration of space flight missions and is starting to envision human presence on the lunar surface and Mars, it becomes legitimate to question the long-term effects of microgravity on bacteria. The effect of long-term exposure to LSMMG on microbial gene expression and physiology in Escherichia coli (E. coli) is being examined using functional genomics, and molecular tech-niques. In previous E. coli short term studies, reproducible changes in transcription were seen but no direct responses to changes in the gravity vector were identified. Instead, absence of shear and a randomized gravity vector appeared to cause local extra-cellular environmental changes, which elicited cellular responses. In order to evaluate the long-term effects of micro-gravity on bacteria, E. coli was grown under simulated microgravity for 1000 generations and gene expression patterns and cellular physiology were analyzed in comparison with short-term exposure. The analysis revealed that the long-term response differed significantly from the short-term exposure and 357 genes were expressed

Imidacloprid intensifies its impact on honeybee and bumblebee cellular immune response when challenged with LPS (lippopolysacharide) of Escherichia coli.

Science.gov (United States)

Walderdorff, Louise; Laval-Gilly, Philippe; Bonnefoy, Antoine; Falla-Angel, Jaïro

2018-05-16

Insect hemocytes play an important role in insects' defense against environmental stressors as they are entirely dependent on their innate immune system for pathogen defense. In recent years a dramatic decline of pollinators has been reported in many countries. The drivers of this declines appear to be associated with pathogen infections like viruses, bacteria or fungi in combination with pesticide exposure. The aim of this study was thus to investigate the impact of imidacloprid, a neonicotinoid insecticide, on the cellular immune response of two pollinators (Apis mellifera and Bombus terrestris) during simultaneous immune activation with LPS (lipopolysaccharide) of Escherichia coli. For this purpose the phagocytosis capacity as well as the production of H 2 O 2 and NO of larval hemocytes, exposed to five different imidacloprid concentrations in vitro, was measured. All used pesticide concentrations showed a weakening effect on phagocytosis with but also without LPS activation. Imidacloprid decreased H 2 O 2 and increased NO production in honeybees. Immune activation by LPS clearly reinforced the effect of imidacloprid on the immune response of hemocytes in all three immune parameters tested. Bumblebee hemocytes appeared more sensitive to imidacloprid during phagocytosis assays while imidacloprid showed a greater impact on honeybee hemocytes during H 2 O 2 and NO production. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mathematical modeling the cross-contamination of Escherichia coli O157:H7 on the surface of ready-to-eat meat product while slicing

Science.gov (United States)

Microbial cross-contamination either at home or production site is one of the major factors of causing contamination of foods and leading to the foodborne illness. The knowledge regarding Escherichia coli O157:H7 surface transfer on ready-to-eat (RTE) deli meat and the slicer used for slicing diffe...

The cellular approach to band structure calculations

International Nuclear Information System (INIS)

Verwoerd, W.S.

1982-01-01

A short introduction to the cellular approach in band structure calculations is given. The linear cellular approach and its potantial applicability in surface structure calculations is given some consideration in particular

Cell surface acid-base properties of Escherichia coli and Bacillus brevis and variation as a function of growth phase, nitrogen source and C:N ratio.

Science.gov (United States)

Hong, Yongsuk; Brown, Derick G

2006-07-01

Potentiometric titration has been conducted to systematically examine the acid-base properties of the cell surfaces of Escherichia coli K-12 and Bacillus brevis as a function of growth phase, nitrogen source (ammonium or nitrate), and carbon to nitrogen (C:N) ratio of the growth substrate. The two bacterial species revealed four distinct proton binding sites, with pK(a) values in the range of 3.08-4.05 (pK(1)), 4.62-5.57 (pK(2)), 6.47-7.30 (pK(3)), and 9.68-10.89 (pK(4)) corresponding to phosphoric/carboxylic, carboxylic, phosphoric, and hydroxyl/amine groups, respectively. Two general observations in the data are that for B. brevis the first site concentration (N(1)), corresponding to phosphoric/carboxylic groups (pK(1)), varied as a function of nitrogen source, while for E. coli the fourth site concentration (N(4)), corresponding to hydroxyl/amine groups (pK(4)), varied as a function of C:N ratio. Correspondingly, it was found that N(1) was the highest of the four site concentrations for B. brevis and N(4) was the highest for E. coli. The concentrations of the remaining sites showed little variation. Finally, comparison between the titration data and a number of cell surface compositional studies in the literature indicates one distinct difference between the two bacteria is that pK(4) of the Gram-negative E. coli can be attributed to hydroxyl groups while that of the Gram-positive B. brevis can be attributed to amine groups.

Dual-species biofilm of Listeria monocytogenes and Escherichia coli on stainless steel surface.

Science.gov (United States)

de Grandi, Aline Zago; Pinto, Uelinton Manoel; Destro, Maria Teresa

2018-04-12

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ B , in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

Science.gov (United States)

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Stress response of Escherichia coli induced by surface streamer discharge in humid air

International Nuclear Information System (INIS)

Doležalová, Eva; Prukner, Václav; Lukeš, Petr; Šimek, Milan

2016-01-01

Inactivation of Escherichia coli by means of surface streamer discharge has been investigated to obtain new insights into the key mechanisms involved, with a particular emphasis placed on the microbial response to plasma-induced stress. The surface streamer discharge was produced in coplanar dielectric barrier discharge electrode geometry, and was driven by an amplitude-modulated ac high voltage in humid synthetic air at atmospheric pressure. The response to plasma-induced stress was evaluated by using conventional cultivation, sublethal injury and resazurin assay and the LIVE/DEAD ® BacLight ™ Bacterial Viability kit. Compared to conventional cultivation, the LIVE/DEAD ® test labels bacteria with damaged membranes, while resazurin assay tracks their metabolic activity. Our results clearly demonstrate that the treated bacteria partly lost their ability to grow properly, i.e. they became injured and culturable, or even viable but nonculturable (VBNC). The ability to develop colonies could have been lost due to damage of the bacterial membrane. Damage of the membranes was mainly caused by the lipid peroxidation, evidencing the key role of oxygen reactive species, in particular ozone. We conclude that the conventional cultivation method overestimates the decontamination efficiency of various plasma sources, and must therefore be complemented by alternative techniques capable of resolving viable but nonculturable bacteria. (paper)

Cysteine Addition Promotes Sulfide Production and 4-Fold Hg(II)-S Coordination in Actively Metabolizing Escherichia coli.

Science.gov (United States)

Thomas, Sara A; Gaillard, Jean-François

2017-04-18

The bacterial uptake of mercury(II), Hg(II), is believed to be energy-dependent and is enhanced by cysteine in diverse species of bacteria under aerobic and anaerobic conditions. To gain insight into this Hg(II) biouptake pathway, we have employed X-ray absorption spectroscopy (XAS) to investigate the relationship between exogenous cysteine, cellular metabolism, cellular localization, and Hg(II) coordination in aerobically respiring Escherichia coli (E. coli). We show that cells harvested in exponential growth phase consistently display mixtures of 2-fold and 4-fold Hg(II) coordination to sulfur (Hg-S 2 and Hg-S 4 ), with added cysteine enhancing Hg-S 4 formation. In contrast, cells in stationary growth phase or cells treated with a protonophore causing a decrease in cellular ATP predominantly contain Hg-S 2 , regardless of cysteine addition. Our XAS results favor metacinnabar (β-HgS) as the Hg-S 4 species, which we show is associated with both the cell envelope and cytoplasm. Additionally, we observe that added cysteine abiotically oxidizes to cystine and exponentially growing E. coli degrade high cysteine concentrations (100-1000 μM) into sulfide. Thermodynamic calculations confirm that cysteine-induced sulfide biosynthesis can promote the formation of dissolved and particulate Hg(II)-sulfide species. This report reveals new complexities arising in Hg(II) bioassays with cysteine and emphasizes the need for considering changes in chemical speciation as well as growth stage.

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

Science.gov (United States)

Wang, Jian-Feng; Xiong, Zhi-Qiang; Li, Shi-Yuan; Wang, Yong

2013-09-01

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli.

Science.gov (United States)

Wu, Hong; Chen, Jinchun; Chen, Guo-Qiang

2016-12-01

E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.

UDP-N-acetylmuramic acid l-alanine ligase (MurC) inhibition in a tolC mutant Escherichia coli strain leads to cell death.

Science.gov (United States)

Humnabadkar, Vaishali; Prabhakar, K R; Narayan, Ashwini; Sharma, Sreevalli; Guptha, Supreeth; Manjrekar, Praveena; Chinnapattu, Murugan; Ramachandran, Vasanthi; Hameed, Shahul P; Ravishankar, Sudha; Chatterji, Monalisa

2014-10-01

The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparative evolutionary analysis of protein complexes in E. coli and yeast

Directory of Open Access Journals (Sweden)

Ranea Juan AG

2010-02-01

Full Text Available Abstract Background Proteins do not act in isolation; they frequently act together in protein complexes to carry out concerted cellular functions. The evolution of complexes is poorly understood, especially in organisms other than yeast, where little experimental data has been available. Results We generated accurate, high coverage datasets of protein complexes for E. coli and yeast in order to study differences in the evolution of complexes between these two species. We show that substantial differences exist in how complexes have evolved between these organisms. A previously proposed model of complex evolution identified complexes with cores of interacting homologues. We support findings of the relative importance of this mode of evolution in yeast, but find that it is much less common in E. coli. Additionally it is shown that those homologues which do cluster in complexes are involved in eukaryote-specific functions. Furthermore we identify correlated pairs of non-homologous domains which occur in multiple protein complexes. These were identified in both yeast and E. coli and we present evidence that these too may represent complex cores in yeast but not those of E. coli. Conclusions Our results suggest that there are differences in the way protein complexes have evolved in E. coli and yeast. Whereas some yeast complexes have evolved by recruiting paralogues, this is not apparent in E. coli. Furthermore, such complexes are involved in eukaryotic-specific functions. This implies that the increase in gene family sizes seen in eukaryotes in part reflects multiple family members being used within complexes. However, in general, in both E. coli and yeast, homologous domains are used in different complexes.

Static allometry of unicellular green algae: scaling of cellular surface area and volume in the genus Micrasterias (Desmidiales).

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Neustupa, J

2016-02-01

The surface area-to-volume ratio of cells is one of the key factors affecting fundamental biological processes and, thus, fitness of unicellular organisms. One of the general models for allometric increase in surface-to-volume scaling involves fractal-like elaboration of cellular surfaces. However, specific data illustrating this pattern in natural populations of the unicellular organisms have not previously been available. This study shows that unicellular green algae of the genus Micrasterias (Desmidiales) have positive allometric surface-to-volume scaling caused by changes in morphology of individual species, especially in the degree of cell lobulation. This allometric pattern was also detected within most of the cultured and natural populations analysed. Values of the allometric S:V scaling within individual populations were closely correlated to the phylogenetic structure of the clade. In addition, they were related to species-specific cellular morphology. Individual populations differed in their allometric patterns, and their position in the allometric space was strongly correlated with the degree of allometric S:V scaling. This result illustrates that allometric shape patterns are an important correlate of the capacity of individual populations to compensate for increases in their cell volumes by increasing the surface area. However, variation in allometric patterns was not associated with phylogenetic structure. This indicates that the position of the populations in the allometric space was not evolutionarily conserved and might be influenced by environmental factors. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

Recombinant protein production data after expression in the bacterium Escherichia coli

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J. Enrique Cantu-Bustos

2016-06-01

Full Text Available Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]. Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP tagged with CusF, using Ag(I metal affinity chromatography.

Spatiotemporal control over molecular delivery and cellular encapsulation from electropolymerized micro- and nanopatterned surfaces.

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Stern, Eric; Jay, Steven M; Demento, Stacey L; Murelli, Ryan P; Reed, Mark A; Malinski, Tadeusz; Spiegel, David A; Mooney, David J; Fahmy, Tarek M

2009-07-13

Bioactive, patterned micro- and nanoscale surfaces that can be spatially engineered for three-dimensional ligand presentation and sustained release of signaling molecules represent a critical advance for the development of next-generation diagnostic and therapeutic devices. Lithography is ideally suited to patterning such surfaces due to its precise, easily scalable, high-throughput nature; however, to date polymers patterned by these techniques have not demonstrated the capacity for sustained release of bioactive agents. We demonstrate here a class of lithographically-defined, electropolymerized polymers with monodisperse micro- and nanopatterned features capable of sustained release of bioactive drugs and proteins. We show that precise control can be achieved over the loading capacity and release rates of encapsulated agents and illustrate this aspect using a fabricated surface releasing a model antigen (ovalbumin) and a cytokine (interleukin-2) for induction of a specific immune response. We further demonstrate the ability of this technique to enable three-dimensional control over cellular encapsulation. The efficacy of the described approach is buttressed by its simplicity, versatility, and reproducibility, rendering it ideally suited for biomaterials engineering.

Genetic diversity and antimicrobial resistance of Escherichia coli from human and animal sources uncovers multiple resistances from human sources.

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A Mark Ibekwe

Full Text Available Escherichia coli are widely used as indicators of fecal contamination, and in some cases to identify host sources of fecal contamination in surface water. Prevalence, genetic diversity and antimicrobial susceptibility were determined for 600 generic E. coli isolates obtained from surface water and sediment from creeks and channels along the middle Santa Ana River (MSAR watershed of southern California, USA, after a 12 month study. Evaluation of E. coli populations along the creeks and channels showed that E. coli were more prevalent in sediment compared to surface water. E. coli populations were not significantly different (P = 0.05 between urban runoff sources and agricultural sources, however, E. coli genotypes determined by pulsed-field gel electrophoresis (PFGE were less diverse in the agricultural sources than in urban runoff sources. PFGE also showed that E. coli populations in surface water were more diverse than in the sediment, suggesting isolates in sediment may be dominated by clonal populations.Twenty four percent (144 isolates of the 600 isolates exhibited resistance to more than one antimicrobial agent. Most multiple resistances were associated with inputs from urban runoff and involved the antimicrobials rifampicin, tetracycline, and erythromycin. The occurrence of a greater number of E. coli with multiple antibiotic resistances from urban runoff sources than agricultural sources in this watershed provides useful evidence in planning strategies for water quality management and public health protection.

Surface-anchored poly(acryloyl-L(D)-valine) with enhanced chirality-selective effect on cellular uptake of gold nanoparticles

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Deng, Jun; Wu, Sai; Yao, Mengyun; Gao, Changyou

2016-01-01

Chirality is one of the ubiquitous phenomena in biological systems. The left handed (L-) amino acids and right handed (D-) sugars are normally found in proteins, and in RNAs and DNAs, respectively. The effect of chiral surfaces at the nanoscale on cellular uptake has, however, not been explored. This study reveals for the first time the molecular chirality on gold nanoparticles (AuNPs) functions as a direct regulator for cellular uptake. Monolayers of 2-mercaptoacetyl-L(D)-valine (L(D)-MAV) and poly(acryloyl-L(D)-valine (L(D)-PAV) chiral molecules were formed on AuNPs surface, respectively. The internalized amount of PAV-AuNPs was several times larger than that of MAV-AuNPs by A549 and HepG2 cells, regardless of the chirality difference. However, the D-PAV-AuNPs were internalized with significantly larger amount than the L-PAV-AuNPs. This chirality-dependent uptake effect is likely attributed to the preferable interaction between the L-phospholipid-based cell membrane and the D-enantiomers. PMID:27531648

Can Escherichia coli fly? The role of flies as transmitters of E. coli to food in an urban slum in Bangladesh.

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Lindeberg, Yrja Lisa; Egedal, Karen; Hossain, Zenat Zebin; Phelps, Matthew; Tulsiani, Suhella; Farhana, Israt; Begum, Anowara; Jensen, Peter Kjaer Mackie

2018-01-01

To investigate the transmission of faecal bacteria by flies to food under natural settings. Over a period of 2 months, paired (exposed and non-exposed) containers with cooked rice were placed on the ground in kitchen areas in an urban slum area in Dhaka, Bangladesh, and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of Escherichia coli and genes of diarrhoeagenic E. coli and Shigella strains. Rice was at greater risk (P E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (P 0·6 × 103 CFU. Genes of diarrhoeagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified: the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala). Flies may transmit large quantities of E. coli to food under field settings. The findings highlight the importance of implementing control measures to minimise exposure of food to flies to ensure food safety. Fly control measures should be considered for the prevention of diarrhoeal diseases caused by E. coli. © 2017 John Wiley & Sons Ltd.

Self-organization of yeast cells on modified polymer surfaces after dewetting: new perspectives in cellular patterning

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Carnazza, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy); Satriano, S [Department of Chemical Sciences, University of Catania, Catania (Italy); Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

2006-08-23

In recent years, biological micro-electro-mechanical systems (commonly referred to as BioMEMS) have found widespread use, becoming increasingly prevalent in diagnostics and therapeutics. Cell-based sensors are nowadays gaining increasing attention, due to cellular built-in natural selectivity and physiologically relevant response to biologically active chemicals. On the other hand, surrogate microbial systems, including yeast models, have become a useful alternative to animal and mammalian cell systems for high-throughput screening for the identification of new pharmacological agents. A main obstacle in biosensor device fabrication is the need for localized geometric confinement of cells, without losing cell viability and sensing capability. Here we illustrate a new approach for cellular patterning using dewetting processes to control cell adhesion and spatial confinement on modified surfaces. By the control of simple system parameters, a rich variety of morphologies, ranging through hexagonal arrays, polygonal networks, bicontinuous structures, and elongated fingers, can be obtained.

Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

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Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

2017-02-01

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

The 70S ribosome modulates the ATPase activity of Escherichia coli YchF.

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Becker, Marion; Gzyl, Katherine E; Altamirano, Alvin M; Vuong, Anthony; Urban, Kirstin; Wieden, Hans-Joachim

2012-10-01

YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF's cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF's catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF's ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria.

ANTIMICROBIAL DRUG RESISTANCE IN STRAINS OF Escherichia coli ISOLATED FROM FOOD SOURCES

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Mohammed Uddin Rasheed

2014-07-01

Full Text Available A variety of foods and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in medicine and agriculture. Antibiotic resistance in Escherichia coli is of particular concern because it is the most common Gram-negative pathogen in humans. Hence this study was conducted to determine the antibiotic sensitivity pattern of E. coli isolated from different types of food items collected randomly from twelve localities of Hyderabad, India. A total of 150 samples comprising; vegetable salad, raw egg-surface, raw chicken, unpasteurized milk, and raw meat were processed microbiologically to isolate E. coli and to study their antibiotic susceptibility pattern by the Kirby-Bauer method. The highest percentages of drug resistance in isolates of E. coli were detected from raw chicken (23.3% followed by vegetable salad (20%, raw meat (13.3%, raw egg-surface (10% and unpasteurized milk (6.7%. The overall incidence of drug resistant E. coli was 14.7%. A total of six (4% Extended Spectrum β-Lactamase (ESBL producers were detected, two each from vegetable salads and raw chicken, and one each from raw egg-surface and raw meat. Multidrug resistant strains of E. coli are a matter of concern as resistance genes are easily transferable to other strains. Pathogen cycling through food is very common and might pose a potential health risk to the consumer. Therefore, in order to avoid this, good hygienic practices are necessary in the abattoirs to prevent contamination of cattle and poultry products with intestinal content as well as forbidding the use of untreated sewage in irrigating vegetables.

Peptidoglycan Hydrolases of Escherichia coli

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van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

EchoBASE: an integrated post-genomic database for Escherichia coli.

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Misra, Raju V; Horler, Richard S P; Reindl, Wolfgang; Goryanin, Igor I; Thomas, Gavin H

2005-01-01

EchoBASE (http://www.ecoli-york.org) is a relational database designed to contain and manipulate information from post-genomic experiments using the model bacterium Escherichia coli K-12. Its aim is to collate information from a wide range of sources to provide clues to the functions of the approximately 1500 gene products that have no confirmed cellular function. The database is built on an enhanced annotation of the updated genome sequence of strain MG1655 and the association of experimental data with the E.coli genes and their products. Experiments that can be held within EchoBASE include proteomics studies, microarray data, protein-protein interaction data, structural data and bioinformatics studies. EchoBASE also contains annotated information on 'orphan' enzyme activities from this microbe to aid characterization of the proteins that catalyse these elusive biochemical reactions.

Modelling for Near-Surface Transport Dynamics of Hydrogen of Plasma Facing Materials by use of Cellular Automaton

International Nuclear Information System (INIS)

Shimura, K.; Terai, T.; Yamawaki, M.

2003-01-01

In this study, the kinetics of desorption of adsorbed hydrogen from an ideal metallic surface is modelled in Cellular Automaton (CA). The modelling is achieved by downgrading the surface to one dimension. The model consists of two parts that are surface migration and desorption. The former is attained by randomly sorting the particles at each time, the latter is realised by modelling the thermally-activated process. For the verification of this model, thermal desorption is simulated then the comparison with the chemical kinetics is carried out. Excellent agreement is observed from the result. The results show that this model is reasonable to express the recombinative desorption of two chemisorbed adatoms. Though, the application of this model is limited to the second-order reaction case. But it can be believed that the groundwork of modelling the transport dynamics of hydrogen through the surface under complex conditions is established

Atypical Enteropathogenic Escherichia coli Strains form Biofilm on Abiotic Surfaces Regardless of Their Adherence Pattern on Cultured Epithelial Cells

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Hebert F. Culler

2014-01-01

Full Text Available The aim of this study was to determine the capacity of biofilm formation of atypical enteropathogenic Escherichia coli (aEPEC strains on abiotic and biotic surfaces. Ninety-one aEPEC strains, isolated from feces of children with diarrhea, were analyzed by the crystal violet (CV assay on an abiotic surface after 24 h of incubation. aEPEC strains representing each HEp-2 cell type of adherence were analyzed after 24 h and 6, 12, and 18 days of incubation at 37°C on abiotic and cell surfaces by CFU/cm2 counting and confocal laser scanning microscopy (CLSM. Biofilm formation on abiotic surfaces occurred in 55 (60.4% of the aEPEC strains. There was no significant difference in biofilm biomass formation on an abiotic versus prefixed cell surface. The biofilms could be visualized by CLSM at various developmental stages. aEPEC strains are able to form biofilm on an abiotic surface with no association with their adherence pattern on HEp-2 cells with the exception of the strains expressing UND (undetermined adherence. This study revealed the capacity of adhesion and biofilm formation by aEPEC strains on abiotic and biotic surfaces, possibly playing a role in pathogenesis, mainly in cases of persistent diarrhea.

ESBL-producing Escherichia coli in Swedish gulls-A case of environmental pollution from humans?

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Clara Atterby

Full Text Available ESBL-producing bacteria are present in wildlife and the environment might serve as a resistance reservoir. Wild gulls have been described as frequent carriers of ESBL-producing E. coli strains with genotypic characteristics similar to strains found in humans. Therefore, potential dissemination of antibiotic resistance genes and bacteria between the human population and wildlife need to be further investigated. Occurrence and characterization of ESBL-producing E. coli in Swedish wild gulls were assessed and compared to isolates from humans, livestock and surface water collected in the same country and similar time-period. Occurrence of ESBL-producing E. coli in Swedish gulls is about three times higher in gulls compared to Swedish community carriers (17% versus 5% and the genetic characteristics of the ESBL-producing E. coli population in Swedish wild gulls and Swedish human are similar. ESBL-plasmids IncF- and IncI1-type carrying ESBL-genes blaCTX-M-15 or blaCTX-M-14 were most common in isolates from both gulls and humans, but there was limited evidence of clonal transmission. Isolates from Swedish surface water harbored similar genetic characteristics, which highlights surface waters as potential dissemination routes between wildlife and the human population. Even in a low-prevalence country such as Sweden, the occurrence of ESBL producing E. coli in wild gulls and the human population appears to be connected and the occurrence of ESBL-producing E. coli in Swedish gulls is likely a case of environmental pollution.

ESBL-producing Escherichia coli in Swedish gulls-A case of environmental pollution from humans?

Science.gov (United States)

Atterby, Clara; Börjesson, Stefan; Ny, Sofia; Järhult, Josef D; Byfors, Sara; Bonnedahl, Jonas

2017-01-01

ESBL-producing bacteria are present in wildlife and the environment might serve as a resistance reservoir. Wild gulls have been described as frequent carriers of ESBL-producing E. coli strains with genotypic characteristics similar to strains found in humans. Therefore, potential dissemination of antibiotic resistance genes and bacteria between the human population and wildlife need to be further investigated. Occurrence and characterization of ESBL-producing E. coli in Swedish wild gulls were assessed and compared to isolates from humans, livestock and surface water collected in the same country and similar time-period. Occurrence of ESBL-producing E. coli in Swedish gulls is about three times higher in gulls compared to Swedish community carriers (17% versus 5%) and the genetic characteristics of the ESBL-producing E. coli population in Swedish wild gulls and Swedish human are similar. ESBL-plasmids IncF- and IncI1-type carrying ESBL-genes blaCTX-M-15 or blaCTX-M-14 were most common in isolates from both gulls and humans, but there was limited evidence of clonal transmission. Isolates from Swedish surface water harbored similar genetic characteristics, which highlights surface waters as potential dissemination routes between wildlife and the human population. Even in a low-prevalence country such as Sweden, the occurrence of ESBL producing E. coli in wild gulls and the human population appears to be connected and the occurrence of ESBL-producing E. coli in Swedish gulls is likely a case of environmental pollution.

Survival of Escherichia coli O157:H7 in needle-tenderized dry cured Westphalian ham.

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Graumann, Gary H; Holley, Richard A

2007-09-15

Westphalian ham is a dry cured, ready-to-eat product that is manufactured without a lethal heat treatment. Hams are preserved by a process that involves curing, fermenting, smoking and drying, which may take 3 months or more to complete. The process can be accelerated by tenderizing the meat with solid needles, to increase the rate of cure-salt diffusion throughout muscle tissues. In this study, intact hams were immersed in a solution containing a five strain cocktail of Escherichia coli O157:H7 at 8 log cfu/mL, to determine whether needle treatment before cure application would internalize organisms from the surface. In two trials, the survival of E. coli O157:H7 on external surfaces and within deep tissues after needle treatment was followed during the ripening of hams. The injured E. coli O157:H7 cells were recovered by plating samples on pre-poured Tryptic Soy Agar plates which were incubated for 3 to 4 h at 35 degrees C, overlaid with Sorbitol MacConkey Agar containing cefixime and tellurite and re-incubated at 35 degrees C for 48 to 72 h. Inoculated-injected hams initially carried E. coli O157:H7 at numbers of 7.3 and 4.6 log cfu/g E. coli O157:H7 on the surface and inside, respectively. After 112 d of ripening, which included 79 d of drying, no E. coli O157:H7 were detected at the surface of hams following enrichment, whereas in deep tissue the organism was recovered at numbers of 3.1 log cfu/g. The Westphalian ham ripening procedure evidently was not adequate to eliminate E. coli O157:H7 internalized by needle tenderization.

Similarities of cellular receptors for interferon and cortisol

International Nuclear Information System (INIS)

Filipic, B.; Schauer, P.; Likar, M.

1977-01-01

Cellular receptors are molecules located on the cell membrane. Their function is to bind different molecules to the cell surface. These molecules can penetrate into the cytoplasm and trigger cellular changes. One kind of such bound molecules are interferons and corticosteroids. Until very recently very little was known about interferon's receptors on the cell surface, mechanisms of interferon's binding to them or about kinetics of such binding. On the basis of results published elsewhere and on the basis of experimental results, the authors suggest: receptors for interferon and cortisol are glycoproteins located on the cell surface, in analogy with PHA receptors they are chemically sialoglycoproteins, binding kinetics of cortisol and interferon is similar, interferon and cortisol compete for cellular receptors, binding of cortisol or interferon is dependent on allosteric configuration of receptor molecules. (author)

Urinary tract infections of Escherichia coli strains of chaperone-usher system.

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Zalewska-Piatek, Beata M

2011-01-01

Urinary tract infections are a very serious health and economic problem affecting millions of people each year worldwide. The most common etiologic agent of this type of bacterial infections, involving the upper and lower urinary tract, are E. coli strains representing approximately 80% of cases. Uropathogenic E. coli strains produce several urovirulence factors which can be divided into two main types, surface virulence factors and exported virulence factors. Surface-exposed structures include mainly extracellular adhesive organelles such as fimbriae/pili necessary in adhesion, invasion, biofilm formation and cytokine induction. Among the surface-exposed polymeric adhesive structures there are three most invasive groups, type 1 pili, type P pili and Dr family of adhesins which are bioassembled via the conserved, among Gram-negative bacteria, chaperone-usher secretion system. Type 1 and P-piliated E. coli cause cystitis and pyelonephritis. The Dr family of adhesins recognizing DAF receptor is responsible for cystitis, pyelonephritis (especially in pregnant women) and diarrhoea (in infants). In addition, Dr-positive E. coli strains carry the risk of recurrent urinary tract infections. Pyelonephritis in pregnant women leads to a series of complications such as bacteremia, urosepsis, acute respiratory distress syndrome and even death. In the era of increasing drug resistance of bacteria, the development of vaccines, drugs termed pilicides and inhibitors of adhesion may be a promising tool in the fight against urogenital infections.

Polyelectrolyte-Functionalized Nanofiber Mats Control the Collection and Inactivation of Escherichia coli

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Katrina A. Rieger

2016-04-01

Full Text Available Quantifying the effect that nanofiber mat chemistry and hydrophilicity have on microorganism collection and inactivation is critical in biomedical applications. In this study, the collection and inactivation of Escherichia coli K12 was examined using cellulose nanofiber mats that were surface-functionalized using three polyelectrolytes: poly (acrylic acid (PAA, chitosan (CS, and polydiallyldimethylammonium chloride (pDADMAC. The polyelectrolyte functionalized nanofiber mats retained the cylindrical morphology and average fiber diameter (~0.84 µm of the underlying cellulose nanofibers. X-ray photoelectron spectroscopy (XPS and contact angle measurements confirmed the presence of polycations or polyanions on the surface of the nanofiber mats. Both the control cellulose and pDADMAC-functionalized nanofiber mats exhibited a high collection of E. coli K12, which suggests that mat hydrophilicity may play a larger role than surface charge on cell collection. While the minimum concentration of polycations needed to inhibit E. coli K12 was 800 µg/mL for both CS and pDADMAC, once immobilized, pDADMAC-functionalized nanofiber mats exhibited a higher inactivation of E. coli K12, (~97%. Here, we demonstrate that the collection and inactivation of microorganisms by electrospun cellulose nanofiber mats can be tailored through a facile polyelectrolyte functionalization process.

Polyelectrolyte-Functionalized Nanofiber Mats Control the Collection and Inactivation of Escherichia coli

Science.gov (United States)

Rieger, Katrina A.; Porter, Michael; Schiffman, Jessica D.

2016-01-01

Quantifying the effect that nanofiber mat chemistry and hydrophilicity have on microorganism collection and inactivation is critical in biomedical applications. In this study, the collection and inactivation of Escherichia coli K12 was examined using cellulose nanofiber mats that were surface-functionalized using three polyelectrolytes: poly (acrylic acid) (PAA), chitosan (CS), and polydiallyldimethylammonium chloride (pDADMAC). The polyelectrolyte functionalized nanofiber mats retained the cylindrical morphology and average fiber diameter (~0.84 µm) of the underlying cellulose nanofibers. X-ray photoelectron spectroscopy (XPS) and contact angle measurements confirmed the presence of polycations or polyanions on the surface of the nanofiber mats. Both the control cellulose and pDADMAC-functionalized nanofiber mats exhibited a high collection of E. coli K12, which suggests that mat hydrophilicity may play a larger role than surface charge on cell collection. While the minimum concentration of polycations needed to inhibit E. coli K12 was 800 µg/mL for both CS and pDADMAC, once immobilized, pDADMAC-functionalized nanofiber mats exhibited a higher inactivation of E. coli K12, (~97%). Here, we demonstrate that the collection and inactivation of microorganisms by electrospun cellulose nanofiber mats can be tailored through a facile polyelectrolyte functionalization process. PMID:28773422

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

Energy Technology Data Exchange (ETDEWEB)

Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

2015-06-15

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

International Nuclear Information System (INIS)

Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

2015-01-01

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli.

Directory of Open Access Journals (Sweden)

Zhen Zhang

Full Text Available Ice nucleation protein (INP is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N to improve its surface display efficiency for human Arginase1 (ARG1. Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1 by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg to L-Ornithine (L-Orn in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.

Transport proteins promoting Escherichia coli pathogenesis

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Tang, Fengyi; Saier, Milton H.

2014-01-01

Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

Transport proteins promoting Escherichia coli pathogenesis.

Science.gov (United States)

Tang, Fengyi; Saier, Milton H

2014-01-01

Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glycan-functionalized diamond nanoparticles as potent E. coli anti-adhesives

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Barras, Alexandre; Martin, Fernando Ariel; Bande, Omprakash; Baumann, Jean-Sébastien; Ghigo, Jean-Marc; Boukherroub, Rabah; Beloin, Christophe; Siriwardena, Aloysius; Szunerits, Sabine

2013-02-01

Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with mannose moieties by a ``click'' chemistry approach, are able to efficiently inhibit E. coli type 1 fimbriae-mediated adhesion to eukaryotic cells with relative inhibitory potency (RIP) of as high as 9259 (bladder cell adhesion assay), which is unprecedented when compared with RIP values previously reported for alternate multivalent mannose-functionalized nanostructures designed to inhibit E. coli adhesion. Also remarkable is that these novel mannose-modified NDs reduce E. coli biofilm formation, a property previously not observed for multivalent glyco-nanoparticles and rarely demonstrated for other multivalent or monovalent mannose glycans. This work sets the stage for the further evaluation of these novel NDs as an anti-adhesive therapeutic strategy against E. coli-derived infections.Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with

Systems-level analysis of Escherichia coli response to silver nanoparticles: the roles of anaerobic respiration in microbial resistance.

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Du, Huamao; Lo, Tat-Ming; Sitompul, Johnner; Chang, Matthew Wook

2012-08-10

Despite extensive use of silver nanoparticles for antimicrobial applications, cellular mechanisms underlying microbial response to silver nanoparticles remain to be further elucidated at the systems level. Here, we report systems-level response of Escherichia coli to silver nanoparticles using transcriptome-based biochemical and phenotype assays. Notably, we provided the evidence that anaerobic respiration is induced upon exposure to silver nanoparticles. Further we showed that anaerobic respiration-related regulators and enzymes play an important role in E. coli resistance to silver nanoparticles. In particular, our results suggest that arcA is essential for resistance against silver NPs and the deletion of fnr, fdnH and narH significantly increases the resistance. We envision that this study offers novel insights into modes of antimicrobial action of silver nanoparticles, and cellular mechanisms contributing to the development of microbial resistance to silver nanoparticles. Copyright © 2012 Elsevier Inc. All rights reserved.

Escherichia coli pathotypes

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Escherichia coli strains are important commensals of the intestinal tract of humans and animals; however, pathogenic strains, including diarrhea-inducing E. coli and extraintestinal pathogenic E. coli. Intestinal E. coli pathotypes may cause a dehydrating watery diarrhea, or more severe diseases su...

Origami interleaved tube cellular materials

International Nuclear Information System (INIS)

Cheung, Kenneth C; Tachi, Tomohiro; Calisch, Sam; Miura, Koryo

2014-01-01

A novel origami cellular material based on a deployable cellular origami structure is described. The structure is bi-directionally flat-foldable in two orthogonal (x and y) directions and is relatively stiff in the third orthogonal (z) direction. While such mechanical orthotropicity is well known in cellular materials with extruded two dimensional geometry, the interleaved tube geometry presented here consists of two orthogonal axes of interleaved tubes with high interfacial surface area and relative volume that changes with fold-state. In addition, the foldability still allows for fabrication by a flat lamination process, similar to methods used for conventional expanded two dimensional cellular materials. This article presents the geometric characteristics of the structure together with corresponding kinematic and mechanical modeling, explaining the orthotropic elastic behavior of the structure with classical dimensional scaling analysis. (paper)

Origami interleaved tube cellular materials

Science.gov (United States)

Cheung, Kenneth C.; Tachi, Tomohiro; Calisch, Sam; Miura, Koryo

2014-09-01

A novel origami cellular material based on a deployable cellular origami structure is described. The structure is bi-directionally flat-foldable in two orthogonal (x and y) directions and is relatively stiff in the third orthogonal (z) direction. While such mechanical orthotropicity is well known in cellular materials with extruded two dimensional geometry, the interleaved tube geometry presented here consists of two orthogonal axes of interleaved tubes with high interfacial surface area and relative volume that changes with fold-state. In addition, the foldability still allows for fabrication by a flat lamination process, similar to methods used for conventional expanded two dimensional cellular materials. This article presents the geometric characteristics of the structure together with corresponding kinematic and mechanical modeling, explaining the orthotropic elastic behavior of the structure with classical dimensional scaling analysis.

Pathogenic Escherichia coli and food handlers in luxury hotels in Nairobi, Kenya.

Science.gov (United States)

Onyango, Abel O; Kenya, Eucharia U; Mbithi, John J N; Ng'ayo, Musa O

2009-11-01

The epidemiology and virulence properties of pathogenic Escherichia coli among food handlers in tourist destination hotels in Kenya are largely uncharacterized. This cross-sectional study among consenting 885 food handlers working in nine luxurious tourist hotels in Nairobi, Kenya determined the epidemiology, virulence properties, antibiotics susceptibility profiles and conjugation abilities of pathogenic Escherichia coli. Pathogenic Escherichia coli was detected among 39 (4.4%) subjects, including 1.8% enteroaggregative Escherichia coli (EAEC) harboring aggR genes, 1.2% enterotoxigenic Escherichia coli (ETEC) expressing both LT and STp toxins, 1.1% enteropathogenic Escherichia coli (EPEC) and 0.2% Shiga-like Escherichia coli (EHEC) both harboring eaeA and stx2 genes respectively. All the pathotypes had increased surface hydrophobicity. Using multivariate analyses, food handlers with loose stools were more likely to be infected with pathogenic Escherichia coli. Majority 53.8% of the pathotypes were resistant to tetracycline with 40.2% being multi-drug resistant. About 85.7% pathotypes trans-conjugated with Escherichia coli K12 F(-) NA(r) LA. The carriage of multi-drug resistant, toxin expressing pathogenic Escherichia coli by this population is of public health concern because exposure to low doses can result in infection. Screening food handlers and implementing public awareness programs is recommended as an intervention to control transmission of enteric pathogens.

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids.

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Lawley, P D; Brookes, P

1968-09-01

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8

Computational determination of the effects of virulent Escherichia coli and salmonella bacteriophages on human gut.

Science.gov (United States)

Mostafa, Marwa Mostafa; Nassef, Mohammad; Badr, Amr

2016-10-01

Salmonella and Escherichia coli are different types of bacteria that cause food poisoning in humans. In the elderly, infants and people with chronic conditions, it is very dangerous if Salmonella or E. coli gets into the bloodstream and then they must be treated by phage therapy. Treating Salmonella and E. coli by phage therapy affects the gut flora. This research paper presents a system for detecting the effects of virulent E. coli and Salmonella bacteriophages on human gut. A method based on Domain-Domain Interactions (DDIs) model is implemented in the proposed system to determine the interactions between the proteins of human gut bacteria and the proteins of bacteriophages that infect virulent E. coli and Salmonella. The system helps gastroenterologists to realize the effect of injecting bacteriophages that infect virulent E. coli and Salmonella on the human gut. By testing the system over Enterobacteria phage 933W, Enterobacteria phage VT2-Sa and Enterobacteria phage P22, it resulted in four interactions between the proteins of the bacteriophages that infect E. coli O157:H7, E. coli O104:H4 and Salmonella typhimurium and the proteins of human gut bacterium strains. Several effects were detected such as: antibacterial activity against a number of bacterial species in human gut, regulation of cellular differentiation and organogenesis during gut, lung, and heart development, ammonia assimilation in bacteria, yeasts, and plants, energizing defense system and its function in the detoxification of lipopolysaccharide, and in the prevention of bacterial translocation in human gut. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Do Surface Porosity and Pore Size Influence Mechanical Properties and Cellular Response to PEEK?

Science.gov (United States)

Torstrick, F Brennan; Evans, Nathan T; Stevens, Hazel Y; Gall, Ken; Guldberg, Robert E

2016-11-01

Despite its widespread use in orthopaedic implants such as soft tissue fasteners and spinal intervertebral implants, polyetheretherketone (PEEK) often suffers from poor osseointegration. Introducing porosity can overcome this limitation by encouraging bone ingrowth; however, the corresponding decrease in implant strength can potentially reduce the implant's ability to bear physiologic loads. We have previously shown, using a single pore size, that limiting porosity to the surface of PEEK implants preserves strength while supporting in vivo osseointegration. However, additional work is needed to investigate the effect of pore size on both the mechanical properties and cellular response to PEEK. (1) Can surface porous PEEK (PEEK-SP) microstructure be reliably controlled? (2) What is the effect of pore size on the mechanical properties of PEEK-SP? (3) Do surface porosity and pore size influence the cellular response to PEEK? PEEK-SP was created by extruding PEEK through NaCl crystals of three controlled ranges: 200 to 312, 312 to 425, and 425 to 508 µm. Micro-CT was used to characterize the microstructure of PEEK-SP. Tensile, fatigue, and interfacial shear tests were performed to compare the mechanical properties of PEEK-SP with injection-molded PEEK (PEEK-IM). The cellular response to PEEK-SP, assessed by proliferation, alkaline phosphatase activity, vascular endothelial growth factor production, and calcium content of osteoblast, mesenchymal stem cell, and preosteoblast (MC3T3-E1) cultures, was compared with that of machined smooth PEEK and Ti6Al4V. Micro-CT analysis showed that PEEK-SP layers possessed pores that were 284 ± 35 µm, 341 ± 49 µm, and 416 ± 54 µm for each pore size group. Porosity and pore layer depth ranged from 61% to 69% and 303 to 391 µm, respectively. Mechanical testing revealed tensile strengths > 67 MPa and interfacial shear strengths > 20 MPa for all three pore size groups. All PEEK-SP groups exhibited > 50% decrease

Glucose-6-phosphate dehydrogenase protects Escherichia coli from tellurite-mediated oxidative stress.

Directory of Open Access Journals (Sweden)

Juan M Sandoval

Full Text Available The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH, which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH. Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P, suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH, better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.

Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA.

Science.gov (United States)

Zheng, Huabao; Wang, Xuan; Yomano, Lorraine P; Shanmugam, Keelnatham T; Ingram, Lonnie O

2012-06-01

Furfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilis YB886, Escherichia coli NC3, and Zymomonas mobilis CP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing the thyA gene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in the de novo pathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression of thyA was no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA in E. coli and to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA in E. coli would be expected to increase the cellular requirement for dTMP. Increased expression of thyA (E. coli, B. subtilis, or Z. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

Effects of storage methods on time-related changes of titanium surface properties and cellular response

International Nuclear Information System (INIS)

Lu Haibin; Zhou Lei; Wan Lei; Li Shaobing; Rong Mingdeng; Guo Zehong

2012-01-01

Titanium implants are sold in the market as storable medical devices. All the implants have a certain shelf life during which they maintain their sterility, but variations of the surface properties through this duration have not been subject to a comprehensive assessment. The aim of this study was to investigate the effects of storage methods on time-related changes of titanium surface properties. Acid-etched titanium discs (Sa = 0.82 µm) were placed in a sealed container (tradition method) or submerged in the ddH 2 O/NaCl solution (0.15 mol L −1 )/CaCl 2 solution (0.15 mol L −1 ), and new titanium discs were used as a control group. SEM and optical profiler showed that surface morphology and roughness did not change within different groups, but the XPS analysis confirmed that the surface chemistry altered by different storage protocols as the storage duration increased, and the contact angle also varied with storage methods. The storage method also affected the protein adsorption capacity and cellular response on the titanium surface. All titanium discs stored in the solution maintained their excellent bioactivity even after four weeks storage time, but titanium discs stored in a traditional manner decreased substantially in an age-dependent manner. Much effort is needed to improve the storage methods in order to maintain the bioactivity of a titanium dental implant. (paper)

Biofilm formation by Escherichia coli is stimulated by synergistic interactions and co-adhesion mechanisms with adherence-proficient bacteria

NARCIS (Netherlands)

Castonguay, MH; van der Schaaf, S; Koester, W; Krooneman, J; Harmsen, H; Landini, P; van der Meer, W.

Laboratory strains of Escherichia coli do not show significant ability to attach to solid surfaces and to form biofilms. We compared the adhesion properties of the E. coli PHL565 laboratory strain to eight environmental E. coli isolates: only four isolates displayed adhesion properties to glass

Adsorption of cellular peptides of Microcystis aeruginosa and two herbicides onto activated carbon. Effect of surface charge and interactions

Czech Academy of Sciences Publication Activity Database

Hnaťuková, Petra; Kopecká, Ivana; Pivokonský, Martin

2011-01-01

Roč. 45, č. 11 (2011), s. 3359-3368 ISSN 0043-1354 R&D Projects: GA AV ČR IAA200600902; GA ČR GPP105/10/P515 Institutional research plan: CEZ:AV0Z20600510 Keywords : cellular organic matter * granular activated carbon * molecular weight distribution * surface charge * cyanobacterial peptides Subject RIV: BK - Fluid Dynamics Impact factor: 4.865, year: 2011

Purification and characterization of Escherichia coli MreB protein.

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Nurse, Pearl; Marians, Kenneth J

2013-02-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.

Purification and Characterization of Escherichia coli MreB Protein*

Science.gov (United States)

Nurse, Pearl; Marians, Kenneth J.

2013-01-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm. PMID:23235161

Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

Science.gov (United States)

Nakayama, Motokazu; Shigemune, Naofumi; Tsugukuni, Takashi; Tokuda, Hajime; Miyamoto, Takahisa

2011-07-01

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium

NARCIS (Netherlands)

Franz, Eelco; Visser, Anna A; Van Diepeningen, Anne D; Klerks, Michel M; Termorshuizen, Aad J; van Bruggen, Ariena H C

The primary objective of this study was to determine the possibility of internalization of GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. Typhimurium) strains MAE 110 (multi-cellular morphology) and 119 (wild type morphology) into lettuce seedlings (Lactuca

Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

Directory of Open Access Journals (Sweden)

Michael eGänzle

2015-06-01

Full Text Available High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

Science.gov (United States)

Gänzle, Michael; Liu, Yang

2015-01-01

High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

E coli enteritis

Science.gov (United States)

... coli; Food poisoning - E. coli; E. coli diarrhea; Hamburger disease ... coleslaw or potato salad) that have been out of the refrigerator too ... reheated Fish or oysters Raw fruits or vegetables that have ...

DNA microarray analysis of fim mutations in Escherichia coli

DEFF Research Database (Denmark)

Schembri, Mark; Ussery, David; Workman, Christopher

2002-01-01

Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

FoldEco: A Model for Proteostasis in E. coli

Directory of Open Access Journals (Sweden)

Evan T. Powers

2012-03-01

Full Text Available To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide questions and shows how to convert these questions to experimentally measurable quantities, such as changes in protein concentrations with chaperone or protease levels, which can then be used to improve our current understanding of proteostasis and refine the model. A web version of FoldEco is available at http://foldeco.scripps.edu.

Identification of genes required for growth of Escherichia coli MG1655 at moderately low pH

Directory of Open Access Journals (Sweden)

Bram Vivijs

2016-10-01

Full Text Available The survival of some pathotypes of E. coli in very low pH environments like highly acidic foods and the stomach has been well documented and contributes to their success as foodborne pathogens. In contrast, the ability of E. coli to grow at moderately low pH has received less attention, although this property can be anticipated to be also very important for the safety of mildly acidic foods. Therefore, the objective of this study was to identify cellular functions required for growth of the non-pathogenic strain E. coli MG1655 at low pH. First, the role of the four E. coli amino acid decarboxylase systems, which are the major cellular mechanisms allowing extreme acid survival, was investigated using mutants defective in each of the systems. Only the lysine decarboxylase (CadA was required for low pH growth. Secondly, a screening of 8544 random transposon insertion mutants resulted in the identification of six genes affecting growth in LB broth acidified to pH 4.50 with HCl. Two of the genes, encoding the transcriptional regulator LeuO and the elongation factor P-β-lysine ligase EpmA, can be linked to CadA production. Two other genes, encoding the diadenosine tetraphosphatase ApaH and the tRNA modification GTPase MnmE, have been previously implicated in the bacterial response to stresses other than low pH. A fifth gene encodes the LPS heptosyltransferase WaaC, and its mutant has a deep rough colony phenotype, which has been linked to reduced acid tolerance in earlier work. Finally, tatC encodes a secA-independent protein translocase that exports a few dozen proteins and thus is likely to have a pleiotropic phenotype. For mnmE, apaH, epmA,and waaC, de novo in frame deletion and genetic complementation confirmed their role in low pH growth, and these deletion mutants were also affected in growth in apple juice and tomato juice. However, the mutants were not affected in survival in gastric simulation medium at pH 2.5, indicating that growth at

Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

Science.gov (United States)

Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

2017-07-01

NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

Incidence of Escherichia coli in black walnut meats.

Science.gov (United States)

Meyer, M T; Vaughn, R H

1969-11-01

Examination of commercially shelled black walnut meats showed inconsistent numbers of total aerobic bacteria, coliforms, and Escherichia coli; variation occurred among different meat sizes and within each meat size. The incidence of E. coli on meats of commercially hulled black walnuts depended on the physical condition of the nuts. Apparently tightly sealed ones contained only a few or none, whereas those with visibly separated sutures and spoiled meats yielded the most. This contamination was in part correlated to a hulling operation. Large numbers of E. coli on the husk of the walnuts contaminated the hulling water, subsequently also contaminating the meats by way of separated sutures. Chlorination of the hulling wash water was ineffective. Attempts were made to decontaminate the walnut meats without subsequent deleterious changes in flavor or texture. A treatment in coconut oil at 100 C followed by removal of excess surface oil by centrifugation was best.

Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli:

DEFF Research Database (Denmark)

Sherlock, Orla; Schembri, Mark; Reisner, A.

2004-01-01

Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA...... binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact...

Oxidative stress inactivates cobalamin-independent methionine synthase (MetE in Escherichia coli.

Directory of Open Access Journals (Sweden)

Elise R Hondorp

2004-11-01

Full Text Available In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE. We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert

Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

International Nuclear Information System (INIS)

Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de

2008-01-01

Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl 2 ) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl 2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl 2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Mattos, Jose Carlos Pelielo de; Motta, Ellen Serri da; Oliveira, Marcia Betania Nunes de; Dantas, Flavio Jose da Silva; Araujo, Adriano Caldeira de [Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Dept. de Biofisica e Biometria. Lab. de Radio e Fotobiologia]. E-mail: jcmattos@uerj.br

2008-12-15

Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl{sub 2}) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl{sub 2} in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl{sub 2} was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms. (author)

In-cell thermodynamics and a new role for protein surfaces.

Science.gov (United States)

Smith, Austin E; Zhou, Larry Z; Gorensek, Annelise H; Senske, Michael; Pielak, Gary J

2016-02-16

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

Effect of antibiotics on cellular stress generated in Shiga toxin-producing Escherichia coli O157:H7 and non-O157 biofilms.

Science.gov (United States)

Angel Villegas, Natalia; Baronetti, José; Albesa, Inés; Etcheverría, Analía; Becerra, M Cecilia; Padola, Nora L; Paraje, M Gabriela

2015-10-01

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, with the main virulence factor of this bacterium being its capacity to secrete Shiga toxins (Stxs). Therefore, the use of certain antibiotics for the treatment of this infection, which induces the liberation of Stxs, is controversial. Reactive oxygen and nitrogen species are also involved in the pathogenesis of different diseases. The purpose of this study was to analyze the effects of antibiotics on biofilms of STEC and the relationships between cellular stress and the release of Stx. To this end, biofilms of reference and clinical strains were treated with antibiotics (ciprofloxacin, fosfomycin and rifaximin) and the production of oxidants, the antioxidant defense system and toxin release were evaluated. Ciprofloxacin altered the prooxidant-antioxidant balance, with a decrease of oxidant metabolites and an increase of superoxide dismutase and catalase activity, being associated with high-levels of Stx production. Furthermore, inhibition of oxidative stress by exogenous antioxidants was correlated with a reduction in the liberation of Stx, indicating the participation of this phenomenon in the release of this toxin. In contrast, fosfomycin and rifaximin produced less alteration with a minimal production of Stx. Our data show that treatment of biofilm-STEC with these antibiotics induces oxidative stress-mediated release of Stx. Copyright © 2015 Elsevier Ltd. All rights reserved.

Escherichia coli biofilms have an organized and complex extracellular matrix structure.

Science.gov (United States)

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

2013-09-10

Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

Development of a cell culture surface conversion technique using alginate thin film for evaluating effect upon cellular differentiation

International Nuclear Information System (INIS)

Nakashima, Y.; Tsusu, K.; Minami, K.; Nakanishi, Y.

2014-01-01

Here, we sought to develop a cell culture surface conversion technique that would not damage living cells. An alginate thin film, formed on a glass plate by spin coating of sodium alginate solution and dipping into calcium chloride solution, was used to inhibit adhesion of cells. The film could be removed by ethylenediaminetetraacetate (EDTA) at any time during cell culture, permitting observation of cellular responses to conversion of the culture surface in real time. Additionally, we demonstrated the validity of the alginate thin film coating method and the performance of the film. The thickness of the alginate thin film was controlled by varying the rotation speed during spin coating. Moreover, the alginate thin film completely inhibited the adhesion of cultured cells to the culture surface, irrespective of the thickness of the film. When the alginate thin film was removed from the culture surface by EDTA, the cultured cells adhered to the culture surface, and their morphology changed. Finally, we achieved effective differentiation of C2C12 myoblasts into myotube cells by cell culture on the convertible culture surface, demonstrating the utility of our novel technique

Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti.

Science.gov (United States)

Zatakia, Hardik M; Arapov, Timofey D; Meier, Veronika M; Scharf, Birgit E

2018-03-15

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli , the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilis IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better

Intra cellular distribution of 8-14C-puromycin aminonucleoside in ultraviolet irradiated Escherichia coli

International Nuclear Information System (INIS)

Sideropoulos, A.S.

1976-01-01

The uptake of 8- 14 C-puromycin aminonucleoside (8- 14 C-PAN) was studied in ultraviolet (UV) irradiated strains of E. coli B/r hcr + and hcr - . The cells took up only 0.1-0.3% of the 8- 14 C-PAN present in the medium when grown in minimal (M9) containing 8- 14 C-PAN. When ethylenedinitrilotetra-acetic acid (EDTA) treated E. coli cells are placed in a medium containing 8- 14 C-PAN, the total concentration of 8- 14 C-PAN in the cell reaches 43-54% of the medium within 30 min of incubation. Almost all 8- 14 C-PAN can be dialyzed from cells exposed in the absence of an energy source, but cells metabolizing in M9 medium during exposure can retain up to 30% of their internal concentration. Bacteria grown in the presence of 8- 14 C-PAN, accumulated the radioactive material intracellulary in three forms, namely, unbound, reversibly bound (dialyzable) and irreversibly bound to the protein (nondialyzable). Approx. 70-77% of the irreversibly bound radioactive material linked with the protein fraction was released by treatment with a protease. Addition of PAN into the post-irradiation medium of EDTA-treated hcr + cells, increased UC induced mutation rates. Antimutagenic purine ribosides decreased the final level of 8- 14 C-PAN accumulated by the cells. Decreases in 8- 14 C-PAN uptake in the presence of antimutagens correspond to reductions in the rate of mutation to streptomycin resistance induced by UV light. Therefore, protein bound PAN appears to be the relevant component in the enhancement of UV induced mutation by this drug

flu, a metastable gene controlling surface properties of Escherichia coli.

OpenAIRE

Diderichsen, B

1980-01-01

flu, a gene of Escherichia coli K-12, was discovered and mapped between his and shiA. It is shown that flu is a metastable gene that changes frequently between the flu+ and flu states. flu+ variants give stable homogeneous suspensions, are piliated, and form glossy colonies. flu variants aggregate, fluff and sediment from suspensions, are nonpiliated, and form frizzy colonies. flu+ and flu variants can be isolated from most strains. Implications of these observations are discussed, and it is ...

Isolation, histopathology and antibiogram of Escherichia coli from pigeons (Columba livia

Directory of Open Access Journals (Sweden)

Pankaj Dutta

2013-04-01

Full Text Available Aim: To know the prevalence of antibiotic resistant Escherichia coli among dead and/or diarrhoic pigeons in and around greater Guwahati. Materials and Methods: Samples were cultured from dead and/or diarrhoic pigeons and identification was done by standard methods. The sensitivity of the isolated E.coli strains to 15 antibiotics of human and veterinary use was also determined. Organs from those dead birds from which E.coli were recovered were processed according to the routine procedure for histopathological studies. Results: Out of 150 pigeons subjected to microbiological investigation, 91(60.67 % samples were found positive for E. coli.The most frequently occurring serotypes were O157 (9.89%, followed by O68, O121 (7.69%, O9, O75, O131 (5.49%, O2, O13, O22 (3.30%. Antibiogram investigation of the isolates revealed that 91isolates (100% exhibited resistance against Ampicillin followed by Nitro-furantoin (73.62%, Tetracycline (65.93 %, Oxytetracycline (62.63 % and Streptomycin (61.54. Gross changes of some birds showed fibrinous pericarditis and perihepatitis and coligranuloma in different organs like liver and serosal surface of intestine. Microscopically, severe congestion and haemorrhages in different organs such as liver, kidney, lung and intestine. In some cases thick layer of fibrinous exudates with large number of heterophills over the surface of liver and heart with early degenerative changes as well as focal necrosis. Conclusion: The result of this study suggests that antimicrobial-resistant pathogenic E.coli is present in pigeons in and around greater Guwahati. Surveillance programs may be introduced to monitor antimicrobial resistance of pathogenic E.coli in pigeons in and around greater guwahati. [Vet World 2013; 6(2.000: 91-94

The role of cellular catalase on the radiosensitization of bacterial vegetative cells by N2O

International Nuclear Information System (INIS)

Watanabe, H.; Takehisa, M.

1983-01-01

The radiosensitizing effect of N 2 O on eight strains of bacteria was measured in dilute suspensions. The dose-modifying factors (DMF) of N 2 O on M. radiodurans R 1 , P. radiora O-1, M. lysodeikticus and B. pumilus E601 (vegetative cells) were 3.4, 2.9, 2.4 and 1.7, respectively. But P. radiora RP-C, P. fluorescens B3-1, E. coli B/r and E. coli K-12 were hardly sensitized by N 2 O. From measurements of catalase activity of each bacterium, it was found that the DMF increases with increased catalase activity, suggesting that cellular catalase promotes the sensitizing action of N 2 O. (author)

Probing cellular behaviors through nanopatterned chitosan membranes

International Nuclear Information System (INIS)

Yang, Chung-Yao; Sung, Chun-Yen; Shuai, Hung-Hsun; Cheng, Chao-Min; Yeh, J Andrew

2013-01-01

This paper describes a high-throughput method for developing physically modified chitosan membranes to probe the cellular behavior of MDCK epithelial cells and HIG-82 fibroblasts adhered onto these modified membranes. To prepare chitosan membranes with micro/nanoscaled features, we have demonstrated an easy-to-handle, facile approach that could be easily integrated with IC-based manufacturing processes with mass production potential. These physically modified chitosan membranes were observed by scanning electron microscopy to gain a better understanding of chitosan membrane surface morphology. After MDCK cells and HIG-82 fibroblasts were cultured on these modified chitosan membranes for various culture durations (i.e. 1, 2, 4, 12 and 24 h), they were investigated to decipher cellular behavior. We found that both cells preferred to adhere onto a flat surface rather than on a nanopatterned surface. However, most (> 80%) of the MDCK cells showed rounded morphology and would suspend in the cultured medium instead of adhering onto the planar surface of negatively nanopatterned chitosan membranes. This means different cell types (e.g. fibroblasts versus epithelia) showed distinct capabilities/preferences of adherence for materials of varying surface roughness. We also showed that chitosan membranes could be re-used at least nine times without significant contamination and would provide us consistency for probing cell–material interactions by permitting reuse of the same substrate. We believe these results would provide us better insight into cellular behavior, specifically, microscopic properties and characteristics of cells grown under unique, nanopatterned cell-interface conditions. (paper)

Effect of surface charge and agglomerate degree of magnetic iron oxide nanoparticles on KB cellular uptake in vitro.

Science.gov (United States)

Ge, Yuqing; Zhang, Yu; Xia, Jingguang; Ma, Ming; He, Shiying; Nie, Fang; Gu, Ning

2009-10-15

We synthesized three types of magnetic iron oxide nanoparticles (MNPs), which were meso-2,3-dimercaptosuccinic acid (DMSA) coated MNPs (DMSA@MNPs, 17.3+/-4.8 nm, negative charge), chitosan (CS) coated MNPs (CS@MNPs, 16.5+/-6.1 nm, positive charge) and magnetic nanoparticles agglomerates, formed by electronic aggregation between DMSA@MNPs and CS (CS-DMSA@MNPs, 85.7+/-72.9 nm, positive charge) respectively. The interactions of these MNPs with Oral Squamous Carcinoma Cell KB were investigated. The results showed that cellular uptakes of MNPs were on the dependence of incubation time, nanoparticles concentration and nanoparticles properties such as surface charge, size, etc. The cellular uptake was enhanced with the increase of incubation time and nanoparticles concentration. Although all MNPs could enter to cells, we observed apparent differences in the magnitude of nanoparticles uptaken. The cellular uptake of CS-DMSA@MNPs by KB cells was the highest and that of DMSA@MNPs was the lowest among the three types of MNPs. The same conclusions were drawn via the reduction of water proton relaxation times T(2)(*), resulting from the different iron load of labeled cells using a 1.5T clinical MR imager. The finding of this study will have implications in the chemical design of nanomaterials for biomedical applications.

In vitro fibroblast and pre-osteoblastic cellular responses on laser surface modified Ti–6Al–4V

International Nuclear Information System (INIS)

Chikarakara, Evans; Vázquez, Mercedes; Bagga, Komal; Brabazon, Dermot; Fitzpatrick, Patricia; Moore, Eric; Levingstone, Tanya; Grehan, Laura; Higginbotham, Clement; Naher, Sumsun

2014-01-01

The success of any implant, dental or orthopaedic, is driven by the interaction of implant material with the surrounding tissue. In this context, the nature of the implant surface plays a direct role in determining the long term stability as physico-chemical properties of the surface affect cellular attachment, expression of proteins, and finally osseointegration. Thus to enhance the degree of integration of the implant into the host tissue, various surface modification techniques are employed. In this work, laser surface melting of titanium alloy Ti–6Al–4V was carried out using a CO 2 laser with an argon gas atmosphere. Investigations were carried out to study the influence of laser surface modification on the biocompatibility of Ti–6Al–4V alloy implant material. Surface roughness, microhardness, and phase development were recorded. Initial knowledge of these effects on biocompatibility was gained from examination of the response of fibroblast cell lines, which was followed by examination of the response of osteoblast cell lines which is relevant to the applications of this material in bone repair. Biocompatibility with these cell lines was analysed via Resazurin cell viability assay, DNA cell attachment assay, and alamarBlue metabolic activity assay. Laser treated surfaces were found to preferentially promote cell attachment, higher levels of proliferation, and enhanced bioactivity when compared to untreated control samples. These results demonstrate the tremendous potential of this laser surface melting treatment to significantly improve the biocompatibility of titanium implants in vivo. (paper)

E. Coli Infections

Science.gov (United States)

E. coli is the name of a type of bacteria that lives in your intestines. Most types of E. coli are harmless. However, some types can make you ... type causes travelers' diarrhea. The worst type of E. coli causes bloody diarrhea, and can sometimes cause kidney ...

Stress analysis of two-dimensional cellular materials with thick cell struts

International Nuclear Information System (INIS)

Lim, Do Hyung; Kim, Han Sung; Kim, Young Ho; Kim, Yoon Hyuk; Al-Hassani, S.T.S.

2008-01-01

Finite element analyses (FEA) were performed to thoroughly validate the collapse criteria of cellular materials presented in our previous companion paper. The maximum stress (von-Mises stress) on the cell strut surface and the plastic collapse stress were computed for two-dimensional (2D) cellular materials with thick cell struts. The results from the FEA were compared with those from theoretical criteria of authors. The FEA results were in good agreement with the theoretical results. The results indicate that when bending moment, axial and shear forces are considered, the maximum stress on the strut surface gives significantly different values in the tensile and compressive parts of the cell wall as well as in the two loading directions. Therefore, for the initial yielding of ductile cellular materials and the fracture of brittle cellular materials, in which the maximum stress on the strut surface is evaluated, it is necessary to consider not only the bending moment but also axial and shear forces. In addition, this study shows that for regular cellular materials with the identical strut geometry for all struts, the initial yielding and the plastic collapse under a biaxial state of stress occur not only in the inclined cell struts but also in the vertical struts. These FEA results support the theoretical conclusion of our previous companion paper that the anisotropic 2D cellular material has a truncated yield surface not only on the compressive quadrant but also on the tensile quadrant

Effects of High Hydrostatic Pressure on Escherichia coli Ultrastructure, Membrane Integrity and Molecular Composition as Assessed by FTIR Spectroscopy and Microscopic Imaging Techniques

Directory of Open Access Journals (Sweden)

María Prieto-Calvo

2014-12-01

Full Text Available High hydrostatic pressure (HHP is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50–900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200–900 cm−1, mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

Effects of high hydrostatic pressure on Escherichia coli ultrastructure, membrane integrity and molecular composition as assessed by FTIR spectroscopy and microscopic imaging techniques.

Science.gov (United States)

Prieto-Calvo, María; Prieto, Miguel; López, Mercedes; Alvarez-Ordóñez, Avelino

2014-12-18

High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50-900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200-900 cm-1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli

Science.gov (United States)

Blum, Shlomo E.; Heller, Elimelech D.; Sela, Shlomo; Elad, Daniel; Edery, Nir; Leitner, Gabriel

2015-01-01

Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide. Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis are largely unknown. This study aimed to identify virulence markers in MPEC through whole genome and phenome comparative analysis. MPEC strains causing acute (VL2874 and P4) or persistent (VL2732) mastitis were compared to an environmental strain (K71) and to the genomes of strains representing different E. coli pathotypes. Intra-mammary challenge in mice confirmed experimentally that the strains studied here have different pathogenic potential, and that the environmental strain K71 is non-pathogenic in the mammary gland. Analysis of whole genome sequences and predicted proteomes revealed high similarity among MPEC, whereas MPEC significantly differed from the non-mammary pathogenic strain K71, and from E. coli genomes from other pathotypes. Functional features identified in MPEC genomes and lacking in the non-mammary pathogenic strain were associated with synthesis of lipopolysaccharide and other membrane antigens, ferric-dicitrate iron acquisition and sugars metabolism. Features associated with cytotoxicity or intra-cellular survival were found specifically in the genomes of strains from severe and acute (VL2874) or persistent (VL2732) mastitis, respectively. MPEC genomes were relatively similar to strain K-12, which was subsequently shown here to be possibly pathogenic in the mammary gland. Phenome analysis showed that the persistent MPEC was the most versatile in terms of nutrients metabolized and acute MPEC the least. Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism. This study

The relationship between cellular adhesion and surface roughness in polystyrene modified by microwave plasma radiation

Directory of Open Access Journals (Sweden)

Biazar E

2011-03-01

Full Text Available Esmaeil Biazar1, Majid Heidari2, Azadeh Asefnezhad2, Naser Montazeri11Department of Chemistry, Islamic Azad University, Tonekabon Branch, Mazandaran; 2Department of Biomaterial Engineering, Faculty of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, IranBackground: Surface modification of medical polymers can improve biocompatibility. Pure polystyrene is hydrophobic and cannot provide a suitable environment for cell cultures. The conventional method for surface modification of polystyrene is treatment with plasma. In this study, conventional polystyrene was exposed to microwave plasma treatment with oxygen and argon gases for 30, 60, and 180 seconds.Methods and results: Attenuated total reflection Fourier transform infrared spectra investigations of irradiated samples indicated clearly the presence of functional groups. Atomic force microscopic images of samples irradiated with inert and active gases indicated nanometric surface topography. Samples irradiated with oxygen plasma showed more roughness (31 nm compared with those irradiated with inert plasma (16 nm at 180 seconds. Surface roughness increased with increasing duration of exposure, which could be due to reduction of the contact angle of samples irradiated with oxygen plasma. Contact angle analysis showed reduction in samples irradiated with inert plasma. Samples irradiated with oxygen plasma showed a lower contact angle compared with those irradiated by argon plasma.Conclusion: Cellular investigations with unrestricted somatic stem cells showed better adhesion, cell growth, and proliferation for samples radiated by oxygen plasma with increasing duration of exposure than those of normal samples.Keywords: surface topography, polystyrene, plasma treatment, argon, oxygen

Measurement uncertainty evaluation of cellular spheroids surface tension in compressing tests using Young-Laplace equation

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Beatrici, Anderson; Santos Baptista, Leandra; Mauro Granjeiro, José

2018-03-01

Regenerative Medicine comprises the Biotechnology, Tissue Engineering and Biometrology for stem cell therapy. Starting from stem cells extracted from the patient, autologous implant, these cells are cultured and differentiated into other tissues, for example, articular cartilage. These cells are reorganized into microspheres (cell spheroids). Such tissue units are recombined into functional tissues constructs that can be implanted in the injured region for regeneration. It is necessary the biomechanical characterization of these constructed to determine if their properties are similar to native tissue. In this study was carried out the modeling of the calculation of uncertainty of the surface tension of cellular spheroids with the use of the Young-Laplace equation. We obtained relative uncertainties about 10%.

Ultraviolet mutagenesis and the SOS response in Escherichia coli: A personal perspective

International Nuclear Information System (INIS)

Witkin, E.M.

1989-01-01

The study of ultraviolet (UV) mutagenesis in Escherichia coli began with the assumption that genes were likely to be changed at the instant of photon absorption. Over many decades, it became clear that postirradiation cellular activities, including enzymatic DNA repair of UV photo products and error-prone modes of tolerating unrepaired DNA lesions can exert profound influences on the mutagenic outcome of irradiation. Current study focusses on the molecular details of radiation-induced translesion DNA replication as the final event in UV mutagenesis

Effect of millimeter waves on survival of UVC-exposed Escherichia coli

International Nuclear Information System (INIS)

Rojavin, M.A.; Ziskin, M.C.

1995-01-01

Bacterial cells of the strain Escherichia coli K12 were exposed to millimeter electromagnetic waves (mm waves) with and without additional exposure to ultraviolet light λ = 254 nm (UVC). The mm waves were produced by a medical microwave generator emitting a 4-GHz-wide band around a 61 GHz center frequency and delivering an irradiation of 1mW/cm 2 and a standard absorption rate (SAR) of 84 W/kg to the bacteria. Exposure to the mm waves alone for up to 30 minutes did not change the survival rate of bacteria. Exposure to mm waves followed by UVC irradiation also did not alter the number of surviving E. coli cells in comparison to UVC-treated controls. When mm waves were applied after the UVC exposure, a dose-dependent increase of up to 30% in the survival of E. coli was observed compared to UVC + sham-irradiated bacteria. Because sham controls and experimental samples were maintained under the same thermal conditions, the effect is not likely to be due to heating, although the possibility of nonuniform distribution of microwave heating in different layers of irradiated bacterial suspension cannot be ruled out. The mechanism for this effect appears to involve certain DNA repair systems that act as cellular targets for mm waves

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on enteroaggregative Escherichia coli and the role of the surface protein dispersin

Energy Technology Data Exchange (ETDEWEB)

Fowlkes, Jason Davidson [ORNL; Doktycz, Mitchel John [ORNL; Allison, David Post [ORNL

2011-01-01

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

E. Coli

Science.gov (United States)

... for the bacteria's medical name Escherichia coli . The strange thing about these bacteria — and lots of other ... In some cases, E. coli poisoning can cause life-threatening kidney problems. What Can Kids Do? Adults ...

Inactivation of Escherichia coli by titanium dioxide photocatalytic oxidation.

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Titanium dioxide in the anatase crystalline form was used as a photocatalyst to generate hydroxyl radicals in a flowthrough water reactor. Experiments were performed on pure cultures of Escherichia coli in dechlorinated tap water and a surface water sample to evaluate the disinfe...

Quantification and Classification of E. coli Proteome Utilization and Unused Protein Costs across Environments.

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Edward J O'Brien

2016-06-01

Full Text Available The costs and benefits of protein expression are balanced through evolution. Expression of un-utilized protein (that have no benefits in the current environment incurs a quantifiable fitness costs on cellular growth rates; however, the magnitude and variability of un-utilized protein expression in natural settings is unknown, largely due to the challenge in determining environment-specific proteome utilization. We address this challenge using absolute and global proteomics data combined with a recently developed genome-scale model of Escherichia coli that computes the environment-specific cost and utility of the proteome on a per gene basis. We show that nearly half of the proteome mass is unused in certain environments and accounting for the cost of this unused protein expression explains >95% of the variance in growth rates of Escherichia coli across 16 distinct environments. Furthermore, reduction in unused protein expression is shown to be a common mechanism to increase cellular growth rates in adaptive evolution experiments. Classification of the unused protein reveals that the unused protein encodes several nutrient- and stress- preparedness functions, which may convey fitness benefits in varying environments. Thus, unused protein expression is the source of large and pervasive fitness costs that may provide the benefit of hedging against environmental change.

Rapid detection of single E. coli bacteria using a graphene-based field-effect transistor device.

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Thakur, Bhawana; Zhou, Guihua; Chang, Jingbo; Pu, Haihui; Jin, Bing; Sui, Xiaoyu; Yuan, Xiaochen; Yang, Ching-Hong; Magruder, Matthew; Chen, Junhong

2018-07-01

Contamination of surface and drinking water due to the presence of Escherichia coli bacteria is a major cause of water-borne disease outbreak. To address unmet challenges for practical pathogen detection in contaminated samples, we report fabrication of thermally reduced graphene oxide-based field-effect transistor (rGO FET) passivated with an ultrathin layer of Al 2 O 3 for real-time detection of E. coli bacteria. The sensor could detect a single E. coli cell within 50 s in a 1 µL sample volume. The ultrathin layer of Al 2 O 3 acted as a barrier between rGO and potential interferents present in the sample. E. coli specific antibodies anchored on gold nanoparticles acted as probes for selective capture of E. coli. The high density of negative charge on the surface of E. coli cells strongly modulates the concentration of majority charge carriers in the rGO monolayer, thereby allowing real-time monitoring of E. coli concentration in a given sample. With a low detection limit of single cell, the FET sensor had a linear range of 1-100 CFU in 1 µL volume of sample (i.e., 10 3 to 10 5 CFU/ mL). The biosensor with good selectivity and rapid detection was further successfully demonstrated for E. coli sensing in river water. The rGO-based FET sensor provides a low cost and label-free approach, and can be mass produced for detection of a broad spectrum of pathogens in water or other liquid media. Copyright © 2018 Elsevier B.V. All rights reserved.

Bacterial resistance of self-assembled surfaces using PPOm-b-PSBMAn zwitterionic copolymer - concomitant effects of surface topography and surface chemistry on attachment of live bacteria.

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Hsiao, Sheng-Wen; Venault, Antoine; Yang, Hui-Shan; Chang, Yung

2014-06-01

Three well-defined diblock copolymers made of poly(sulfobetaine methacrylate) (poly(SBMA)) and poly(propylene oxide) (PPO) groups were synthesized by atom transfer radical polymerization (ATRP) method. They were physically adsorbed onto three types of surfaces having different topography, including smooth flat surface, convex surface, and indented surface. Chemical state of surfaces was characterized by XPS while the various topographies were examined by SEM and AFM. Hydrophilicity of surfaces was dependent on both the surface chemistry and the surface topography, suggesting that orientation of copolymer brushes can be tuned in the design of surfaces aimed at resisting bacterial attachment. Escherichia coli, Staphylococcus epidermidis, Streptococcus mutans and Escherichia coli with green fluorescent protein (E. coli GFP) were used in bacterial tests to assess the resistance to bacterial attachment of poly(SBMA)-covered surfaces. Results highlighted a drastic improvement of resistance to bacterial adhesion with the increasing of poly(SBMA) to PPO ratio, as well as an important effect of surface topography. The chemical effect was directly related to the length of the hydrophilic moieties. When longer, more water could be entrapped, leading to improved anti-bacterial properties. The physical effect impacted on the orientation of the copolymer brushes, as well as on the surface contact area available. Convex surfaces as well as indented surfaces wafer presented the best resistance to bacterial adhesion. Indeed, bacterial attachment was more importantly reduced on these surfaces compared with smooth surfaces. It was explained by the non-orthogonal orientation of copolymer brushes, resulting in a more efficient surface coverage of zwitterionic molecules. This work suggests that not only the control of surface chemistry is essential in the preparation of surfaces resisting bacterial attachment, but also the control of surface topography and orientation of antifouling

Antibacterial Activities and Possible Modes of Action of Acacia nilotica (L. Del. against Multidrug-Resistant Escherichia coli and Salmonella

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Muhammad Bilal Sadiq

2017-01-01

Full Text Available Medicinal plants are frequently used for the treatment of various infectious diseases. The objective of this study was to evaluate the antibacterial activity and mode of action of Acacia nilotica and the antibiogram patterns of foodborne and clinical strains of Escherichia coli and Salmonella. The mechanism of action of acacia extracts against E. coli and Salmonella was elucidated by observing morphological damages including cell integrity and cell membrane permeability, as well as changes in cell structures and growth patterns in kill-time experiments. The clinical isolates of E. coli and Salmonella were found resistant to more of the tested antibiotics, compared to food isolates. Minimum inhibitory concentration and minimum bactericidal concentration of acacia leaf extracts were in the ranges of 1.56–3.12 mg/mL and 3.12–6.25 mg/mL, respectively, whereas pods and bark extracts showed somewhat higher values of 3.12–6.25 mg/mL and 6.25–12.5 mg/mL, respectively, against all tested pathogens. The release of electrolytes and essential cellular constituents (proteins and nucleic acids indicated that acacia extracts damaged the cellular membrane of the pathogens. These changes corresponded to simultaneous reduction in the growth of viable bacteria. This study indicates that A. nilotica can be a potential source of new antimicrobials, effective against antibiotic-resistant strains of pathogens.

Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli

DEFF Research Database (Denmark)

Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili

2017-01-01

Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production econo...

Curli fimbriae are conditionally required in Escherichia coli O157:H7 for initial attachment and biofilm formation.

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Carter, Michelle Qiu; Louie, Jacqueline W; Feng, Doris; Zhong, Wayne; Brandl, Maria T

2016-08-01

Several species of enteric pathogens produce curli fimbriae, which may affect their interaction with surfaces and other microbes in nonhost environments. Here we used two Escherichia coli O157:H7 outbreak strains with distinct genotypes to understand the role of curli in surface attachment and biofilm formation in several systems relevant to fresh produce production and processing. Curli significantly enhanced the initial attachment of E. coli O157:H7 to spinach leaves and stainless steel surfaces by 5-fold. Curli was also required for E. coli O157:H7 biofilm formation on stainless steel and enhanced biofilm production on glass by 19-27 fold in LB no-salt broth. However, this contribution was not observed when cells were grown in sterile spinach lysates. Furthermore, both strains of E. coli O157:H7 produced minimal biofilms on polypropylene in LB no-salt broth but considerable amounts in spinach lysates. Under the latter conditions, curli appeared to slightly increase biofilm production. Importantly, curli played an essential role in the formation of mixed biofilm by E. coli O157:H7 and plant-associated microorganisms in spinach leaf washes, as revealed by confocal microscopy. Little or no E. coli O157:H7 biofilms were detected at 4 °C, supporting the importance of temperature control in postharvest and produce processing environments. Published by Elsevier Ltd.

A new multicompartmental reaction-diffusion modeling method links transient membrane attachment of E. coli MinE to E-ring formation.

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Arjunan, Satya Nanda Vel; Tomita, Masaru

2010-03-01

Many important cellular processes are regulated by reaction-diffusion (RD) of molecules that takes place both in the cytoplasm and on the membrane. To model and analyze such multicompartmental processes, we developed a lattice-based Monte Carlo method, Spatiocyte that supports RD in volume and surface compartments at single molecule resolution. Stochasticity in RD and the excluded volume effect brought by intracellular molecular crowding, both of which can significantly affect RD and thus, cellular processes, are also supported. We verified the method by comparing simulation results of diffusion, irreversible and reversible reactions with the predicted analytical and best available numerical solutions. Moreover, to directly compare the localization patterns of molecules in fluorescence microscopy images with simulation, we devised a visualization method that mimics the microphotography process by showing the trajectory of simulated molecules averaged according to the camera exposure time. In the rod-shaped bacterium Escherichia coli, the division site is suppressed at the cell poles by periodic pole-to-pole oscillations of the Min proteins (MinC, MinD and MinE) arising from carefully orchestrated RD in both cytoplasm and membrane compartments. Using Spatiocyte we could model and reproduce the in vivo MinDE localization dynamics by accounting for the previously reported properties of MinE. Our results suggest that the MinE ring, which is essential in preventing polar septation, is largely composed of MinE that is transiently attached to the membrane independently after recruited by MinD. Overall, Spatiocyte allows simulation and visualization of complex spatial and reaction-diffusion mediated cellular processes in volumes and surfaces. As we showed, it can potentially provide mechanistic insights otherwise difficult to obtain experimentally. The online version of this article (doi:10.1007/s11693-009-9047-2) contains supplementary material, which is available to

Surface thiolation of silicon for antifouling application.

Science.gov (United States)

Zhang, Xiaoning; Gao, Pei; Hollimon, Valerie; Brodus, DaShan; Johnson, Arion; Hu, Hongmei

2018-02-07

Thiol groups grafted silicon surface was prepared as previously described. 1H,1H,2H,2H-perfluorodecanethiol (PFDT) molecules were then immobilized on such a surface through disulfide bonds formation. To investigate the contribution of PFDT coating to antifouling, the adhesion behaviors of Botryococcus braunii (B. braunii) and Escherichia coli (E. coli) were studied through biofouling assays in the laboratory. The representative microscope images suggest reduced B. braunii and E. coli accumulation densities on PFDT integrated silicon substrate. However, the antifouling performance of PFDT integrated silicon substrate decreased over time. By incubating the aged substrate in 10 mM TCEP·HCl solution for 1 h, the fouled PFDT coating could be removed as the disulfide bonds were cleaved, resulting in reduced absorption of algal cells and exposure of non-fouled silicon substrate surface. Our results indicate that the thiol-terminated substrate can be potentially useful for restoring the fouled surface, as well as maximizing the effective usage of the substrate.

Factors determinating the shape of survival curves of Escherichia coli cells irradiated by ionizing radiation with different LET. Peculiarities of genom organization and the shape of survival curves

International Nuclear Information System (INIS)

Krasavin, E.A.

1984-01-01

The basic biological mechanisms realized on molecular, cellular and population levels and stipulating the shape of dependence of the cell suriival (S) on the dose (D) are considered. One of possible causes of nonlinear S(D) dependence are the peculiarities of DNA degradation in E. coli cells. The mechanisms of genetic control of different types of degradation are discussed. Some regularities of the genetic recombination and replication of DNA in E. coli are considered. The conclusion is made that one of the basic stipulating for the shoulder on the survival curves in E. coli are the peculiarities of the chromosome replication

Inactivation of E. Coli in Water Using Photocatalytic, Nanostructured Films Synthesized by Aerosol Routes

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Pratim Biswas

2013-03-01

Full Text Available TiO2 nanostructured films were synthesized by an aerosol chemical vapor deposition (ACVD method with different controlled morphologies: columnar, granular, and branched structures for the photocatalytic inactivation of Escherichia coli (E. coli in water. Effects of film morphology and external applied voltage on inactivation rate were investigated. As-prepared films were characterized using scanning electron microscopy (SEM, transmission electron microscopy (TEM, X-ray diffractometry (XRD, and UV-VIS. Photocatalytic and photoelectrochemical inactivation of E. coli using as-prepared TiO2 films were performed under irradiation of UVA light (note: UVA has a low efficiency to inactivate E. coli. Inactivation rate constants for each case were obtained from their respective inactivation curve through a 2 h incubation period. Photocatalytic inactivation rate constants of E. coli are 0.02/min (using columnar films, and 0.08/min (using branched films. The inactivation rate constant for the columnar film was enhanced by 330% by applied voltage on the film while that for the branched film was increased only by 30%. Photocatalytic microbial inactivation rate of the columnar and the branched films were also compared taking into account their different surface areas. Since the majority of the UV radiation that reaches the Earth’s surface is UVA, this study provides an opportunity to use sunlight to efficiently decontaminate drinking water.

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis.

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Zhao, Xiao-wei; Yang, Yong-xin; Huang, Dong-wei; Cheng, Guang-long; Zhao, Hui-ling

2015-01-01

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and a-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.

E. Coli and Pregnancy

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... chat Live Help Fact Sheets Share Escherichia coli (E. coli) Friday, 01 September 2017 In every pregnancy, a ... risk. This sheet talks about whether exposure to E. coli may increase the risk for birth defects over ...

Chromosomal features of Escherichia coli serotype O2:K2, an avian pathogenic E. coli.

Science.gov (United States)

Jørgensen, Steffen L; Kudirkiene, Egle; Li, Lili; Christensen, Jens P; Olsen, John E; Nolan, Lisa; Olsen, Rikke H

2017-01-01

Escherichia coli causing infection outside the gastrointestinal system are referred to as extra-intestinal pathogenic E. coli. Avian pathogenic E. coli is a subgroup of extra-intestinal pathogenic E. coli and infections due to avian pathogenic E. coli have major impact on poultry production economy and welfare worldwide. An almost defining characteristic of avian pathogenic E. coli is the carriage of plasmids, which may encode virulence factors and antibiotic resistance determinates. For the same reason, plasmids of avian pathogenic E. coli have been intensively studied. However, genes encoded by the chromosome may also be important for disease manifestation and antimicrobial resistance. For the E. coli strain APEC_O2 the plasmids have been sequenced and analyzed in several studies, and E. coli APEC_O2 may therefore serve as a reference strain in future studies. Here we describe the chromosomal features of E. coli APEC_O2. E. coli APEC_O2 is a sequence type ST135, has a chromosome of 4,908,820 bp (plasmid removed), comprising 4672 protein-coding genes, 110 RNA genes, and 156 pseudogenes, with an average G + C content of 50.69%. We identified 82 insertion sequences as well as 4672 protein coding sequences, 12 predicated genomic islands, three prophage-related sequences, and two clustered regularly interspaced short palindromic repeats regions on the chromosome, suggesting the possible occurrence of horizontal gene transfer in this strain. The wildtype strain of E. coli APEC_O2 is resistant towards multiple antimicrobials, however, no (complete) antibiotic resistance genes were present on the chromosome, but a number of genes associated with extra-intestinal disease were identified. Together, the information provided here on E. coli APEC_O2 will assist in future studies of avian pathogenic E. coli strains, in particular regarding strain of E. coli APEC_O2, and aid in the general understanding of the pathogenesis of avian pathogenic E. coli .

Spatial Variability of Escherichia coli in Rivers of Northern Coastal Ecuador

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Gouthami Rao

2015-02-01

Full Text Available The use of contaminated surface water continues to be a pressing issue in areas of the world where people lack improved drinking water sources. In northern coastal Ecuador, many communities rely on untreated surface water as their primary source of drinking water. We undertook a study to explore how microscale river hydrodynamics affect microbial water quality at community water collection locations at three rivers with varying stream velocity and turbidity profiles. To examine how the distance from river shore and physiochemical water quality variables affect microbial contamination levels in the rivers; we collected a total of 355 water samples within six villages on three rivers; and tested for Escherichia coli concentrations using the IDEXX Quanti-tray method. We found that log10 E. coli concentrations decreased with increasing distance from shore (β = −0.017; p = 0.003. Water in the main channel had E. coli concentrations on average 0.12 log10 lower than within eddies along the river shore and 0.27 log10 lower between the sample closest to shore and any sample >6 m from the shore. Higher E. coli concentrations were also significantly associated with increased turbidity (β = 0.003; p < 0.0001 and decreased dissolved oxygen levels (β = −0.310; p < 0.0001. The results of this study can help inform community members about the safest locations to collect drinking water and also provide information on watershed scale transport of microbial contaminants between villages.

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation.

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Meadows, Adam L; Karnik, Rahi; Lam, Harry; Forestell, Sean; Snedecor, Brad

2010-03-01

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000 L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations--such as glucose overbatching and insufficient aeration--on growth, metabolism, and titer. (c) 2009 Elsevier Inc. All rights reserved.

Human Meningitis-Associated Escherichia coli

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KIM, KWANG SIK

2016-01-01

E. coli is the most common Gram-negative bacillary organism causing meningitis and E. coli meningitis continues to be an important cause of mortality and morbidity throughout the world. Our incomplete knowledge of its pathogenesis contributes to such mortality and morbidity. Recent reports of E. coli strains producing CTX-M-type or TEM-type extended-spectrum β-lactamases create a challenge. Studies using in vitro and in vivo models of the blood-brain barrier have shown that E. coli meningitis follows a high-degree of bacteremia and invasion of the blood-brain barrier. E. coli invasion of the blood-brain barrier, the essentials step in the development of E. coli meningitis, requires specific microbial and host factors as well as microbe- and host-specific signaling molecules. Blockade of such microbial and host factors contributing to E. coli invasion of the blood-brain barrier is shown to be efficient in preventing E. coli penetration into the brain. The basis for requiring a high-degree of bacteremia for E. coli penetration of the blood-brain barrier, however, remains unclear. Continued investigation on the microbial and host factors contributing to a high-degree of bacteremia and E. coli invasion of the blood-brain barrier is likely to identify new targets for prevention and therapy of E. coli meningitis. PMID:27223820

Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants ▿

OpenAIRE

Reyes, Daniel; Vilchez, Samuel; Paniagua, Margarita; Colque-Navarro, Patricia; Weintraub, Andrej; Möllby, Roland; Kühn, Inger

2010-01-01

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

Macromolecular cell surface engineering for accelerated and reversible cellular aggregation.

OpenAIRE

Amaral, A. J.; Pasparakis, G.

2015-01-01

We report the synthesis of two simple copolymers that induce rapid cell aggregation within minutes in a fully reversible manner. The polymers can act as self-supporting "cellular glues" or as "drivers" of 3D cell spheroids/aggregates formation at minute concentrations.

Effect of bile on growth, peritoneal absorption, and blood clearance of Escherichia coli in E coli peritonitis

International Nuclear Information System (INIS)

Andersson, R.; Schalen, C.; Tranberg, K.G.

1991-01-01

The effect of intraperitoneal bile on growth, peritoneal absorption, and clearance of Escherichia coli was determined in E coli peritonitis in the rat. In E coli peritonitis, intraperitoneal bacterial counts gradually decreased, whereas they increased (after 2 hours) with subsequent development of bacteremia in E coli plus bile peritonitis. After an intraperitoneal injection of labeled bacteria, blood radioactivity was only initially lower in E coli plus bile peritonitis compared with E coli peritonitis. Clearance from blood was lower in E coli plus bile peritonitis than in E coli peritonitis. Organ localization was similar in E coli peritonitis and E coli plus bile peritonitis with decreased splenic, increased pulmonary, and unchanged hepatic uptakes compared with controls. Impaired peritoneal absorption of bacteria, together with impaired local host defense, is likely to enhance the noxious effect of bile in E coli peritonitis

Inactivation of Escherichia coli in broth and sausage by combined high pressure and Lactobacillus casei cell extract.

Science.gov (United States)

Chung, Hyun-Jung; Yousef, Ahmed E

2010-10-01

The purpose of this study was to investigate the effect of combined high pressure and Lactobacillus casei cell extract (CE) on Escherichia coli O157 strains with variation in pressure resistance in broth and sausage. Pressure-resistant (O157:H7 and O157:H12) and -sensitive (O157-M1 and O157-M2) E. coli strains were used. Pressure treatment at 350 MPa for 20 min in broth caused 1.1-1.2 logs reduction in O157:H12 and O157:H7 and 4.1-5.5 logs reduction in the O157-M1 and O157-M2. When high pressure was treated in the presence of CE (32 CEAU/mL), the combination treatment caused a significant inactivation in the pressure-resistant O157:H7 strains resulting in the viability loss of 4.3-4.6 logs and the synergistic effect increased with increase in treatment time (p casei CE may cause considerable damage to cellular components of E. coli during the high pressure treatment. The synergy between high pressure processing and Lb. casei OSY-LB6A CE against pressure-resistant E. coli O157 strains suggests the feasibility of using this combination to minimize the risk of transmission of E. coli O157 by food.

Physical Property Control on the Cellular Uptake Pathway and Spatial Distribution of Nanoparticles in Cells.

Science.gov (United States)

Ahn, Sungsook; Seo, Eunseok; Kim, Ki Hean; Lee, Sang Joon

2015-06-01

Nanoparticles have been developed in broad biomedical research in terms of effective cellular interactions to treat and visualize diseased cells. Considering the charge and polar functional groups of proteins that are embedded in cellular membranes, charged nanoparticles have been strategically developed to enhance electrostatic cellular interactions. In this study, we show that cellular uptake efficiency, pathway, and spatial distribution of gold nanoparticles in a cell are significantly modulated based on the surface condition of gold nanoparticles and human cancer cells that were tuned by controlling the pH of the medium and by introducing an electron beam. Cellular uptake efficiency is increased when electrostatic attraction is induced between the cells and the gold nanoparticles. Cell surface modification changes the cellular uptake pathways of the gold nanoparticles and concentrates the gold nanoparticles at the membrane region. Surface modification of the gold nanoparticles also contributes to deep penetration and homogeneous spatial distributions in a cell.

The Response Surface Methodology speeds up the search for optimal parameters in the photoinactivation of E. coli by Photodynamic Therapy.

Science.gov (United States)

Amaral, Larissa S; Azevedo, Eduardo B; Perussi, Janice R

2018-02-27

Antimicrobial Photodynamic Inactivation (a-PDI) is based on the oxidative destruction of biological molecules by reactive oxygen species generated by the photo-excitation of a photosensitive molecule. When the a-PDT is performed along with the use of mathematical models, the optimal conditions for maximum inactivation are easily found. Experimental designs allow a multivariate analysis of the experimental parameters. This is usually made using a univariate approach, which demands a large number of experiments, being time and money consuming. This paper presents the use of the response surface methodology for improving the search for the best conditions to reduce E. coli survival levels by a-PDT using methylene blue (MB) and toluidine blue (TB) as photosensitizers and white light. The goal was achieved by analyzing the effects and interactions of the three main parameters involved in the process: incubation time (IT), photosensitizer concentration (C PS ), and light dose (LD). The optimization procedure began with a full 2 3 factorial design, followed by a central composite one, in which the optimal conditions were estimated. For MB, C PS was the most important parameter followed by LD and IT whereas, for TB, the main parameter was LD followed by C PS and IT. Using the estimated optimal conditions for inactivation, MB was able to inactivate 99.999999% CFU mL -1 of E. coli with IT of 28 min, LD of 31 J cm -2 , and C PS of 32 μmol L -1 , while TB required 18 min, 39 J cm -2 , and 37 μmol L -1 . The feasibility of using the response surface methodology with a-PDT was demonstrated, enabling enhanced photoinactivation efficiency and fast results with a minimal number of experiments. Copyright © 2018. Published by Elsevier B.V.

Conjugation in Escherichia coli

Science.gov (United States)

Boyer, Herbert

1966-01-01

Boyer, Herbert (Yale University, New Haven, Conn.). Conjugation in Escherichia coli. J. Bacteriol. 91:1767–1772. 1966.—The sex factor of Escherichia coli K-12 was introduced into an E. coli B/r strain by circumventing the host-controlled modification and restriction incompatibilities known to exist between these closely related strains. The sexual properties of the constructed F+ B strain and its Hfr derivatives were examined. These studies showed that the E. coli strain B/r F+ and Hfr derivatives are similar to the E. coli strain K-12 F+ and Hfr derivatives. However, the site of sex factor integration was found to be dependent on the host genome. PMID:5327905

Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

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Park Sung

2008-12-01

Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism.

Science.gov (United States)

Möller, Jens; Lühmann, Tessa; Chabria, Mamta; Hall, Heike; Vogel, Viola

2013-10-07

To clear pathogens from host tissues or biomaterial surfaces, phagocytes have to break the adhesive bacteria-substrate interactions. Here we analysed the mechanobiological process that enables macrophages to lift-off and phagocytose surface-bound Escherichia coli (E. coli). In this opsonin-independent process, macrophage filopodia hold on to the E. coli fimbriae long enough to induce a local protrusion of a lamellipodium. Specific contacts between the macrophage and E. coli are formed via the glycoprotein CD48 on filopodia and the adhesin FimH on type 1 fimbriae (hook). We show that bacterial detachment from surfaces occurrs after a lamellipodium has protruded underneath the bacterium (shovel), thereby breaking the multiple bacterium-surface interactions. After lift-off, the bacterium is engulfed by a phagocytic cup. Force activated catch bonds enable the long-term survival of the filopodium-fimbrium interactions while soluble mannose inhibitors and CD48 antibodies suppress the contact formation and thereby inhibit subsequent E. coli phagocytosis.

Investigation on Selective Laser Melting AlSi10Mg Cellular Lattice Strut: Molten Pool Morphology, Surface Roughness and Dimensional Accuracy

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Xuesong Han

2018-03-01

Full Text Available AlSi10Mg inclined struts with angle of 45° were fabricated by selective laser melting (SLM using different scanning speed and hatch spacing to gain insight into the evolution of the molten pool morphology, surface roughness, and dimensional accuracy. The results show that the average width and depth of the molten pool, the lower surface roughness and dimensional deviation decrease with the increase of scanning speed and hatch spacing. The upper surface roughness is found to be almost constant under different processing parameters. The width and depth of the molten pool on powder-supported zone are larger than that of the molten pool on the solid-supported zone, while the width changes more significantly than that of depth. However, if the scanning speed is high enough, the width and depth of the molten pool and the lower surface roughness almost keep constant as the density is still high. Therefore, high dimensional accuracy and density as well as good surface quality can be achieved simultaneously by using high scanning speed during SLMed cellular lattice strut.

Investigation on Selective Laser Melting AlSi10Mg Cellular Lattice Strut: Molten Pool Morphology, Surface Roughness and Dimensional Accuracy.

Science.gov (United States)

Han, Xuesong; Zhu, Haihong; Nie, Xiaojia; Wang, Guoqing; Zeng, Xiaoyan

2018-03-07

AlSi10Mg inclined struts with angle of 45° were fabricated by selective laser melting (SLM) using different scanning speed and hatch spacing to gain insight into the evolution of the molten pool morphology, surface roughness, and dimensional accuracy. The results show that the average width and depth of the molten pool, the lower surface roughness and dimensional deviation decrease with the increase of scanning speed and hatch spacing. The upper surface roughness is found to be almost constant under different processing parameters. The width and depth of the molten pool on powder-supported zone are larger than that of the molten pool on the solid-supported zone, while the width changes more significantly than that of depth. However, if the scanning speed is high enough, the width and depth of the molten pool and the lower surface roughness almost keep constant as the density is still high. Therefore, high dimensional accuracy and density as well as good surface quality can be achieved simultaneously by using high scanning speed during SLMed cellular lattice strut.

Design parameters to control synthetic gene expression in Escherichia coli.

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Mark Welch

Full Text Available BACKGROUND: Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. PRINCIPAL FINDINGS: To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. CONCLUSION: The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.

Genes under positive selection in Escherichia coli

DEFF Research Database (Denmark)

Petersen, Lise; Bollback, Jonathan P; Dimmic, Matt

2007-01-01

We used a comparative genomics approach to identify genes that are under positive selection in six strains of Escherichia coli and Shigella flexneri, including five strains that are human pathogens. We find that positive selection targets a wide range of different functions in the E. coli genome......, including cell surface proteins such as beta barrel porins, presumably because of the involvement of these genes in evolutionary arms races with other bacteria, phages, and/or the host immune system. Structural mapping of positively selected sites on trans-membrane beta barrel porins reveals...... that the residues under positive selection occur almost exclusively in the extracellular region of the proteins that are enriched with sites known to be targets of phages, colicins, or the host immune system. More surprisingly, we also find a number of other categories of genes that show very strong evidence...

Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

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Asma Rehman

Full Text Available Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4 vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

An oral Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus delivered by Escherichia coli elicits immune responses in dogs.

Science.gov (United States)

Dahiya, S S; Saini, M; Kumar, P; Gupta, P K

2011-01-01

A Sindbis virus replicon-based DNA vaccine containing VP2 gene of canine parvovirus (CPV) was delivered by Escherichia coli to elicit immune responses. The orally immunized dogs developed CPV-specific serum IgG and virus neutralizing antibody responses. The cellular immune responses analyzed using lymphocyte proliferation test and flow cytometry indicated CPV-specific sensitization of both CD3+CD4+ and CD3+CD8+ lymphocytes. This study demonstrated that the oral CPV DNA vaccine delivered by E. coli can be considered as a promising approach for vaccination of dogs against CPV.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

Science.gov (United States)

Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Attachment of Escherichia coli and enterococci to particles in runoff.

Science.gov (United States)

Soupir, Michelle L; Mostaghimi, Saied; Dillaha, Theo

2010-01-01

Association of Escherichia coli and enterococci with particulates present in runoff from erodible soils has important implications for modeling the fate and transport of bacteria from agricultural sources and in the selection of management practices to reduce bacterial movement to surface waters. Three soils with different textures were collected from the Ap horizon (silty loam, silty clay loam, and loamy fine sand), placed in portable box plots, treated with standard cowpats, and placed under a rainfall simulator. Rainfall was applied to the plots until saturation-excess flow occurred for 30 min, and samples were collected 10, 20, and 30 min after initiation of the runoff event. The attachment of E. coli and enterococci to particles present in runoff was determined by a screen filtration and centrifugation procedure. Percentage of E. coli and enterococci attached to particulates in runoff ranged from 28 to 49%, with few statistically significant differences in attachment among the three soils. Similar partitioning release patterns were observed between E. coli and enterococci from the silty loam (r = 0.57) and silty clay loam soils (r = 0.60). At least 60% of all attached E. coli and enterococci were associated particles within an 8- to 62-microm particle size category. The results indicate that the majority of fecal bacteria attach to and are transported with manure colloids in sediment-laden flow regardless of the soil texture.

Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress.

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Grégory Francius

Full Text Available The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM and electrokinetics (electrophoresis. Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus. From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO(3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700-900 kPa and ∼100-300 kPa respectively. Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

Science.gov (United States)

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Expression of ultraviolet-induced restriction alleviation in Escherichia coli K-12

International Nuclear Information System (INIS)

Thoms, B.; Wackernagel, W.

1983-01-01

Ultraviolet-induced restriction alleviation is an SOS function which partially relieves the K-12-specific DNA restriction in Escherichia coli. Restriction alleviation is determined by observing elevated survival of unmodified phage lambda in cells irradiated with ultraviolet prior to infection. The authors demonstrate that restriction of lambda is also relieved when log-phase cells are irradiated as late as 50 min after adsorption of lambda. At this time more than 60% of the lambda DNA is already released as acid-soluble material from the cells. Experiments involving reextraction of lambda DNA from infected cells and a mild detergent treatment removing adsorbed phages from the cellular surface showed that only a small specific fraction of all lambda infections is destined to escape restriction due to restriction alleviation. This fraction (10-20%) has a retarded mode of DNA injection (60 min or longer) after adsorption which allows the expression of the restriction alleviation function before the phage DNA is exposed to restriction endonucleases. This behaviour of a fraction of lambda phages explains why the SOS function restriction alleviation could initially be discovered. The authors show that the retarded mode of DNA injection is not required for another SOS function acting on lambda DNA, the increased repair of ultraviolet-irradiated DNA (Weigle reactivation). (Auth.)

Short-Term Pretreatment of Sub-Inhibitory Concentrations of Gentamycin Inhibits the Swarming Motility of Escherichia Coli by Down-Regulating the Succinate Dehydrogenase Gene

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Yijing Zhuang

2016-09-01

Full Text Available Background/Aims: Motility is a feature of many pathogens that contributes to the migration and dispersion of the infectious agent. Whether gentamycin has a post-antibiotic effect (PAE on the swarming and swimming motility of Escherichia coli (E. coli remains unknown. In this study, we aimed to examine whether short-term pretreatment of sub-inhibitory concentrations of gentamycin alter motility of E. coli and the mechanisms involved therein. Methods: After exposure to sub-inhibitory concentrations (0.8 μg/ml of gentamicin, the swarming and swimming motility of E. coli was tested in semi-solid media. Real-time PCR was used to detect the gene expression of succinate dehydrogenase (SDH. The production of SDH and fumarate by E. coli pretreated with or without gentamycin was measured. Fumarate was added to swarming agar to determine whether fumarate could restore the swarming motility of E. coli. Results: After pretreatment of E. coli with sub-inhibitory concentrations of gentamycin, swarming motility was repressed in the absence of growth inhibition. The expression of all four subunits of SDH was down-regulated, and the intracellular concentration of SDH and fumarate, produced by E. coli, were both decreased. Supplementary fumarate could restore the swarming motility inhibited by gentamycin. A selective inhibitor of SDH (propanedioic acid could strongly repress the swarming motility. Conclusion: Sub-inhibitory concentrations of gentamycin inhibits the swarming motility of E. coli. This effect is mediated by a reduction in cellular fumarate caused by down-regulation of SDH. Gentamycin may be advantageous for treatment of E. coli infections.

Impact of O-glycosylation on the molecular and cellular adhesion properties of the Escherichia coli autotransporter protein Ag43.

Science.gov (United States)

Reidl, Sebastian; Lehmann, Annika; Schiller, Roswitha; Salam Khan, A; Dobrindt, Ulrich

2009-08-01

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (alpha(43)) stabilizes its conformation and increases adhesion to Hep-2 cells. We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the alpha(43) domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the alpha(43) domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many alpha(43) domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.

Hygienic-sanitary quality of vegetables and evaluation of treatments for the elimination of indigenous E. coli and E. coli O157:H7 from the surface of leaves of lettuce (Lactuca sativa L.

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Ytana oliveira Santos

2010-12-01

Full Text Available The purpose of this study is to evaluate the hygienic-sanitary quality of vegetables and irrigation water and assess the effectiveness of lemon juice and vinegar in reducing E. coli strains inoculated on lettuce. One hundred and forty samples of vegetables and 45 samples of irrigation water were investigated for thermotolerant coliforms and Salmonella spp. In order to verify the effectiveness of natural household sanitizers in reducing E. coli in inoculated lettuce, four treatment solutions were tested: fresh lemon juice, alcohol vinegar, lemon juice-vinegar mixture, and lemon juice-vinegar-water mixture. The microbiological analysis revealed high rates of contamination by thermotolerant coliforms and identified the presence of E. coli in 32% of the tested vegetable samples and 56% of the water samples. While no significant statistical difference (p < 0, 05 was identified in the tested solutions, the treatment with a combination of lemon juice and vinegar resulted in the highest Decimal Reductions (DR of E. coli O157: H7 while the treatment with vinegar alone was the most effective against the indigenous E. coli strain

Self-assembled monolayers-based immunosensor for detection of Escherichia coli using electrochemical impedance spectroscopy

International Nuclear Information System (INIS)

Geng Ping; Zhang Xinai; Meng Weiwei; Wang Qingjiang; Zhang Wen; Jin Litong; Feng Zhen; Wu Zirong

2008-01-01

An electrochemical impedance immunosensor for the detection of Escherichia coli was developed by immobilizing anti-E. coli antibodies at an Au electrode. The immobilization of antibodies at the Au electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the self-assembled monolayer (SAM). The surface characteristics of the immunosensor before and after the binding reaction of antibodies with E. coli were characterized by atomic force microscopy (AFM). The immobilization of antibodies and the binding of E. coli cells to the electrode could increase the electro-transfer resistance, which was directly detected by electrochemical impedance spectroscopy (EIS) in the presence of Fe(CN) 6 3- /Fe(CN) 6 4- as a redox probe. A linear relationship between the electron-transfer resistance and the logarithmic value of E. coli concentration was found in the range of E. coli cells from 3.0 x 10 3 to 3.0 x 10 7 cfu mL -1 with the detection limit of 1.0 x 10 3 cfu mL -1 . With preconcentration and pre-enrichment steps, it was possible to detect E. coli concentration as low as 50 cfu/mL in river water samples

The effect of sub-minimum inhibitory concentration of ciprofloxacin concentrations on enteroaggregative Escherichia coli and the role of the surface protein dispersin

Energy Technology Data Exchange (ETDEWEB)

Mortensen, Ninell P [ORNL; Fowlkes, Jason Davidson [ORNL; Trevino-Dopatka, Sonia [ORNL; Maggart, Michael J [ORNL; Boisen, Nadia [University of Virginia School of Medicine; Doktycz, Mitchel John [ORNL; Nataro, James [University of Virginia School of Medicine; Allison, David P [ORNL

2011-01-01

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.

Preharvest internalization of Escherichia coli O157:H7 into lettuce leaves, as affected by insect and physical damage.

Science.gov (United States)

Erickson, Marilyn C; Liao, Jean; Payton, Alison S; Riley, David G; Webb, Cathy C; Davey, Lindsey E; Kimbrel, Sophia; Ma, Li; Zhang, Guodong; Flitcroft, Ian; Doyle, Michael P; Beuchat, Larry R

2010-10-01

Environmental pests may serve as reservoirs and vectors of zoonotic pathogens to leafy greens; however, it is unknown whether insect pests feeding on plant tissues could redistribute these pathogens present on the surface of leaves to internal sites. This study sought to differentiate the degree of tissue internalization of Escherichia coli O157:H7 when applied at different populations on the surface of lettuce and spinach leaves, and to ascertain whether lettuce-infesting insects or physical injury could influence the fate of either surface or internalized populations of this enteric pathogen. No internalization of E. coli O157:H7 occurred when lettuce leaves were inoculated with 4.4 log CFU per leaf, but it did occur when inoculated with 6.4 log CFU per leaf. Internalization was statistically greater when spinach leaves were inoculated on the abaxial (underside) than when inoculated on the adaxial (topside) side, and when the enteric pathogen was spread after surface inoculation. Brief exposure (∼18 h) of lettuce leaves to insects (5 cabbage loopers, 10 thrips, or 10 aphids) prior to inoculation with E. coli O157:H7 resulted in significantly reduced internalized populations of the pathogen within these leaves after approximately 2 weeks, as compared with leaves not exposed to insects. Surface-contaminated leaves physically injured through file abrasions also had significantly reduced populations of both total and internalized E. coli O157:H7 as compared with nonabraded leaves 2 weeks after pathogen exposure.

Study on E. coli and Salmonella biofilms from fresh fruits and vegetables.

Science.gov (United States)

Amrutha, Balagopal; Sundar, Kothandapani; Shetty, Prathapkumar Halady

2017-04-01

Foodborne outbreaks associated with fresh fruits and vegetables are on the rise worldwide. Biofilm formation is one of the important traits of pathogens making them strongly attached to substrates as well as express virulence phenotypes. Present study investigates the biofilm forming ability of E. coli and Salmonella sp. isolated from fresh fruits and vegetables. A total of 53 strains, including 35 E. coli and 18 Salmonella sp. isolated from different fruit and vegetable samples were taken into account for the study. Initial screening for biofilm formation was done using Congo Red agar plate test. Results revealed that 22.8% E. coli and 22.2% Salmonella sp. were potential biofilm formers. However, the MTP (Micro-Titre Plate) assay suggested more isolates of both E. coli and Salmonella sp. were moderate to strong biofilm producers. Agar plate diffusion assay with Agrobacterium tumefaciens NTL-4 showed the production of quorum signaling molecules (AHLs) by three isolates of E. coli and one Salmonella sp. Two E. coli isolates showed a significant amount of EPS production indicating higher biofilm forming potential. The Presence of LUX R homologue gene ( sdi A) in two of the Salmonella isolates were confirmed by PCR which demonstrated their potential pathogenicity. Results of the work underline the biofilm forming and potentially virulent capacities of isolates from the surface of fruits and vegetables.

Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia.

Directory of Open Access Journals (Sweden)

Sohyun Cho

Full Text Available Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal, pathogenic, or antimicrobial resistant (AR bacteria is a major environmental and public health concern. However, very little data is available on prevalence of these bacteria in surface water throughout a watershed. This study aimed to characterize Escherichia coli present in the Upper Oconee Watershed, a mixed-use watershed in Athens, GA, USA for potential pathogenicity and AR. E. coli were enumerated by colony counts, cultured by enrichment and direct plating, and characterized by phylo-groups, diarrheagenic pathotypes, and antimicrobial susceptibility. From the analysis, 99.3% (455/458 of the total samples were positive for E. coli resulting in 496 isolates. E. coli counts were as high as 1.2×104 CFU/100 ml, which is above the United States Environmental Protection Agency (U.S. EPA threshold for recreational water (235 CFU/100 ml based on a one-time measurement. Phylo-groups B2 (31.7%; 157/496 and B1 (30.8%; 153/496 were the most prevalent among the isolates. Enteropathogenic E. coli (EPEC (19/496 and Shiga toxin-producing E. coli (STEC (1/496 were the only diarrheagenic pathotypes detected. AR was observed in 6.9% (34/496 of the isolates, 15 of which were multidrug resistant (MDR; resistance to two or more classes of antimicrobials. Tetracycline resistance was most often detected (76.5%; 26/34, followed by ampicillin (32.4%; 11/34, streptomycin (23.5%; 8/34, sulfisoxazole (23.5%; 8/34, and nalidixic acid (14.7%; 5/34. Results from this study showed that E. coli is prevalent in high levels in the Upper Oconee Watershed, suggesting possible widespread fecal contamination. The presence of pathogenic, AR E. coli in the watershed indicates that environmental water can serve as a reservoir of resistant bacteria that may be transferred to humans through drinking and recreational activities.

Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia.

Science.gov (United States)

Cho, Sohyun; Hiott, Lari M; Barrett, John B; McMillan, Elizabeth A; House, Sandra L; Humayoun, Shaheen B; Adams, Eric S; Jackson, Charlene R; Frye, Jonathan G

2018-01-01

Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal, pathogenic, or antimicrobial resistant (AR) bacteria is a major environmental and public health concern. However, very little data is available on prevalence of these bacteria in surface water throughout a watershed. This study aimed to characterize Escherichia coli present in the Upper Oconee Watershed, a mixed-use watershed in Athens, GA, USA for potential pathogenicity and AR. E. coli were enumerated by colony counts, cultured by enrichment and direct plating, and characterized by phylo-groups, diarrheagenic pathotypes, and antimicrobial susceptibility. From the analysis, 99.3% (455/458) of the total samples were positive for E. coli resulting in 496 isolates. E. coli counts were as high as 1.2×104 CFU/100 ml, which is above the United States Environmental Protection Agency (U.S. EPA) threshold for recreational water (235 CFU/100 ml based on a one-time measurement). Phylo-groups B2 (31.7%; 157/496) and B1 (30.8%; 153/496) were the most prevalent among the isolates. Enteropathogenic E. coli (EPEC) (19/496) and Shiga toxin-producing E. coli (STEC) (1/496) were the only diarrheagenic pathotypes detected. AR was observed in 6.9% (34/496) of the isolates, 15 of which were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). Tetracycline resistance was most often detected (76.5%; 26/34), followed by ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and nalidixic acid (14.7%; 5/34). Results from this study showed that E. coli is prevalent in high levels in the Upper Oconee Watershed, suggesting possible widespread fecal contamination. The presence of pathogenic, AR E. coli in the watershed indicates that environmental water can serve as a reservoir of resistant bacteria that may be transferred to humans through drinking and recreational activities.

Cellular membrane trafficking of mesoporous silica nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Fang, I-Ju [Iowa State Univ., Ames, IA (United States)

2012-01-01

This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulf some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine

Expanded flux variability analysis on metabolic network of Escherichia coli

Institute of Scientific and Technical Information of China (English)

CHEN Tong; XIE ZhengWei; OUYANG Qi

2009-01-01

Flux balance analysis,based on the mass conservation law in a cellular organism,has been extensively employed to study the interplay between structures and functions of cellular metabolic networks.Consequently,the phenotypes of the metabolism can be well elucidated.In this paper,we introduce the Expanded Flux Variability Analysis (EFVA) to characterize the intrinsic nature of metabolic reactions,such as flexibility,modularity and essentiality,by exploring the trend of the range,the maximum and the minimum flux of reactions.We took the metabolic network of Escherichia coli as an example and analyzed the variability of reaction fluxes under different growth rate constraints.The average variability of all reactions decreases dramatically when the growth rate increases.Consider the noise effect on the metabolic system,we thus argue that the microorganism may practically grow under a suboptimal state.Besides,under the EFVA framework,the reactions are easily to be grouped into catabolic and anabolic groups.And the anabolic groups can be further assigned to specific biomass constitute.We also discovered the growth rate dependent essentiality of reactions.

Differential actions of chlorhexidine on the cell wall of Bacillus subtilis and Escherichia coli.

Directory of Open Access Journals (Sweden)

Hon-Yeung Cheung

Full Text Available Chlorhexidine is a chlorinated phenolic disinfectant used commonly in mouthwash for its action against bacteria. However, a comparative study of the action of chlorhexidine on the cell morphology of gram-positive and gram-negative bacteria is lacking. In this study, the actions of chlorhexidine on the cell morphology were identified with the aids of electron microscopy. After exposure to chlorhexidine, numerous spots of indentation on the cell wall were found in both Bacillus subtilis and Escherichia coli. The number of indentation spots increased with time of incubation and increasing chlorhexidine concentration. Interestingly, the dented spots found in B. subtilis appeared mainly at the hemispherical caps of the cells, while in E. coli the dented spots were found all over the cells. After being exposed to chlorhexidine for a prolonged period, leakage of cellular contents and subsequent ghost cells were observed, especially from B subtilis. By using 2-D gel/MS-MS analysis, five proteins related to purine nucleoside interconversion and metabolism were preferentially induced in the cell wall of E. coli, while three proteins related to stress response and four others in amino acid biosynthesis were up-regulated in the cell wall materials of B. subtilis. The localized morphological damages together with the biochemical and protein analysis of the chlorhexidine-treated cells suggest that chlorhexidine may act on the differentially distributed lipids in the cell membranes/wall of B. subtilis and E. coli.

Phenotypic Profiling of Antibiotic Response Signatures in Escherichia coli Using Raman Spectroscopy

Science.gov (United States)

Athamneh, A. I. M.; Alajlouni, R. A.; Wallace, R. S.; Seleem, M. N.

2014-01-01

Identifying the mechanism of action of new potential antibiotics is a necessary but time-consuming and costly process. Phenotypic profiling has been utilized effectively to facilitate the discovery of the mechanism of action and molecular targets of uncharacterized drugs. In this research, Raman spectroscopy was used to profile the phenotypic response of Escherichia coli to applied antibiotics. The use of Raman spectroscopy is advantageous because it is noninvasive, label free, and prone to automation, and its results can be obtained in real time. In this research, E. coli cultures were subjected to three times the MICs of 15 different antibiotics (representing five functional antibiotic classes) with known mechanisms of action for 30 min before being analyzed by Raman spectroscopy (using a 532-nm excitation wavelength). The resulting Raman spectra contained sufficient biochemical information to distinguish between profiles induced by individual antibiotics belonging to the same class. The collected spectral data were used to build a discriminant analysis model that identified the effects of unknown antibiotic compounds on the phenotype of E. coli cultures. Chemometric analysis showed the ability of Raman spectroscopy to predict the functional class of an unknown antibiotic and to identify individual antibiotics that elicit similar phenotypic responses. Results of this research demonstrate the power of Raman spectroscopy as a cellular phenotypic profiling methodology and its potential impact on antibiotic drug development research. PMID:24295982

Adenylate kinase amplification of ATP bioluminescence for hygiene monitoring in the food and beverage industry.

Science.gov (United States)

Corbitt, A J; Bennion, N; Forsythe, S J

2000-06-01

Fourteen food residues, Escherichia coli O157:H7 and Staphylococcus aureus on stainless steel surfaces were detected using a combined assay with adenylate kinase as a cellular marker and ATP bioluminescence. The limit of sensitivity ranged from 0.02 to 708 microg for minced meat and broccoli, respectively. Both methods gave the same detection limit (105 cfu) for E. coli and Staph. aureus on stainless steel surfaces. The combined adenylate kinase-ATP assay is applicable to monitor the hygiene of work surfaces, especially those prone to contamination by meat and vegetable residues.

Facile synthesis of silver nanoparticles and its antibacterial activity against Escherichia coli and unknown bacteria on mobile phone touch surfaces/computer keyboards

Science.gov (United States)

Reddy, T. Ranjeth Kumar; Kim, Hyun-Joong

2016-07-01

In recent years, there has been significant interest in the development of novel metallic nanoparticles using various top-down and bottom-up synthesis techniques. Kenaf is a huge biomass product and a potential component for industrial applications. In this work, we investigated the green synthesis of silver nanoparticles (AgNPs) by using kenaf ( Hibiscus cannabinus) cellulose extract and sucrose, which act as stabilizing and reducing agents in solution. With this method, by changing the pH of the solution as a function of time, we studied the optical, morphological and antibacterial properties of the synthesized AgNPs. In addition, these nanoparticles were characterized by Ultraviolet-visible spectroscopy, transmission electron microscopy (TEM), field-emission scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy and energy-dispersive X-ray spectroscopy (EDX). As the pH of the solution varies, the surface plasmon resonance peak also varies. A fast rate of reaction at pH 10 compared with that at pH 5 was identified. TEM micrographs confirm that the shapes of the particles are spherical and polygonal. Furthermore, the average size of the nanoparticles synthesized at pH 5, pH 8 and pH 10 is 40.26, 28.57 and 24.57 nm, respectively. The structure of the synthesized AgNPs was identified as face-centered cubic (fcc) by XRD. The compositional analysis was determined by EDX. FTIR confirms that the kenaf cellulose extract and sucrose act as stabilizing and reducing agents for the silver nanoparticles. Meanwhile, these AgNPs exhibited size-dependent antibacterial activity against Escherichia coli ( E. coli) and two other unknown bacteria from mobile phone screens and computer keyboard surfaces.

Changes in the cellular energy state affect the activity of the bacterial phosphotransferase system

DEFF Research Database (Denmark)

Rohwer, J.M.; Jensen, Peter Ruhdal; Shinohara, Y.

1996-01-01

The effect of different cellular free-energy states on the uptake of methyl alfa-D-glucopyranoside, an analoque of glucose, by Escherichia coli phosphoenolpyruvate:carbohydrate phosphotransferase system was investigated. The intracellular ATP/ADP ratio was varied by changing the expression...... of the atp operon, which codes for the H+-ATPase, or by adding an uncoupler of oxidative phosphorylation or an inhibitor of respiration. Corresponding initial phosphotransferase uptake rates were determined using an improved uptake assay that works with growing cells in steady state. The results show...... that the initial uptake rate was decreased under conditions of lowered intracellular ATP/ADP ratios, irrespective of which method was used to change the cellular energy state.. When either the expression of the atp operon was changed or 2,4-dinitrophenol was added to wild-type cells, the relationship between...

Safety assessment of greenhouse hydroponic tomatoes irrigated with reclaimed and surface water.

Science.gov (United States)

Lopez-Galvez, Francisco; Allende, Ana; Pedrero-Salcedo, Francisco; Alarcon, Juan Jose; Gil, Maria Isabel

2014-11-17

The impact of reclaimed and surface water on the microbiological safety of hydroponic tomatoes was assessed. Greenhouse tomatoes were irrigated with reclaimed and surface water and grown on two hydroponic substrates (coconut fiber and rock wool). Water samples (n=208) were taken from irrigation water, with and without the addition of fertilizers and drainage water, and hydroponic tomatoes (n=72). Samples were analyzed for indicator microorganisms, generic Escherichia coli and Listeria spp., and pathogenic bacteria such as Salmonella spp. and Shiga-toxigenic E. coli (STEC), using multiplex real-time PCR (RT-PCR) after enrichment. The correlation between climatological parameters such as temperature and the levels of microorganisms in water samples was also determined. In irrigation water, generic E. coli counts were higher in reclaimed than in surface water whereas Listeria spp. numbers increased after adding the fertilizers in both water sources. In drainage water, no clear differences in E. coli and Listeria numbers were observed between reclaimed and surface water. No positive samples for STEC were found in irrigation water. Presumptive positives for Salmonella spp. were found in 7.7% of the water samples and 62.5% of these samples were reclaimed water. Salmonella-positive samples by RT-PCR could not be confirmed by conventional methods. Higher concentrations of E. coli were associated with Salmonella-presumptive positive samples. Climatological parameters, such as temperature, were not correlated with the E. coli and Listeria spp. counts. Tomato samples were negative for bacterial pathogens, while generic E. coli and Listeria spp. counts were below the detection limit. The prevalence of presumptive Salmonella spp. found in irrigation water (reclaimed and surface water) was high, which might present a risk of contamination. The absence of pathogens on greenhouse hydroponic tomatoes indicates that good agricultural practices (GAP) were in place, avoiding the

Impact of modified diamond-like carbon coatings on the spatial organization and disinfection of mixed-biofilms composed of Escherichia coli and Pantoea agglomerans industrial isolates.

Science.gov (United States)

Gomes, L C; Deschamps, J; Briandet, R; Mergulhão, F J

2018-07-20

This work investigated the effects of diamond-like carbon (DLC) coatings on the architecture and biocide reactivity of dual-species biofilms mimicking food processing contaminants. Biofilms were grown using industrial isolates of Escherichia coli and Pantoea agglomerans on bare stainless steel (SST) and on two DLC surface coatings (a-C:H:Si:O designated by SICON® and a-C:H:Si designated by SICAN) in order to evaluate their antifouling activities. Quantification and spatial organization in single- and dual-species biofilms were examined by confocal laser scanning microscopy (CLSM) using a strain specific labelling procedure. Those assays revealed that the E. coli isolate exhibited a higher adhesion to the modified surfaces and a decreased susceptibility to disinfectant in presence of P. agglomerans than alone in axenic culture. While SICON® reduced the short-term growth of E. coli in axenic conditions, both DLC surfaces increased the E. coli colonization in presence of P. agglomerans. However, both modified surfaces triggered a significantly higher log reduction of E. coli cells within mixed-species biofilms, thus the use of SICON® and SICAN surfaces may be a good approach to facilitate the disinfection process in critical areas of food processing plants. This study presents a new illustration of the importance of interspecies interactions in surface-associated community functions, and of the need to evaluate the effectiveness of hygienic strategies with relevant multi-species consortia. Copyright © 2018 Elsevier B.V. All rights reserved.

Annual Surveillance Summary: Escherichia coli (E. coli) Infections in the Military Health System (MHS), 2015

Science.gov (United States)

2017-03-01

Annual Surveillance Summary: Escherichia coli ( E . coli ) Infections in the Military Health System (MHS...or position of the Department of the Navy, Department of Defense, nor the U.S. Government. i i E . coli in the MHS: Annual Summary 2015 Prepared...March 2017 EpiData Center Department NMCPHC-EDC-TR-187-2017 ii ii E . coli in the MHS: Annual Summary 2015 Prepared March 2017 EpiData

The Effects of Sugars on the Biofilm Formation of Escherichia coli 185p on Stainless Steel and Polyethylene Terephthalate Surfaces in a Laboratory Model.

Science.gov (United States)

Khangholi, Mahdi; Jamalli, Ailar

2016-09-01

Bacteria utilize various methods in order to live in protection from adverse environmental conditions. One such method involves biofilm formation; however, this formation is dependent on many factors. The type and concentration of substances such as sugars that are present in an environment can be effective facilitators of biofilm formation. First, the physico-chemical properties of the bacteria and the target surface were studied via the MATS and contact angle measurement methods. Additionally, adhesion to different surfaces in the presence of various concentrations of sugars was compared in order to evaluate the effect of these factors on the biofilm formation of Escherichia coli , which represents a major food contaminant . Results showed that the presence of sugars has no effect on the bacterial growth rate; all three concentrations of sugars were hydrophilic and demonstrated a high affinity toward binding to the surfaces. The impact of sugars and other factors on biofilm formation can vary depending on the type of bacteria present.

Role of major surface structures of Escherichia coli O157:H7 in initial attachment to biotic and abiotic surfaces

Science.gov (United States)

Infection by human pathogens through fresh, minimally processed produce and solid plant-derived foods is a major concern of U.S. and global food industry and public health services. The enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent food borne pathogen that causes severe disease...

Protective effects of indigenous Escherichia coli against a pathogenic E. coli challenge strain in pigs.

Science.gov (United States)

Vahjen, W; Cuisiniere, T; Zentek, J

2017-10-13

To investigate the inhibitory effect of indigenous enterobacteria on pathogenic Escherichia coli, a challenge trial with postweaning pigs was conducted. A pathogenic E. coli strain was administered to all animals and their health was closely monitored thereafter. Faecal samples were taken from three healthy and three diarrhoeic animals. Samples were cultivated on MacConkey agar and isolates were subcultured. A soft agar overlay assay was used to determine the inhibitory activity of the isolates. A total of 1,173 enterobacterial isolates were screened for their ability to inhibit the E. coli challenge strain. Colony forming units of enterobacteria on MacConkey agar were not different between healthy and diarrhoeic animals in the original samples. Furthermore, numbers of isolates per animal were also not significantly different between healthy (482 isolates) and diarrhoeic animals (691 isolates). A total of 43 isolates (3.7%) with inhibitory activity against the pathogenic E. coli challenge strain were detected. All inhibitory isolates were identified as E. coli via MALDI-TOF. The isolates belonged to the phylotypes A, C and E. Many isolates (67.4%) were commensal E. coli without relevant porcine pathogenic factors, but toxin- and fimbrial genes (stx2e, fae, estIb, elt1a, fas, fan) were detected in 14 inhibitory isolates. Healthy animals showed significantly (P=0.003) more inhibitory isolates (36 of 482 isolates; 7.5%) than diseased animals (7 of 691 isolates; 1.0%). There were no significant correlations regarding phylotype or pathogenic factors between healthy and diseased animals. This study has shown that a small proportion of indigenous E. coli is able to inhibit in vitro growth of a pathogenic E. coli strain in pigs. Furthermore, healthy animals possess significantly more inhibitory E. coli strains than diarrhoeic animals. The inhibition of pathogenic E. coli by specific indigenous E. coli strains may be an underlying principle for the containment of pathogenic

Shiga toxin-producing Escherichia coli (STEC) O22:H8 isolated from cattle reduces E. coli O157:H7 adherence in vitro and in vivo.

Science.gov (United States)

Martorelli, L; Albanese, A; Vilte, D; Cantet, R; Bentancor, A; Zolezzi, G; Chinen, I; Ibarra, C; Rivas, M; Mercado, E C; Cataldi, A

2017-09-01

Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. Our group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. The three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpf O113 . E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 10 8 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. These results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Enterohemorrhagic Escherichia coli (EHEC

Directory of Open Access Journals (Sweden)

Abdullah Kilic

2011-08-01

Full Text Available Escherichia coli is a bacterium that is commonly found in the gut of humans and warm-blooded animals. Most strains of E. coli are harmless for human. E. coli O157:H7 is the most common member of a group of pathogenic E. coli strains known variously as enterohaemorrhagic, verocytotoxin-producing, or Shiga-toxin-producing organisms. EHEC bacterium is the major cause of haemorrhagic colitis and haemolytic uraemic syndrome. The reservoir of this pathogen appears to be mainly cattle and other ruminants such as camels. It is transmitted to humans primarily through consumption of contaminated foods. [TAF Prev Med Bull 2011; 10(4.000: 387-388

Impact of dry chilling on the genetic diversity of Escherichia coli on beef carcasses and on the survival of E. coli and E. coli O157.

Science.gov (United States)

Visvalingam, Jeyachchandran; Liu, Yang; Yang, Xianqin

2017-03-06

The objective of this study was to examine the effect of dry chilling on the genetic diversity of naturally occurring Escherichia coli on beef carcasses, and to examine whether two populations of E. coli recovered from carcasses during chilling and E. coli O157 differed in their response to desiccation. Isolates of E. coli were obtained from beef carcasses during a 67h dry chilling process and were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA). Ten E. coli genotypes found only at 0h (group A) and found more than once (group B), as well as five strains of E. coli O157 (group C) were inoculated on stainless steel coupons and their survival was examined after exposure to 75 and 100% relative humidity (RH) at 0 or 35°C for 67h. A total of 450 E. coli isolates were obtained, with 254, 49, 49, 51, 23, 20, and 4 from 0, 1, 2, 4, 6, 8 and 24h of chilling, respectively. No E. coli were recovered at 67h. MLVA of the isolates revealed 173 distinct genotypes. Genetic diversity of E. coli isolates, defined as ratio of the number of isolates to the number of genotypes, remained between 2.3 and 1.3 during the 24h of chilling. All strains inoculated on stainless steel coupons and exposed to 75% RH at 35°C were completely inactivated, irrespective of their groups. Inactivation of E. coli of the three groups was not significantly (P>0.05) different by exposure to 75% RH at 0°C. The findings indicate that the genetic diversity of E. coli on beef carcasses was not affected by dry chilling. In addition, inactivation of E. coli genotypes and E. coli O157 by desiccation on stainless steel simulating dry chilling conditions did not differ significantly (P>0.05). Thus, dry chilling may be used as an effective antimicrobial intervention for beef carcasses. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Alkaline phosphatase labeled SERS active sandwich immunoassay for detection of Escherichia coli

Science.gov (United States)

Bozkurt, Akif Goktug; Buyukgoz, Guluzar Gorkem; Soforoglu, Mehmet; Tamer, Ugur; Suludere, Zekiye; Boyaci, Ismail Hakki

2018-04-01

In this study, a sandwich immunoassay method utilizing enzymatic activity of alkaline phosphatase (ALP) on 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for Escherichia coli (E. coli) detection was developed using surface enhanced Raman spectroscopy (SERS). For this purpose, spherical magnetic gold coated core-shell nanoparticles (MNPs-Au) and rod shape gold nanoparticles (Au-NRs) were synthesized and modified for immunomagnetic separation (IMS) of E. coli from the solution. In order to specify the developed method to ALP activity, Au-NRs were labeled with this enzyme. After successful construction of the immunoassay, BCIP substrate was added to produce the SERS-active product; 5-bromo-4-chloro-3-indole (BCI). A good linearity (R2 = 0.992) was established between the specific SERS intensity of BCI at 600 cm- 1 and logarithmic E. coli concentration in the range of 1.7 × 101-1.7 × 106 cfu mL- 1. LOD and LOQ values were also calculated and found to be 10 cfu mL- 1 and 30 cfu mL- 1, respectively.

YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop.

Science.gov (United States)

Tapader, Rima; Bose, Dipro; Pal, Amit

2017-04-01

YghJ, also known as SslE (Secreted and surface associated lipoprotein) is a cell surface associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the gene is known to be conserved among both pathogenic and commensal Escherichia coli isolates, the expression and secretion of YghJ was found to be higher among diverse E. coli pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and hence facilitates colonization of these enteric pathogens to intestinal epithelial cells. Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study, we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue damage in mouse ileum and is also able to induce significant fluid accumulation in a dose dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal pathogenic E. coli. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Development of a Portable SPR Bioanalyzer for Sensitive Detection of Escherichia coli O157:H7

Directory of Open Access Journals (Sweden)

Shun Wang

2016-11-01

Full Text Available The purpose of this study was to develop a portable surface plasmon resonance (SPR bioanalyzer for the sensitive detection of Escherichia coli O157:H7 in comparison with an enzyme-linked immunosorbent assay (ELISA. The experimental setup mainly consisted of an integrated biosensor and a homemade microfluidic cell with a three-way solenoid valve. In order to detect Escherichia coli O157:H7 using the SPR immunoassay, 3-mercaptopropionic acid (3-MPA was chemisorbed onto a gold surface via covalent bond for the immobilization of biological species. 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC and N-hydroxysuccinimide (NHS were used as crosslinker reagents to enable the reaction between 3-MPA and Escherichia coli O157:H7 antibodies by covalent –CO–NH– amide bonding. The experimental results were obtained from the Escherichia coli O157:H7 positive samples prepared by 10-, 20-, 40-, 80-, and 160-fold dilution respectively, which show that a good linear relationship with the correlation coefficient R of 0.982 existed between the response units from the portable SPR bioanalyzer and the concentration of Escherichia coli O157:H7 positive samples. Moreover, the theoretical detection limit of 1.87 × 103 cfu/mL was calculated from the positive control samples. Compared with the Escherichia coli O157:H7 ELISA kit, the sensitivity of this portable SPR bioanalyzer is four orders of magnitude higher than the ELISA kit. The results demonstrate that the portable SPR bioanalyzer could provide an alternative method for the quantitative and sensitive determination of Escherichia coli O157:H7 in field.

Response of Escherichia coli to Prolonged Berberine Exposure.

Science.gov (United States)

Budeyri Gokgoz, Nilay; Avci, Fatma Gizem; Yoneten, Kubra Karaosmanoglu; Alaybeyoglu, Begum; Ozkirimli, Elif; Sayar, Nihat Alpagu; Kazan, Dilek; Sariyar Akbulut, Berna

2017-07-01

Berberine is a plant-derived alkaloid possessing antimicrobial activity; unfortunately, its efflux through multidrug resistance pumps reduces its efficacy. Cellular life span of Escherichia coli is generally shorter with prolonged berberine exposure; nevertheless, about 30% of the cells still remain robust following this treatment. To elucidate its mechanism of action and to identify proteins that could be involved in development of antimicrobial resistance, protein profiles of E. coli cells treated with berberine for 4.5 and 8 hours were compared with control cells. A total of 42 proteins were differentially expressed in cells treated with berberine for 8 hours when compared to control cells. In both 4.5 and 8 hours of berberine-treated cells, carbohydrate and peptide uptake regimens remained unchanged, although amino acid maintenance regimen switched from transport to synthesis. Defect in cell division persisted and this condition was confirmed by images obtained from scanning electron microscopy. Universal stress proteins were not involved in stress response. The significant increase in the abundance of elongation factors could suggest the involvement of these proteins in protection by exhibiting chaperone activities. Furthermore, the involvement of the outer membrane protein OmpW could receive special attention as a protein involved in response to antimicrobial agents, since the expression of only this porin protein was upregulated after 8 hours of exposure.

Silver-Palladium Surfaces Inhibit Biofilm Formation

DEFF Research Database (Denmark)

Chiang, Wen-Chi; Schroll, Casper; Hilbert, Lisbeth Rischel

2009-01-01

Undesired biofilm formation is a major concern in many areas. In the present study, we investigated biofilm-inhibiting properties of a silver-palladium surface that kills bacteria by generating microelectric fields and electrochemical redox processes. For evaluation of the biofilm inhibition...... efficacy and study of the biofilm inhibition mechanism, the silver-sensitive Escherichia coli J53 and the silver-resistant E. coli J53[pMG101] strains were used as model organisms, and batch and flow chamber setups were used as model systems. In the case of the silver-sensitive strain, the silver......-palladium surfaces killed the bacteria and prevented biofilm formation under conditions of low or high bacterial load. In the case of the silver-resistant strain, the silver-palladium surfaces killed surface-associated bacteria and prevented biofilm formation under conditions of low bacterial load, whereas under...

Exposure to the proton scavenger glycine under alkaline conditions induces Escherichia coli viability loss.

Directory of Open Access Journals (Sweden)

Donna Vanhauteghem

Full Text Available Our previous work described a clear loss of Escherichia coli (E. coli membrane integrity after incubation with glycine or its N-methylated derivatives N-methylglycine (sarcosine and N,N-dimethylglycine (DMG, but not N,N,N-trimethylglycine (betaine, under alkaline stress conditions. The current study offers a thorough viability analysis, based on a combination of real-time physiological techniques, of E. coli exposed to glycine and its N-methylated derivatives at alkaline pH. Flow cytometry was applied to assess various physiological parameters such as membrane permeability, esterase activity, respiratory activity and membrane potential. ATP and inorganic phosphate concentrations were also determined. Membrane damage was confirmed through the measurement of nucleic acid leakage. Results further showed no loss of esterase or respiratory activity, while an instant and significant decrease in the ATP concentration occurred upon exposure to either glycine, sarcosine or DMG, but not betaine. There was a clear membrane hyperpolarization as well as a significant increase in cellular inorganic phosphate concentration. Based on these results, we suggest that the inability to sustain an adequate level of ATP combined with a decrease in membrane functionality leads to the loss of bacterial viability when exposed to the proton scavengers glycine, sarcosine and DMG at alkaline pH.

Exposure to the Proton Scavenger Glycine under Alkaline Conditions Induces Escherichia coli Viability Loss

Science.gov (United States)

Vanhauteghem, Donna; Janssens, Geert Paul Jules; Lauwaerts, Angelo; Sys, Stanislas; Boyen, Filip; Cox, Eric; Meyer, Evelyne

2013-01-01

Our previous work described a clear loss of Escherichia coli (E. coli) membrane integrity after incubation with glycine or its N-methylated derivatives N-methylglycine (sarcosine) and N,N-dimethylglycine (DMG), but not N,N,N-trimethylglycine (betaine), under alkaline stress conditions. The current study offers a thorough viability analysis, based on a combination of real-time physiological techniques, of E. coli exposed to glycine and its N-methylated derivatives at alkaline pH. Flow cytometry was applied to assess various physiological parameters such as membrane permeability, esterase activity, respiratory activity and membrane potential. ATP and inorganic phosphate concentrations were also determined. Membrane damage was confirmed through the measurement of nucleic acid leakage. Results further showed no loss of esterase or respiratory activity, while an instant and significant decrease in the ATP concentration occurred upon exposure to either glycine, sarcosine or DMG, but not betaine. There was a clear membrane hyperpolarization as well as a significant increase in cellular inorganic phosphate concentration. Based on these results, we suggest that the inability to sustain an adequate level of ATP combined with a decrease in membrane functionality leads to the loss of bacterial viability when exposed to the proton scavengers glycine, sarcosine and DMG at alkaline pH. PMID:23544135

Studying of cellular interaction of hairpin-like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds with plant pathogenic fungus Fusarium solani using microscopy techniques.

Science.gov (United States)

Vasilchenko, Alexey S; Yuryev, Mikhail; Ryazantsev, Dmitry Yu; Zavriev, Sergey K; Feofanov, Alexey V; Grishin, Eugene V; Rogozhin, Eugene A

2016-11-01

An interaction of recombinant hairpin-like cationic peptide EcAMP1 with conidia of plant pathogenic fungus Fusarium solani at the cellular level was studied by a combination of microscopic methods. EcAMP1 is from barnyard grass (Echinochloa crusgalli L.), and obtained by heterologous expression in Escherichia coli system. As a result, a direct relationship between hyphal growth inhibition and increasing active peptide concentration, time of incubation and fungal physiological condition has been determined. Dynamics of accumulation and redistribution of the peptide studied on fungal cellular cover and inside the conidia cells has been shown. The dynamics are dependent on time of coupling, as well as, a dissimilarity of EcAMP1 binding with cover of fungal conidia and its stepwise accumulation and diffuse localization in the cytoplasm. Correlation between structural disruption of fungal conidia and the presence of morphological changes has also been found. The correlation was found under the influence of peptide high concentrations at concentrations above 32 μM. The results indicate the presence of a binding of EcAMP1 with the surface of fungal conidia, thus, demonstrating a main specificity for its antifungal action at the cellular level. These results, however, cannot exclude the existence of attendant EcAMP1 action based on its intracellular localization on some specific targets. SCANNING 38:591-598, 2016. © 2016 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

A yigP mutant strain is a small colony variant of E. coli and shows pleiotropic antibiotic resistance.

Science.gov (United States)

Xia, Hui; Tang, Qiongwei; Song, Jie; Ye, Jiang; Wu, Haizhen; Zhang, Huizhan

2017-12-01

Small colony variants (SCVs) are a commonly observed subpopulation of bacteria that have a small colony size and distinctive biochemical characteristics. SCVs are more resistant than the wild type to some antibiotics and usually cause persistent infections in the clinic. SCV studies have been very active during the past 2 decades, especially Staphylococcus aureus SCVs. However, fewer studies on Escherichia coli SCVs exist, so we studied an E. coli SCV during an experiment involving the deletion of the yigP locus. PCR and DNA sequencing revealed that the SCV was attributable to a defect in the yigP function. Furthermore, we investigated the antibiotic resistance profile of the E. coli SCV and it showed increased erythromycin, kanamycin, and d-cycloserine resistance, but collateral sensitivity to ampicillin, polymyxin, chloramphenicol, tetracycline, rifampin, and nalidixic acid. We tried to determine the association between yigP and the pleiotropic antibiotic resistance of the SCV by analyzing biofilm formation, cellular morphology, and coenzyme Q (Q 8 ) production. Our results indicated that impaired Q 8 biosynthesis was the primary factor that contributed to the increased resistance and collateral sensitivity of the SCV. This study offers a novel genetic basis for E. coli SCVs and an insight into the development of alternative antimicrobial strategies for clinical therapy.

Utilization of evolutionary model, bioinformatics and heuristics for development of a multiplex Escherichia coli O157:H7 PCR assay

Science.gov (United States)

Introduction: Escherichia coli O157:H7 is a devastating foodborne pathogen causing many foodborne outbreaks worldwide with significant morbidity and mortality. The plasticity of the E. coli O157:H7 genome, inconsistent expression of surface antigens, and sharing of genetic elements with other non-...

Cell-wall recycling and synthesis in Escherichia coli and Pseudomonas aeruginosa - their role in the development of resistance.

Science.gov (United States)

Dhar, Supurna; Kumari, Hansi; Balasubramanian, Deepak; Mathee, Kalai

2018-01-01

The bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria. Cell-wall synthesis and recycling are critical cellular processes essential for cell growth, elongation and division. Both de novo synthesis and recycling involve an array of enzymes across all cellular compartments, namely the outer membrane, periplasm, inner membrane and cytoplasm. Due to the exclusivity of peptidoglycan in the bacterial cell-wall, these players are the target of choice for many antibacterial agents. Our current understanding of cell-wall biochemistry and biogenesis in Gram-negative organisms stems mostly from studies of Escherichia coli. An incomplete knowledge on these processes exists for the opportunistic Gram-negative pathogen, Pseudomonas aeruginosa. In this review, cell-wall synthesis and recycling in the various cellular compartments are compared and contrasted between E. coli and P. aeruginosa. Despite the fact that there is a remarkable similarity of these processes between the two bacterial species, crucial differences alter their resistance to β-lactams, fluoroquinolones and aminoglycosides. One of the common mediators underlying resistance is the amp system whose mechanism of action is closely associated with the cell-wall recycling pathway. The activation of amp genes results in expression of AmpC β-lactamase through its cognate regulator AmpR which further regulates multi-drug resistance. In addition, other cell-wall recycling enzymes also contribute to antibiotic resistance. This comprehensive summary of the information should spawn new ideas on how to effectively target cell-wall processes to combat the growing resistance to existing antibiotics.

Localized surface plasmon enhanced cellular imaging using random metallic structures

Science.gov (United States)

Son, Taehwang; Lee, Wonju; Kim, Donghyun

2017-02-01

We have studied fluorescence cellular imaging with randomly distributed localized near-field induced by silver nano-islands. For the fabrication of nano-islands, a 10-nm silver thin film evaporated on a BK7 glass substrate with an adhesion layer of 2-nm thick chromium. Micrometer sized silver square pattern was defined using e-beam lithography and then the film was annealed at 200°C. Raw images were restored using electric field distribution produced on the surface of random nano-islands. Nano-islands were modeled from SEM images. 488-nm p-polarized light source was set to be incident at 60°. Simulation results show that localized electric fields were created among nano-islands and that their average size was found to be 135 nm. The feasibility was tested using conventional total internal reflection fluorescence microscopy while the angle of incidence was adjusted to maximize field enhancement. Mouse microphage cells were cultured on nano-islands, and actin filaments were selectively stained with FITC-conjugated phalloidin. Acquired images were deconvolved based on linear imaging theory, in which molecular distribution was sampled by randomly distributed localized near-field and blurred by point spread function of far-field optics. The optimum fluorophore distribution was probabilistically estimated by repetitively matching a raw image. The deconvolved images are estimated to have a resolution in the range of 100-150 nm largely determined by the size of localized near-fields. We also discuss and compare the results with images acquired with periodic nano-aperture arrays in various optical configurations to excite localized plasmonic fields and to produce super-resolved molecular images.

The absence of the luxS gene increases swimming motility and flagella synthesis in Escherichia coli K12

International Nuclear Information System (INIS)

Ling, Hua; Kang, Aram; Tan, Mui Hua; Qi, Xiaobao; Chang, Matthew Wook

2010-01-01

Research highlights: → This paper provides the first evidence that luxS deletion enhances swimming motility and flagella synthesis in Escherichia coli K12 based on motility, transcriptome, and scanning electron microscopy analyses. → A conceptual genetic regulatory network underlying the increased flagella synthesis was constructed based on the transcriptome and network component analyses, and previously known regulatory relations. → The genetic regulatory network suggests that the increased flagella synthesis and motility might be contributed to by increased flhDC transcription level and/or decreased c-di-GMP concentration in luxS-deficient E. coli. -- Abstract: Despite the significant role of S-ribosylhomocysteinase (LuxS) in the activated methyl cycle pathway and quorum sensing, the connectivity between luxS and other cellular functions remains incomplete. Herein, we show that luxS deletion significantly increases swimming motility and flagella synthesis in Escherichia coli K12 using motility, transcriptome, and scanning electron microscopy assays. Further, based on the transcriptome and network component analyses, and known regulatory relations, we propose a conceptual genetic regulatory network underlying the increased flagella synthesis in response to luxS deletion.

The absence of the luxS gene increases swimming motility and flagella synthesis in Escherichia coli K12

Energy Technology Data Exchange (ETDEWEB)

Ling, Hua; Kang, Aram; Tan, Mui Hua; Qi, Xiaobao [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459 (Singapore); Chang, Matthew Wook, E-mail: Matthewchang@ntu.edu.sg [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459 (Singapore)

2010-10-29

Research highlights: {yields} This paper provides the first evidence that luxS deletion enhances swimming motility and flagella synthesis in Escherichia coli K12 based on motility, transcriptome, and scanning electron microscopy analyses. {yields} A conceptual genetic regulatory network underlying the increased flagella synthesis was constructed based on the transcriptome and network component analyses, and previously known regulatory relations. {yields} The genetic regulatory network suggests that the increased flagella synthesis and motility might be contributed to by increased flhDC transcription level and/or decreased c-di-GMP concentration in luxS-deficient E. coli. -- Abstract: Despite the significant role of S-ribosylhomocysteinase (LuxS) in the activated methyl cycle pathway and quorum sensing, the connectivity between luxS and other cellular functions remains incomplete. Herein, we show that luxS deletion significantly increases swimming motility and flagella synthesis in Escherichia coli K12 using motility, transcriptome, and scanning electron microscopy assays. Further, based on the transcriptome and network component analyses, and known regulatory relations, we propose a conceptual genetic regulatory network underlying the increased flagella synthesis in response to luxS deletion.

Hierarchical structure and modules in the Escherichia coli transcriptional regulatory network revealed by a new top-down approach

Directory of Open Access Journals (Sweden)

Buer Jan

2004-12-01

Full Text Available Abstract Background Cellular functions are coordinately carried out by groups of genes forming functional modules. Identifying such modules in the transcriptional regulatory network (TRN of organisms is important for understanding the structure and function of these fundamental cellular networks and essential for the emerging modular biology. So far, the global connectivity structure of TRN has not been well studied and consequently not applied for the identification of functional modules. Moreover, network motifs such as feed forward loop are recently proposed to be basic building blocks of TRN. However, their relationship to functional modules is not clear. Results In this work we proposed a top-down approach to identify modules in the TRN of E. coli. By studying the global connectivity structure of the regulatory network, we first revealed a five-layer hierarchical structure in which all the regulatory relationships are downward. Based on this regulatory hierarchy, we developed a new method to decompose the regulatory network into functional modules and to identify global regulators governing multiple modules. As a result, 10 global regulators and 39 modules were identified and shown to have well defined functions. We then investigated the distribution and composition of the two basic network motifs (feed forward loop and bi-fan motif in the hierarchical structure of TRN. We found that most of these network motifs include global regulators, indicating that these motifs are not basic building blocks of modules since modules should not contain global regulators. Conclusion The transcriptional regulatory network of E. coli possesses a multi-layer hierarchical modular structure without feedback regulation at transcription level. This hierarchical structure builds the basis for a new and simple decomposition method which is suitable for the identification of functional modules and global regulators in the transcriptional regulatory network of E

Succinic acid production from xylose mother liquor by recombinant Escherichia coli strain.

Science.gov (United States)

Wang, Honghui; Pan, Jiachuan; Wang, Jing; Wang, Nan; Zhang, Jie; Li, Qiang; Wang, Dan; Zhou, Xiaohua

2014-11-02

Succinic acid (1,4-butanedioic acid) is identified as one of important building-block chemicals. Xylose mother liquor is an abundant industrial residue in xylitol biorefining industry. In this study, xylose mother liquor was utilized to produce succinic acid by recombinant Escherichia coli strain SD121, and the response surface methodology was used to optimize the fermentation media. The optimal conditions of succinic acid fermentation were as follows: 82.62 g L -1 total initial sugars, 42.27 g L -1 MgCO 3 and 17.84 g L -1 yeast extract. The maximum production of succinic acid was 52.09 ± 0.21 g L -1 after 84 h with a yield of 0.63 ± 0.03 g g -1 total sugar, approaching the predicted value (53.18 g L -1 ). It was 1.78-fold of the production of that obtained with the basic medium. This was the first report on succinic acid production from xylose mother liquor by recombinant E. coli strains with media optimization using response surface methodology. This work suggested that the xylose mother liquor could be an alternative substrate for the economical production of succinic acid by recombinant E. coli strains.

Is epidermal growth factor involved in development of duodenal polyps in familial polyposis coli?

DEFF Research Database (Denmark)

Poulsen, Steen Seier

1988-01-01

Duodenal adenomas are a frequent extracolonic manifestation in patients with familial polyposis coli (FPC). Epidermal growth factor (EGF), a polypeptide that stimulates cellular growth and differentiation, is localized in Paneth cells in the small intestine. In two patients with FPC, we found EGF...... immunoreactivity in duodenal adenomas. Numerous EGF immunoreactive Paneth cells were localized, not as usually, in the bottom of the crypts, but scattered along the crypts alone or in clusters. We do not know whether EGF is involved in the development of duodenal polyps in FPC patients, or whether the present...

The survival of Escherichia coli O157:H7 in the presence of Penicillium expansum and Glomerella cingulata in wounds on apple surfaces.

Science.gov (United States)

Riordan, D C; Sapers, G M; Annous, B A

2000-12-01

The survival of Escherichia coli O157:H7 in the presence of one of two plant pathogens, Penicillium expansum and Glomerella cingulata, in wounds on apples was observed during 14 days storage at room temperature (RT) and at 4 degrees C. The aim of this work was to determine if changes in apple physiology caused by the proliferation of fungal decay organisms would foster the survival of E. coli O157:H7. Trials were performed where (A) plant pathogens (4 log10 spores) were added to apple wounds 4 days before the wounds were inoculated with E. coli O157:H7 (3 log10 CFU g(-1) apple) (both RT and 4 degrees C storage), (B) plant pathogens and E. coli O157:H7 were added on the same day (both RT and 4 degrees C storage), and (C) E. coli O157:H7 was added 2 days (RT storage) and 4 days (4 degrees C storage) before plant pathogens. In all trials E. coli O157:H7 levels generally declined to cingulata at RT E. coli O157:H7 numbers increased from 3.18 to 4.03 log10 CFU g(-1) in the apple wound during trial A, from 3.26 to 6.31 log10 CFU g(-1) during trial B, and from 3.22 to 6.81 log10 CFU g(-1) during trial C. This effect is probably a consequence of the attendant rise in pH from 4.1 to approximately 6.8, observed with the proliferation of G. cingulata rot. Control apples (inoculated with E. coli O157:H7 only) were contaminated with opportunistic decay organisms at RT during trials A and B, leading to E. coli O157:H7 death. However, E. coli O157:H7 in control apples in trial C, where no contamination occurred, increased from 3.22 to 5.97 log10 CFU g(-1). The fact that E. coli O157:H7 can proliferate in areas of decay and/or injury on fruit highlights the hazards associated with the use of such fruit in the production of unpasteurized juice.

Analyses of Biofilm on Implant Abutment Surfaces Coating with Diamond-Like Carbon and Biocompatibility.

Science.gov (United States)

Huacho, Patricia Milagros Maquera; Nogueira, Marianne N Marques; Basso, Fernanda G; Jafelicci Junior, Miguel; Francisconi, Renata S; Spolidorio, Denise M P

2017-01-01

The aim of this study was to evaluate the surface free energy (SFE), wetting and surface properties as well as antimicrobial, adhesion and biocompatibility properties of diamond-like carbon (DLC)-coated surfaces. In addition, the leakage of Escherichia coli through the abutment-dental implant interface was also calculated. SFE was calculated from contact angle values; R a was measured before and after DLC coating. Antimicrobial and adhesion properties against E. coli and cytotoxicity of DLC with human keratinocytes (HaCaT) were evaluated. Further, the ability of DLC-coated surfaces to prevent the migration of E. coli into the external hexagonal implant interface was also evaluated. A sterile technique was used for the semi-quantitative polymerase chain reaction (semi-quantitative PCR). The surfaces showed slight decreases in cell viability (p0.05). It was concluded that DLC was shown to be a biocompatible material with mild cytotoxicity that did not show changes in R a, SFE, bacterial adhesion or antimicrobial properties and did not inhibit the infiltration of E. coli into the abutment-dental implant interface.

Pilot study of whole-blood gamma interferon response to the Vibrio cholerae toxin B subunit and resistance to enterotoxigenic Escherichia coli-associated diarrhea.

Science.gov (United States)

Flores, Jose; DuPont, Herbert L; Paredes-Paredes, Mercedes; Aguirre-Garcia, M Magdalena; Rojas, Araceli; Gonzalez, Alexei; Okhuysen, Pablo C

2010-05-01

Enterotoxigenic Escherichia coli (ETEC), which produces heat-labile toxin (LT), is a common cause of travelers' diarrhea (TD). The B subunit of ETEC LT is immunologically related to the B subunit of Vibrio cholerae toxin (CT). In this pilot study we evaluated the whole-blood gamma interferon response to CT B in 17 U.S. adults traveling to Mexico. Only one of nine subjects who demonstrated a cellular immune response as determined by whole-blood gamma interferon production to CT B on arrival to Mexico developed diarrhea, whereas five of eight without a cellular response developed diarrhea. Markers of the cellular immune response to ETEC LT could help in identifying individuals immune to ETEC LT, and these markers deserve additional study.

Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells

International Nuclear Information System (INIS)

Hossain, Sk Tofajjen; Mukherjee, Samir Kumar

2013-01-01

Highlights: • Toxic effect of CdS NPs on the growth and cell division in E. coli was studied. • CdS NPs affected cell surface topology and cell division. • Downregulation of both FtsZ and FtsQ was observed due to NPs exposure. • CdS NPs affected HeLa cell morphology with fragmented nuclei. • All such effects might be due to elevated oxidative stress. -- Abstract: The present study endeavours to assess the toxic effect of synthesized CdS nanoparticles (NPs) on Escherichia coli and HeLa cells. The CdS NPs were characterized by DLS, XRD, TEM and AFM studies and the average size of NPs was revealed as ∼3 nm. On CdS NPs exposure bacterial cells changed morphological features to filamentous form and damage of the cell surface was found by AFM study. The expression of two conserved cell division components namely ftsZ and ftsQ in E. coli was decreased both at transcriptional and translational levels upon CdS NPs exposure. CdS NPs inhibited proper cell septum formation without affecting the nucleoid segregation. Viability of HeLa cells declined with increasing concentration of CdS NPs and the IC 50 value was found to be 4 μg/mL. NPs treated HeLa cells showed changed morphology with condensed and fragmented nuclei. Increased level of reactive oxygen species (ROS) was found both in E. coli and HeLa cells on CdS NPs exposure. The inverse correlation between declined cell viabilities and elevated ROS level suggested that oxidative stress seems to be the key event by which NPs induce toxicity both in E. coli and HeLa cells

Cellular immobilization within microfluidic microenvironments: dielectrophoresis with polyelectrolyte multilayers.

Science.gov (United States)

Forry, Samuel P; Reyes, Darwin R; Gaitan, Michael; Locascio, Laurie E

2006-10-25

The development of biomimetic microenvironments will improve cell culture techniques by enabling in vitro cell cultures that mimic in vivo behavior; however, experimental control over attachment, cellular position, or intercellular distances within such microenvironments remains challenging. We report here the rapid and controllable immobilization of suspended mammalian cells within microfabricated environments using a combination of electronic (dielectrophoresis, DEP) and chemical (polyelectrolyte multilayers, PEMS) forces. While cellular position within the microsystem is rapidly patterned via intermittent DEP trapping, persistent adhesion after removal of electronic forces is enabled by surface treatment with PEMS that are amenable to cellular attachment. In contrast to DEP trapping alone, persistent adhesion enables the soluble microenvironment to be systematically varied, facilitating the use of soluble probes of cell state and enabling cellular characterization in response to various soluble stimuli.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

Science.gov (United States)

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

Science.gov (United States)

Polkinghorne, Adam; Vaughan, Lloyd

2011-01-01

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth. Copyright © 2010 Elsevier Ltd. All rights reserved.

Reducing Escherichia coli growth on a composite biomaterial by a surface immobilized antimicrobial peptide

Energy Technology Data Exchange (ETDEWEB)

Buckholtz, Gavin A.; Reger, Nina A. [Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 (United States); Anderton, William D.; Schimoler, Patrick J. [Orthopaedic Biomechanics Research Laboratory, Allegheny General Hospital, Pittsburgh, PA 15212 (United States); Roudebush, Shana L.; Meng, Wilson S. [Division of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282 (United States); Miller, Mark C. [Orthopaedic Biomechanics Research Laboratory, Allegheny General Hospital, Pittsburgh, PA 15212 (United States); Gawalt, Ellen S., E-mail: gawalte@duq.edu [Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219 (United States)

2016-08-01

A new composite bioceramic consisting of calcium aluminum oxide (CaAlO) and hydroxyapatite (HA) was functionalized with the synthetic antimicrobial peptide Inverso-CysHHC10. CaAlO is a bioceramic that can be mold cast easily and quickly at room temperature. Improved functionality was previously achieved through surface reactions. Here, composites containing 0–5% HA (by mass) were prepared and the elastic modulus and modulus of rupture were mechanically similar to non-load bearing bone. The addition of hydroxyapatite resulted in increased osteoblast attachment (> 180%) and proliferation (> 140%) on all composites compared to 100% CaAlO. Antimicrobial peptide (AMP) immobilization was achieved using an interfacial alkene-thiol click reaction. The linked AMP persisted on the composite (> 99.6% after 24 h) and retained its activity against Escherichia coli based on N-phenylnaphthylamine uptake and bacterial turbidity tests. Overall, this simple scaffold system improves osteoblast activity and reduces bacterial activity. - Highlights: • Calcium aluminum oxide and hydroxyapatite were cast into a composite material. • Osteoblast attachment and proliferation were significantly increased on composites. • An active antimicrobial peptide was linked to and remained stable on the composite. • Bacterial turbidity and NPN uptake tests showed modified composites had an effect equal to a 10 μM Inverso-CysHHC10 solution. • Antimicrobial peptide linkage did not affect the increased osteoblast performance.

Reducing Escherichia coli growth on a composite biomaterial by a surface immobilized antimicrobial peptide

International Nuclear Information System (INIS)

Buckholtz, Gavin A.; Reger, Nina A.; Anderton, William D.; Schimoler, Patrick J.; Roudebush, Shana L.; Meng, Wilson S.; Miller, Mark C.; Gawalt, Ellen S.

2016-01-01

A new composite bioceramic consisting of calcium aluminum oxide (CaAlO) and hydroxyapatite (HA) was functionalized with the synthetic antimicrobial peptide Inverso-CysHHC10. CaAlO is a bioceramic that can be mold cast easily and quickly at room temperature. Improved functionality was previously achieved through surface reactions. Here, composites containing 0–5% HA (by mass) were prepared and the elastic modulus and modulus of rupture were mechanically similar to non-load bearing bone. The addition of hydroxyapatite resulted in increased osteoblast attachment (> 180%) and proliferation (> 140%) on all composites compared to 100% CaAlO. Antimicrobial peptide (AMP) immobilization was achieved using an interfacial alkene-thiol click reaction. The linked AMP persisted on the composite (> 99.6% after 24 h) and retained its activity against Escherichia coli based on N-phenylnaphthylamine uptake and bacterial turbidity tests. Overall, this simple scaffold system improves osteoblast activity and reduces bacterial activity. - Highlights: • Calcium aluminum oxide and hydroxyapatite were cast into a composite material. • Osteoblast attachment and proliferation were significantly increased on composites. • An active antimicrobial peptide was linked to and remained stable on the composite. • Bacterial turbidity and NPN uptake tests showed modified composites had an effect equal to a 10 μM Inverso-CysHHC10 solution. • Antimicrobial peptide linkage did not affect the increased osteoblast performance.

Influence of culture conditions on Escherichia coli O157:H7 biofilm formation by atomic force microscopy

International Nuclear Information System (INIS)

Oh, Y.J.; Jo, W.; Yang, Y.; Park, S.

2007-01-01

Biofilms are complex microbial communities that are resistant against attacks by bacteriophages and removal by drugs and chemicals. In this study, biofilms of Escherichia coli O157:H7, a bacterial pathogen, were investigated using atomic force microscopy (AFM) in terms of the dynamic transition of morphology and surface properties of bacterial cells over the development of biofilms. The physical and topographical properties of biofilms are different, depending on nutrient availability. Compared to biofilms formed in a high nutrient medium, biofilms form faster and a higher number of bacterial cells were recovered on glass surface in a low nutrient medium. We demonstrate that AFM can obtain high-resolution images and the elastic information about biofilms. As E. coli biofilm becomes mature, the magnitude of the force between a tip and the surface of the biofilm gets stronger, suggesting that extracellular polymeric substances (EPSs), sticky components of biofilms, accumulate over the surface of cells upon the initial attachment of bacterial cells to surfaces

Applying graphene oxide nano-film over a polycarbonate nanoporous membrane to monitor E. coli by infrared spectroscopy.

Science.gov (United States)

Singh, Krishna Pal; Dhek, Neeraj Singh; Nehra, Anuj; Ahlawat, Sweeti; Puri, Anu

2017-01-05

Nano-biosensors are excellent monitoring tools for rapid, specific, sensitive, inexpensive, in-field, on-line, and/or real-time detection of pathogens in foods, soil, air, and water samples. A variety of nano-materials (metallic, polymeric, and/or carbon-based) were employed to enhance the efficacy, efficiency, and sensitivity of these nano-biosensors, including graphene-based materials, especially graphene oxide (GO)-based materials. GO bears many oxygen-bearing groups, enabling ligand conjugation at the high density critical for sensitive detection. We have fabricated GO-modified nano-porous polycarbonate track-etched (PCTE) membranes that were conjugated to an Escherichia coli-specific antibody (Ab) and used to detect E. coli. The random distribution of nanopores on the PCTE membrane surface and the bright coating of the GO onto the membrane were confirmed by scanning electron microscope. Anti-E. coli β-gal Abs were conjugated to the GO surface via 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride-N-hydroxysuccinimide chemistry; antibody coating was confirmed by the presence of a characteristic IR peak near 1600cm(-1). A non-corresponding Ab (anti-Pseudomonas) was used as a negative control under identical conditions. When E. coli interacted anti-E.coli β-gal with Ab-coated GO-nano-biosensor units, we observed a clear shift in the IR peak from 3373.14 to 3315cm(-1); in contrast, we did not observe any shift in IR peaks when the GO unit was coated with the non-corresponding Ab (anti-Pseudomonas). Therefore, the detection of E. coli using the described GO-nano-sensor unit is highly specific, is highly selective and can be applied for real-time monitoring of E. coli with a detection limit between 100μg/mL and 10μg/mL, similar to existing detection systems. Copyright © 2016 Elsevier B.V. All rights reserved.

T4 bacteriophage conjugated magnetic particles for E. coli capturing: Influence of bacteriophage loading, temperature and tryptone.

Science.gov (United States)

Liana, Ayu Ekajayanthi; Marquis, Christopher P; Gunawan, Cindy; Gooding, J Justin; Amal, Rose

2017-03-01

This work demonstrates the use of bacteriophage conjugated magnetic particles (Fe 3 O 4 ) for the rapid capturing and isolation of Escherichia coli. The investigation of T4 bacteriophage adsorption to silane functionalised Fe 3 O 4 with amine (NH 2 ), carboxylic (COOH) and methyl (CH 3 ) surface functional groups reveals the domination of net electrostatic and hydrophobic interactions in governing bacteriophage adsorption. The bare Fe 3 O 4 and Fe 3 O 4 -NH 2 with high T4 loading captured 3-fold more E. coli (∼70% capturing efficiency) compared to the low loading T4 on Fe 3 O 4 -COOH, suggesting the significance of T4 loading in E. coli capturing efficiency. Importantly, it is further revealed that E. coli capture is highly dependent on the incubation temperature and the presence of tryptone in the media. Effective E. coli capturing only occurs at 37°C in tryptone-containing media with the absence of either conditions resulted in poor bacteria capture. The incubation temperature dictates the capturing ability of Fe 3 O 4 /T4, whereby T4 and E. coli need to establish an irreversible binding that occurred at 37°C. The presence of tryptophan-rich tryptone in the suspending media was also critical, as shown by a 3-fold increase in E. coli capture efficiency of Fe 3 O 4 /T4 in tryptone-containing media compared to that in tryptone-free media. This highlights for the first time that successful bacteria capturing requires not only an optimum tailoring of the particle's surface physicochemical properties for favourable bacteriophage loading, but also an in-depth understanding of how factors, such as temperature and solution chemistry influence the subsequent bacteriophage-bacteria interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibitory effect of 2‑mercaptoethane sulfonate on the formation of Escherichia coli biofilms in vitro.

Science.gov (United States)

Chen, Sheng; He, Nianhai; Yu, Jialin; Li, Luquan; Sun, Fengjun; Hu, Ying; Deng, Rui; Zhong, Shiming; Shen, Leilei

2015-10-01

The biofilms (BF) formed by Escherichia coli (E. coli) is an important cause of chronic and recurrent infections due to its capacity to persist on medical surfaces and indwelling devices, demonstrating the importance of inhibiting the formation of E. coli BF and reducing BF infection. Although 2‑mercaptoethane sulfonate (MESNA) exhibits a marked mucolytic effect clinically, the effect of MESNA on the inhibition of E. coli BF formation remains to be elucidated. The present study investigated whether MESNA inhibits the formation of E. coli BF in vitro. The minimum inhibitory concentration of MESNA on E. coli was determined to be 10 mg/ml. Subsequently, the effect of MESNA on BF early adhesion, extracellular polysaccharide (EPS) and extracellular protein were detected. The effect of a subinhibitory concentration of MESNA on BF formation was evaluated, and the inhibitory potency of MESNA against matured BF was assayed. The results revealed that MESNA inhibited early stage adhesion and formation of the E. coli BF, destroyed the mature BF membrane and reduced the EPS and extracellular proteins levels of the BF. In addition, the present study investigated the effects of MESNA on the expression of EPS‑ and adhesion protein‑associated genes using quantitative polymerase chain reaction analysis, which demonstrated that MESNA effectively inhibited the expression of these genes. These results suggested that MESNA possesses anti‑BF formation capability on E. coli in vitro and may be used as a potential reagent for the clinical treatment of E. coli BF‑associated infections.

Low-intensity electromagnetic irradiation of 70.6 and 73 GHz frequencies enhances the effects of disulfide bonds reducer on Escherichia coli growth and affects the bacterial surface oxidation-reduction state

International Nuclear Information System (INIS)

Torgomyan, Heghine; Trchounian, Armen

2011-01-01

Highlights: → Low intensity 70.6 and 73 GHz electromagnetic irradiation (EMI) strongly suppressed Escherichia coli growth at 73 GHz and pH 7.3. → Reducer DL-dithiothreitol had bactericidal effect and disturbed the SH-groups number. → EMI enhanced E. coli sensitivity toward dithiothreitol. → EMI decreased the SH-groups number of membrane disturbed by ATP and N,N'-dicyclohexycarbodiimide. → The changed membrane oxidation-reduction state could be the primary mechanisms in EMI effects. -- Abstract: Low-intensity electromagnetic irradiation (EMI) of 70.6 and 73 GHz frequencies (flux capacity - 0.06 mW cm -2 ) had bactericidal effects on Escherichia coli. This EMI (1 h) exposure suppressed the growth of E. coli K-12(λ). The pH value (6.0-8.0) did not significantly affect the growth. The lag-phase duration was prolonged, and the growth specific rate was inhibited, and these effects were more noticeable after 73 GHz irradiation. These effects were enhanced by the addition of DL-dithiothreitol (DTT), a strong reducer of disulfide bonds in surface membrane proteins, which in its turn also has bactericidal effect. Further, the number of accessible SH-groups in membrane vesicles was markedly decreased by EMI that was augmented by N,N'-dicyclohexycarbodiimide and DTT. These results indicate a change in the oxidation-reduction state of bacterial cell membrane proteins that could be the primary membranous mechanism in the bactericidal effects of low-intensity EMI of the 70.6 and 73 GHz frequencies.

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

Directory of Open Access Journals (Sweden)

Mai Nguyen-Chi

2014-07-01

Full Text Available Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.

Energy buffering of DNA structure fails when Escherichia coli runs out of substrate

DEFF Research Database (Denmark)

Jensen, Peter Ruhdal; Loman, Leine; Petra, Bob

1995-01-01

To study how changes in the (ATP)/(ADP) ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H+-ATPase was modulated around the wild-type level. A relatively large drop in the (ATP)/(ADP) ratio from the normal ratio resulted in a small increase in the linking...... number of our reporter plasmid (corresponding to a small decrease in negative supercoiling). However, when cells depleted their carbon and energy source, the ensuing drop in energy state was accompanied by a strong increase in linking number. This increase was not due to reduced transcription of the DNA...... in the absence of growth substrate, since rifampin had virtually no effect on the plasmid linking number. To examine whether DNA supercoiling depends more strongly on the cellular energy state at low (ATP)/(ADP) ratios than at high ratios, we used cells that were already at a low energy state after substrate...

99mTechnetium labelled Escherichia coli

International Nuclear Information System (INIS)

Diniz, S.O.F.; Cardoso, V.N.; Resende, B.M.; Nunan, E.A.; Simal, C.J.R.

1999-01-01

Samples of a culture of unlabeled Escherichia coli were incubated with different concentrations of stannous chloride for various time periods. 99m Tc (26.0 MBq) was added to each preparation and the results showed a labelling yield of 98% for E. coli. Since the bacterial viability of 99m Tc-E. coli and E. coli did not show any statistical differences, these results demonstrate that labelling of E. coli with 99m Tc does not modify the bacterial viability, and the radiolabelled bacteria may be a good model to study bacterial translocation

Graphene-interfaced electrical biosensor for label-free and sensitive detection of foodborne pathogenic E. coli O157:H7.

Science.gov (United States)

Pandey, Ashish; Gurbuz, Yasar; Ozguz, Volkan; Niazi, Javed H; Qureshi, Anjum

2017-05-15

E. coli O157:H7 is an enterohemorrhagic bacteria responsible for serious foodborne outbreaks that causes diarrhoea, fever and vomiting in humans. Recent foodborne E. coli outbreaks has left a serious concern to public health. Therefore, there is an increasing demand for a simple, rapid and sensitive method for pathogen detection in contaminated foods. In this study, we developed a label-free electrical biosensor interfaced with graphene for sensitive detection of pathogenic bacteria. This biosensor was fabricated by interfacing graphene with interdigitated microelectrodes of capacitors that were biofunctionalized with E. coli O157:H7 specific antibodies for sensitive pathogenic bacteria detection. Here, graphene nanostructures on the sensor surface provided superior chemical properties such as high carrier mobility and biocompatibility with antibodies and bacteria. The sensors transduced the signal based on changes in dielectric properties (capacitance) through (i) polarization of captured cell-surface charges, (ii) cells' internal bioactivity, (iii) cell-wall's electronegativity or dipole moment and their relaxation and (iv) charge carrier mobility of graphene that modulated the electrical properties once the pathogenic E. coli O157:H7 captured on the sensor surface. Sensitive capacitance changes thus observed with graphene based capacitors were specific to E. coli O157:H7 strain with a sensitivity as low as 10-100 cells/ml. The proposed graphene based electrical biosensor provided advantages of speed, sensitivity, specificity and in-situ bacterial detection with no chemical mediators, represents a versatile approach for detection of a wide variety of other pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4

Directory of Open Access Journals (Sweden)

Karlsson Mattias

2012-01-01

Full Text Available Abstract Background Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs, and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation. Results Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR-1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR-1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB. Conclusions The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR-1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1

A microfluidic device for rapid screening of E. coli O157:H7 based on IFAST and ATP bioluminescence assay for water analysis

CSIR Research Space (South Africa)

Ngamsom, B

2017-08-01

Full Text Available We present a simple microfluidic system for rapid screening of Escherichia coli (E. coli) O157:H7 employing the specificity of immunomagnetic separation (IMS) via immiscible filtration assisted by surface tension (IFAST), and the sensitivity...

Elimination of Escherichia coli O157:H7 in meats by gamma irradiation

International Nuclear Information System (INIS)

Thayer, D.W.; Boyd, G.

1993-01-01

Undercooked and raw meat has been linked to outbreaks of hemorrhagic diarrhea due to the presence of Escherichia coli O157:H7; therefore, treatment with ionizing radiation was investigated as a potential method for the elimination of this organism. Response-surface methods were used to study the effects of irradiation dose (0 to 2.0 kGy), temperature (-20 to +20 degrees C), and atmosphere (air and vacuum) on E. coli O157:H7 in mechanically deboned chicken meat. Differences in irradiation dose and temperature significantly affected the results. Ninety percent of the viable E. coli in chicken meat was eliminated by doses of 0.27 kGy at +5 degrees C and 0.42 kGy at -5 degrees C. Small, but significant, differences in radiation resistance by E. coli were found when finely ground lean beef rather than chicken was the substrate. Unlike nonirradiated samples, no measurable verotoxin was found in finely ground lean beef which had been inoculated with 10(4.8) CFU of E. coli O157:H7 per g, irradiated at a minimum dose of 1.5 kGy, and temperature abused at 35 degrees C for 20 h. Irradiation is an effective method to control this food-borne pathogen

GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

Science.gov (United States)

Goyal, Megha; Chaudhuri, Tapan K

2015-07-01

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli. Copyright © 2015 Elsevier Ltd. All rights reserved.

The oxygen effect in E. coli cells

International Nuclear Information System (INIS)

Myasnik, M.N.; Skvortsov, V.G.; Sokolov, V.A.

1982-01-01

In experiments on E. coli strains deficient in some stages of DNA repair from radiation damages, it was demonstrated that the value of the oxygen effect, under optimal conditions for manifestation thereof, decreases in the following order: E. coli WP2 (the wild type) → E. coli WP2 exr - and E. coli B → E. coli WP2 uvr A6 → E. coli WP2 rec Al and E. coli WP2 hcr - exr - . It was detected that 0.14 M NaCl solution sensitizes the anoxic cells of some E. coli strains to the effect of γ-radiation. It was established that mutation of the uvr A-gene increases sharply the sensitivity of cells to iradiation under the anoxic conditions in the presence of NaCl, the reverse'' oxygen effect being observed

Hemolytic porcine intestinal Escherichia coli without virulence-associated genes typical of intestinal pathogenic E. coli.

Science.gov (United States)

Schierack, Peter; Weinreich, Joerg; Ewers, Christa; Tachu, Babila; Nicholson, Bryon; Barth, Stefanie

2011-12-01

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.

escherichia coli serotypes confirmed in experimental mammary ...

African Journals Online (AJOL)

DJFLEX

VARIATIONS IN VIRULENCE OF THREE (3) ESCHERICHIA COLI. SEROTYPES CONFIRMED IN ... ows are susceptible to E. coli infection because. E. coli exist in the .... Coli infections in mice: A laboratory animal model for research in.

Co-Expression of ORFCma with PHB Depolymerase (PhaZCma ) in Escherichia coli Induces Efficient Whole-Cell Biodegradation of Polyesters.

Science.gov (United States)

Lee, Ming-Chieh; Liu, En-Jung; Yang, Cheng-Han; Hsiao, Li-Jung; Wu, Tzong-Ming; Li, Si-Yu

2018-04-01

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZ Cma , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORF Cma (a putative periplasmic substrate binding protein that is within the same operon of phaZ Cma ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZ Cma has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORF Cma can potentially be a universal element for enhancing the secretion of recombinant protein. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Autodisplay of the La/SSB protein on LPS-free E. coli for the diagnosis of Sjögren's syndrome.

Science.gov (United States)

Yoo, Gu; Dilkaute, Carina; Bong, Ji-Hong; Song, Hyun-Woo; Lee, Misu; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2017-05-01

The objective of this study was to present an immunoassay for the diagnosis of Sjögren's syndrome based on the autodisplayed La/SSB protein on the outer membrane of intact E. coli (strain UT-5600) and LPS-free E. coli (ClearColi™). As the first step, an autodisplay vector (pCK002) was transfected into intact E. coli and LPS-free E. coli for comparison of efficiency of autdisplay of La/SSB. The maximal level of La/SSB expression was estimated to be similar for LPS-free E. coli and intact E. coli at different optimal induction periods. Intact E. coli was found to grow twofold faster than LPS-free E. coli, and the maximal level of expression for LPS-free E. coli was obtained with a longer induction period. When the zeta potential was measured, both intact E. coli and LPS-free E. coli showed negative values, and the autodisplay of negatively charged La/SSB protein (pIE. coli and LPS-free E. coli resulted in a slight change in zeta potential values. E. coli with autodisplayed La/SSB protein was used for an immunoassay of anti-La/SSB antibodies for the diagnosis of Sjögren's syndrome. The surface of E. coli with the autodisplayed antigen was modified with rabbit serum and papain to prevent false positive signals because of nonspecific binding of unrelated antibodies from human serum. LPS-free E. coli with autodisplayed La/SSB protein yielded sensitivity and selectivity of 81.6% and 78.6%, respectively. The Bland-Altman test showed that the immunoassays based on LPS-free E. coli and intact E. coli with autodisplayed La/SSB protein were statistically equivalent to a clinical immunoassay for detection of anti-La/SSB antibodies (confidence coefficient 95%). Copyright © 2017 Elsevier Inc. All rights reserved.

Sequential effect of phages and cold nitrogen plasma against Escherichia coli O157:H7 biofilms on different vegetables.

Science.gov (United States)

Cui, Haiying; Bai, Mei; Yuan, Lu; Surendhiran, Duraiarasan; Lin, Lin

2018-03-02

Escherichia coli O157:H7 (E. coli O157:H7) is one of the most common pathogens in fresh vegetables and fruits, and most of the diseases produced by E. coli O157:H7 are associated with biofilms. Cold nitrogen plasma (CNP) is a cold sterilization technique which has no residue. However to completely eliminate the biofilm on the surface of vegetables the processing power and time of CNP have to be enhanced, which will impact on the quality of fruits and vegetables. Thus the sequential treatment of CNP and phage techniques was engineered in this study. Compared to treatment performed separately, sequential treatment not only had more mild treatment conditions as 400W CNP treatment for 2min and 5% phage treatment for 30min, but also exhibited more remarkable effect on eradicating E. coli O157:H7 biofilms in vitro and on vegetables. The population of E. coli O157:H7 was approximately reduced by 2logCFU/cm 2 after individual treatment of 5% phages for 30min or 500W CNP for 3min. While the sequential treatment of CNP (400W, 2min) and phages (5%, 30min) reduced the E. coli O157:H7 viable count in biofilm by 5.71logCFU/cm 2 . Therefore, the sequential treatment holds a great promise to improve the current treatment systems of bacterial contamination on different vegetable surfaces. Copyright © 2018 Elsevier B.V. All rights reserved.

Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

DEFF Research Database (Denmark)

Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.

2015-01-01

The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehens...

Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

Science.gov (United States)

Gómez-Aldapa, Carlos A; Segovia-Cruz, Jesús A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Salas-Rangel, Laura P; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

2016-10-01

The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander. Copyright © 2016 Elsevier Ltd. All rights reserved.

One-pot synthesis of gold nanoclusters with bright red fluorescence and good biorecognition abilities for visualization fluorescence enhancement detection of E. coli.

Science.gov (United States)

Liu, Jiali; Lu, Lili; Xu, Suying; Wang, Leyu

2015-03-01

A facile one-pot strategy was developed for the synthesis of lysozyme functionalized fluorescence gold nanoclusters (AuNCs). The lysozymes added to reduce Au(3+) ions and stabilize the AuNCs during the synthesis were coated on the AuNCs surface and retained their specific recognition ability for bacteria such as Escherichia coli (E. coli). Based on such ability, these AuNCs were specifically attached onto the surface of E. coli, which resulted in great red fluorescence enhancement. Nevertheless, the bovine serum albumin (BSA) stabilized AuNCs could not recognize E. coli and no fluorescence enhancement was observed. Upon the addition of E. coli, the red fluorescence intensity of lysozyme-AuNCs was enhanced linearly over the range of 2.4×10(4) -6.0×10(6) CFU/mL of E. coli with high sensitivity (LOD=2.0×10(4) CFU/mL, S/N=3). The visualization fluorescence evolution may enable the rapid and real-time detection of bacteria. This study may be extended to other functional proteins such as antibody, enzyme, and peptide functionalized nanoclusters while retaining the bioactivity of coating proteins and find wide applications in the fields of biochemistry and biomedicine. Copyright © 2014 Elsevier B.V. All rights reserved.

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation.

Science.gov (United States)

Wu, Qiong; Liu, Ming-Chao; Yang, Jun; Wang, Jiu-Feng; Zhu, Yao-Hong

2016-02-15

Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. coli-induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. coli-induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. coli-induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. coli-induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity of L. rhamnosus, the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Persistence of Escherichia coli O157:H7 and total Escherichia coli in feces and feedlot surface manure from cattle fed diets with or without corn or sorghum wet distillers grains with solubles

Science.gov (United States)

Feeding corn wet distillers grains with solubles (WDGS) to cattle can increase the load of Escherichia coli O157:H7 in feces and on hides, but the mechanisms are not fully understood. The objective of these experiments was to examine a role for the persistence of E. coli O157:H7 in the feces and fee...

Wave-induced mass transport affects daily Escherichia coli fluctuations in nearshore water

Science.gov (United States)

Ge, Zhongfu; Whitman, Richard L.; Nevers, Meredith B.; Phanikumar, Mantha S.

2012-01-01

Characterization of diel variability of fecal indicator bacteria concentration in nearshore waters is of particular importance for development of water sampling standards and protection of public health. Significant nighttime increase in Escherichia coli (E. coli) concentration in beach water, previously observed at marine sites, has also been identified in summer 2000 from fixed locations in waist- and knee-deep waters at Chicago 63rd Street Beach, an embayed, tideless, freshwater beach with low currents at night (approximately 0.015 m s–1). A theoretical model using wave-induced mass transport velocity for advection was developed to assess the contribution of surface waves to the observed nighttime E. coli replenishment in the nearshore water. Using average wave conditions for the summer season of year 2000, the model predicted an amount of E. coli transported from water of intermediate depth, where sediment resuspension occurred intermittently, that would be sufficient to have elevated E. coli concentration in the surf and swash zones as observed. The nighttime replenishment of E. coli in the surf and swash zones revealed here is an important phase in the cycle of diel variations of E. coli concentration in nearshore water. According to previous findings in Ge et al. (Environ. Sci. Technol. 2010, 44, 6731–6737), enhanced current circulation in the embayment during the day tends to displace and deposit material offshore, which partially sets up the system by the early evening for a new period of nighttime onshore movement. This wave-induced mass transport effect, although facilitating a significant base supply of material shoreward, can be perturbed or significantly influenced by high currents (orders of magnitude larger than a typical wave-induced mass transport velocity), current-induced turbulence, and tidal forcing.

Identification and Prevalence of Escherichia coli and Escherichia coli O157: H7 in Foods

Directory of Open Access Journals (Sweden)

Ancuta Mihaela Rotar

2013-11-01

Full Text Available The objective of this study is to investigate the incidence of Escherichia coli in animal and non-animal foods, and mainly the incidence of the serotype O157: H7 producing verotoxin. The presence of common Escherichia coli and Escherichia coli O157: H7 in various foods (of animal and non animal origin was performed in Transylvania area. We analyzed a total of one hundred forty-one samples of minced meat, one hundred twenty-six samples of meat , twenty six samples of meat products, five samples of alcoholic beverages, three samples of seafood, one hundred samples of cheese from pasteurized milk, seventeen samples of butter, four samples of vegetables and one sample of milk powder, using the standard cultural method and Vidas Eco method for E. coli O157: H7 strains. E. coli was identified in 50 samples of minced meat, 55 samples of meat prepared, 4 samples of meat products, 2 samples of alcoholic beverages, 25 samples of cheese from pasteurized milk, 6 samples of butter and 1 sample of vegetables. In this study were not been identified any foods contaminated with the E. coli O157: H7 serotype. The results of this reasearch have demostrated that E. coli wich represents a hygienic indicator of recent food contamination, can be destroyed with heat treatment and hygienic handling of foods. Our country over the years has been among the few countries where the incidence of the E. coli O157: H7 serotype has been minimal.

Methods for generation of reporter phages and immobilization of active bacteriophages on a polymer surface

Science.gov (United States)

Morgan, Mark Thomas (Inventor); Kothapalli, Aparna (Inventor); Applegate, Bruce Michael (Inventor); Perry, Lynda Louise (Inventor)

2012-01-01

Novel reporter bacteriophages are provided. Provided are compositions and methods that allow bacteriophages that are used for specific detection or killing of E. coli 0157:H7 to be propagated in nonpathogenic E. coli, thereby eliminating the safety and security risks of propagation in E. coli 0157:H7. Provided are compositions and methods for attaching active bacteriophages to the surface of a polymer in order to kill target bacteria with which the phage comes into contact. Provided are modified bacteriophages immobilized to a surface, which capture E. coli 0157:H7 and cause the captured cells to emit light or fluorescence, allowing detection of the bacteria in a sample.

The asymptomatic bacteriuria Escherichia coli strain 83972 outcompetes uropathogenic E. coli strains in human urine

DEFF Research Database (Denmark)

Hancock, Viktoria; Ulett, G.C.; Schembri, M.A.

2006-01-01

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract....... The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals with E. coli 83972 has been used successfully as an alternative approach for the treatment of patients who are refractory...... to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics of E. coli 83972 in human...

Removal of Microbial Contamination from Surface by Plasma

Science.gov (United States)

Feng, Xinxin; Liu, Hongxia; Shen, Zhenxing; Wang, Taobo

2018-01-01

Microbial contamination is closely associated with human and environmental health, they can be tested on food surfaces, medical devices, packing material and so on. In this paper the removal of the microbial contamination from surface using plasma treatment is investigated. The Escherichia coli (E. coli) has been chosen as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. Oxygen gas was as the working gas. The plasma RF power, plasma exposition time, gas flow and the concentration of organic pollutant were varied in order to see the effect of the plasma treatment on the Gram-negative germ removal. After the treatment, the microbial abatement was evaluated by the standard plate count method. This proved a positive effect of the plasma treatment on Gram-negative germ removal. The kinetics and mathematical model of removal were studied after plasma treatment, and then the removing course of E. coli was analyzed. This work is meaningful for deepening our understanding of the fundamental scientific principles regarding microbial contamination from surface by plasma.

Cellular automaton simulation of the diffusive motion of bacteria and their adhesion to nanostructures on a solid surface.

Science.gov (United States)

Yamamoto, Takehiro; Emura, Chie; Oya, Masashi

2016-12-01

The growth of a biofilm begins with the adhesion of bacteria to a solid surface. Consequently, biofilm growth can be managed by the control of bacterial adhesion. Recent experimental studies have suggested that bacterial adhesion can be controlled by modifying a solid surface using nanostructures. Computational prediction and analysis of bacterial adhesion behavior are expected to be useful for the design of effective arrangements of nanostructures for controlling bacterial adhesion. The present study developed a cellular automaton (CA) model for bacterial adhesion simulation that could describe both the diffusive motion of bacteria and dependence of their adhesion patterns on the distance between nanostructures observed in experimental studies. The diffusive motion was analyzed by the moment scaling spectrum theory, and the present model was confirmed to describe subdiffusion behavior due to obstacles. Adhesion patterns observed in experimental studies can be successfully simulated by introducing CA rules to describe a mechanism by which bacteria tend to move to increase the area of contact with nanostructures. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of data from the literature on the transport and survival of Escherichia coli and thermotolerant coliforms in aquifers under saturated conditions.

NARCIS (Netherlands)

Foppen, J W A; Schijven, J F

2006-01-01

Escherichia coli and thermotolerant coliforms are of major importance as indicators of fecal contamination of water. Due to its negative surface charge and relatively low die-off or inactivation rate coefficient, E. coli is able to travel long distances underground and is therefore also a useful

Antibacterial Derivatives of Ciprofloxacin to Inhibit Growth of Necrotizing Fasciitis Associated Penicillin Resistant Escherichia coli

Directory of Open Access Journals (Sweden)

Ronald Bartzatt

2013-01-01

Full Text Available Escherichia coli (E. coli is associated with necrotizing fasciitis (type I and can induce enough damage to tissue causing hypoxia. Three ester derivatives of the broad-spectrum antibiotic ciprofloxacin were placed into bacteria culture simultaneously with the parent ciprofloxacin (drug 1 to ascertain the level of antibacterial activity. The n-propyl (drug 2, n-pentyl (drug 3, and n-octyl (drug 4 esters of ciprofloxacin were synthesized under mixed phase conditions and by microwave excitation. The formation of ester derivatives of ciprofloxacin modified important molecular properties such as Log P and polar surface area which improves tissue penetration, yet preserved strong antibacterial activity. The Log P values for drugs 1, 2, 3, and 4 became −0.701, 0.437, 1.50, and 3.02, respectively. The polar surface areas for drugs 1, 2, 3, and 4 were determined to be 74.6 Angstroms2, 63.6 Angstroms2, 63.6 Angstroms2, and 63.6 Angstroms2, respectively. These values of Log P and polar surface area improved tissue penetration, as indicated by the determination of dermal permeability coefficient (Kp and subsequently into the superficial fascial layer. All drugs induced greater than 60% bacterial cell death at concentrations less than 1.0 micrograms/milliliter. The ester derivatives of ciprofloxacin showed strong antibacterial activity toward penicillin resistant E. coli.

New evidence on the role of catalase in Escherichia coli-mediated biocorrosion

International Nuclear Information System (INIS)

Baeza, S.; Vejar, N.; Gulppi, M.; Azocar, M.; Melo, F.; Monsalve, A.; Pérez-Donoso, J.; Vásquez, C.C.; Pavez, J.; Zagal, J.H.; Zhou, X.; Thompson, G.E.; Páez, M.A.

2013-01-01

Highlights: ► MIC on stainless by catalase deficient Escherichia coli bacteria reveals the enzyme influence. ► The localized damage was greater in the presence of the wild E. coli. ► Catalase assists oxygen generation by disproportionation of H 2 O 2 to H 2 O and O 2 . - Abstract: The role of catalase on the microbiologically influenced corrosion mechanism by Escherichia coli (E. coli) has been examined, employing wild type and catalase-deficient cells. The bacteria were cultured for different times in the presence of AISI 316L stainless steel samples. The morphologies of the metallic surfaces covered by biofilms were studied by optical microscopy. The localized corrosion catalyzed by the bacteria was followed by scanning electron microscopy after immersion in the bacterial culture for different times. Susceptibility to corrosion was further investigated by potentiodynamic measurements. It was found that wild type E. coli is more aggressive than the mutant one, suggesting a role for catalase in increasing the kinetics of the cathodic reaction and, consequently, the global corrosion process. This correlates with oxygen uptake kinetics, as determined by differential pulse voltammetry on a pyrolytic graphite electrode modified with cobalt phthalocyanine, which was higher in the presence of wild type E. coli. When H 2 O 2 was deliberately added to the culture medium, wild type E. coli catalyzed oxygen disproportionation more efficiently than the mutant derivative, thus limiting H 2 O 2 accumulation in the medium and, hence, bacterial poisoning. In fact, the reduced adhesion of mutant cells to the metal substrate is apparently the result of H 2 O 2 accumulation in the culture broth. Thus, the rapid consumption of oxygen and peroxide in the presence of wild type E. coli is associated with the catalysis of H 2 O 2 disproportionation to water and oxygen. On the stainless steel, however, a dual mechanism of oxygen reduction, i.e. through formation of hydrogen peroxide

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

Science.gov (United States)

Serda, Rita E.; Blanco, Elvin; Mack, Aaron; Stafford, Susan J.; Amra, Sarah; Li, Qingpo; van de Ven, Anne L.; Tanaka, Takemi; Torchilin, Vladimir P.; Wiktorowicz, John E.; Ferrari, Mauro

2014-01-01

Mass transport of drug delivery vehicles is guided by particle properties, such as shape, composition and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light chain variable region, fibrinogen, and complement component 1 compared to their anionic counterparts. The anionic-surface favored equal accumulation of microparticles in the liver and spleen, while cationic-surfaces favored preferential accumulation in the spleen. Immunohistochemistry supported macrophage internalization of both anionic and cationic silicon microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution. PMID:21303614

Effects of Plant-Derived Extracts, Other Antimicrobials, and Their Combinations against Escherichia coli O157:H7 in Beef Systems.

Science.gov (United States)

Ko, Kyung Yuk; Geornaras, Ifigenia; Paik, Hyun-Dong; Kim, Kee-Tae; Sofos, John N

2015-06-01

The antimicrobial effects of thyme oil (TO), grapefruit seed extract (GSE), and basil essential oil, alone or in combination with cetylpyridinium chloride (CPC), sodium diacetate, or lactic acid, were evaluated against Escherichia coli O157:H7 in a moisture-enhanced beef model system. The model system was composed of a nonsterile beef homogenate to which NaCl (0.5%) and sodium tripolyphosphate (0.25%) were added, together with the tested antimicrobial ingredients. Beef homogenate treatments were inoculated (ca. 3 log CFU/ml) with rifampin-resistant E. coli O157:H7 (eight-strain mixture) and incubated at 15 °C (48 h). The most effective individual treatments were TO (0.25 or 0.5%) and GSE (0.5 or 1.0%), which immediately reduced (P extracts with CPC (0.02 or 0.04%) and sodium diacetate (0.25%) had an additive effect with respect to antibacterial activity. In a second study, antimicrobial interventions were evaluated for their efficacy in reducing surface contamination of E. coli O157:H7 on beef cuts and to determine the effect of these surface treatments on subsequent internalization of the pathogen during blade tenderization. Beef cuts (10 by 8 by 3.5 cm) were inoculated (ca. 4 log CFU/g) on one side with the rifampin-resistant E. coli O157:H7 strain mixture and were then spray treated (20 lb/in(2), 10 s) with water, GSE (5 and 10%), lactic acid (5%), or CPC (5%). Untreated (control) and spray-treated surfaces were then subjected to double-pass blade tenderization. Surface contamination (4.4 log CFU/g) of E. coli O157:H7 was reduced (P < 0.05) to 3.4 (5% CPC) to 4.1 (water or 5% GSE) log CFU/g following spray treatment. The highest and lowest transfer rates of pathogen cells from the surface to deeper tissues of blade-tenderized sections were obtained in the untreated control and CPC-treated samples, respectively.

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNAHis pair.

Science.gov (United States)

Englert, Markus; Vargas-Rodriguez, Oscar; Reynolds, Noah M; Wang, Yane-Shih; Söll, Dieter; Umehara, Takuya

2017-11-01

Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNA His recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNA His pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus. E. coli was genetically engineered to use a C. crescentus HisRS•tRNA His pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo. A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNA His pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNA His pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNA His CUA elevated its suppression efficiency by 2-fold. The C. crescentus HisRS•tRNA His pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNA His is orthogonal in MEOV1 cells. E. coli tRNA His CUA is an efficient amber suppressor in MEOV1. We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Pathologic Cellular Events in Smoking-Related Pancreatitis

Energy Technology Data Exchange (ETDEWEB)

Thrower, Edwin [Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT 06520 (United States); Veterans Affairs Connecticut Healthcare, West Haven, CT 06516 (United States)

2015-04-29

Pancreatitis, a debilitating inflammatory disorder, results from pancreatic injury. Alcohol abuse is the foremost cause, although cigarette smoking has recently surfaced as a distinct risk factor. The mechanisms by which cigarette smoke and its toxins initiate pathological cellular events leading to pancreatitis, have not been clearly defined. Although cigarette smoke is composed of more than 4000 compounds, it is mainly nicotine and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which have been extensively studied with respect to pancreatic diseases. This review summarizes these research findings and highlights cellular pathways which may be of relevance in initiation and progression of smoking-related pancreatitis.

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice.

Science.gov (United States)

Balagué, Claudia E; de Ruiz, Clara Silva; Rey, Rosario; de Duffard, Ana María Evangelista; Nader-Macías, María Elena

2004-11-01

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.

Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point.

Science.gov (United States)

Wang, Lu; Lai, Luhua; Ouyang, Qi; Tang, Chao

2011-01-25

Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT). In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15)N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT) strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

Flux balance analysis of ammonia assimilation network in E. coli predicts preferred regulation point.

Directory of Open Access Journals (Sweden)

Lu Wang

Full Text Available Nitrogen assimilation is a critical biological process for the synthesis of biomolecules in Escherichia coli. The central ammonium assimilation network in E. coli converts carbon skeleton α-ketoglutarate and ammonium into glutamate and glutamine, which further serve as nitrogen donors for nitrogen metabolism in the cell. This reaction network involves three enzymes: glutamate dehydrogenase (GDH, glutamine synthetase (GS and glutamate synthase (GOGAT. In minimal media, E. coli tries to maintain an optimal growth rate by regulating the activity of the enzymes to match the availability of the external ammonia. The molecular mechanism and the strategy of the regulation in this network have been the research topics for many investigators. In this paper, we develop a flux balance model for the nitrogen metabolism, taking into account of the cellular composition and biosynthetic requirements for nitrogen. The model agrees well with known experimental results. Specifically, it reproduces all the (15N isotope labeling experiments in the wild type and the two mutant (ΔGDH and ΔGOGAT strains of E. coli. Furthermore, the predicted catalytic activities of GDH, GS and GOGAT in different ammonium concentrations and growth rates for the wild type, ΔGDH and ΔGOGAT strains agree well with the enzyme concentrations obtained from western blots. Based on this flux balance model, we show that GS is the preferred regulation point among the three enzymes in the nitrogen assimilation network. Our analysis reveals the pattern of regulation in this central and highly regulated network, thus providing insights into the regulation strategy adopted by the bacteria. Our model and methods may also be useful in future investigations in this and other networks.

Low-intensity electromagnetic irradiation of 70.6 and 73 GHz frequencies enhances the effects of disulfide bonds reducer on Escherichia coli growth and affects the bacterial surface oxidation-reduction state

Energy Technology Data Exchange (ETDEWEB)

Torgomyan, Heghine [Department of Biophysics of Biology Faculty, Yerevan State University, Yerevan 0025 (Armenia); Trchounian, Armen, E-mail: Trchounian@ysu.am [Department of Biophysics of Biology Faculty, Yerevan State University, Yerevan 0025 (Armenia)

2011-10-14

Highlights: {yields} Low intensity 70.6 and 73 GHz electromagnetic irradiation (EMI) strongly suppressed Escherichia coli growth at 73 GHz and pH 7.3. {yields} Reducer DL-dithiothreitol had bactericidal effect and disturbed the SH-groups number. {yields} EMI enhanced E. coli sensitivity toward dithiothreitol. {yields} EMI decreased the SH-groups number of membrane disturbed by ATP and N,N'-dicyclohexycarbodiimide. {yields} The changed membrane oxidation-reduction state could be the primary mechanisms in EMI effects. -- Abstract: Low-intensity electromagnetic irradiation (EMI) of 70.6 and 73 GHz frequencies (flux capacity - 0.06 mW cm{sup -2}) had bactericidal effects on Escherichia coli. This EMI (1 h) exposure suppressed the growth of E. coli K-12({lambda}). The pH value (6.0-8.0) did not significantly affect the growth. The lag-phase duration was prolonged, and the growth specific rate was inhibited, and these effects were more noticeable after 73 GHz irradiation. These effects were enhanced by the addition of DL-dithiothreitol (DTT), a strong reducer of disulfide bonds in surface membrane proteins, which in its turn also has bactericidal effect. Further, the number of accessible SH-groups in membrane vesicles was markedly decreased by EMI that was augmented by N,N'-dicyclohexycarbodiimide and DTT. These results indicate a change in the oxidation-reduction state of bacterial cell membrane proteins that could be the primary membranous mechanism in the bactericidal effects of low-intensity EMI of the 70.6 and 73 GHz frequencies.

Structure-function analysis of the self-recognizing Antigen 43 autotransporter protein from Escherichia coli

DEFF Research Database (Denmark)

Klemm, Per; Hjerrild, L.; Gjermansen, Morten

2004-01-01

Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Expression of Ag43 confers aggregation and fluffing of cells, promotes biofilm formation and is associated with enhanced resistance to antimicrobial agents. Ag43 is an autotransporter protein and consi......Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Expression of Ag43 confers aggregation and fluffing of cells, promotes biofilm formation and is associated with enhanced resistance to antimicrobial agents. Ag43 is an autotransporter protein......-clumping variants, we have pinpointed the region of the protein responsible for autoaggregation to be located within the N-terminal one-third of the passenger domain. Our data suggest that ionic interactions between charged residues residing in interacting pairs of Ag43(alpha) domains may be important for the self...

Amplification of the uvrA gene product of Escherichia coli to 7% of cellular protein by linkage to the p/sub L/ promoter of pKC30

International Nuclear Information System (INIS)

Yoakum, G.H.; Yeung, A.T.; Mattes, W.B.; Grossman, L.

1982-01-01

Researchers have constructed a hybrid pKC30-uvrA plasmid (pGHY5003) in which transcription of the uvrA gene can be induced under p/sub L/ control to amplify the uvrA gene product to 7% of cellular protein. To construct pGHY5003, researchers developed a genetic selection using the basal level of expression (30 0 C) from p/sub L/ in thermosensitive cI857 lysogens to isolate appropriately tailored repair genes inserted at the Hpa I site of pKC30 from recombinant DNA mixtures with a variety of products. In addition, a post-uv-irradiation radiolabeling method was adapted to screen inserts for temperature-inducible polypeptide synthesis directed by transcription under p/sub L/ control rapidly. This should prove generally useful for isolating genes inserted at the Hpa I site of plasmid pKC30 with the following characteristics: (1) genetically functional hybrid plasmids selected from a large population of exonucleolytically tailored fragments ligated into Hpa I of pKC30 and (2) production of high-level amplification for the gene product of interest by screening for post-uv-irradiation temperature inducibility of polypeptides synthesized from hybrid plasmids. The level of amplification obtained for the uvrA gene product from pGHY5003 is approximately 10,000-fold higher than estimates of the level of uvrA protein in logarithmic phase Escherichia coli

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface*

Science.gov (United States)

Fantappiè, Laura; Irene, Carmela; De Santis, Micaela; Armini, Alessandro; Gagliardi, Assunta; Tomasi, Michele; Parri, Matteo; Cafardi, Valeria; Bonomi, Serena; Ganfini, Luisa; Zerbini, Francesca; Zanella, Ilaria; Carnemolla, Chiara; Bini, Luca; Grandi, Alberto; Grandi, Guido

2017-01-01

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides. PMID:28483926

Silver nanoparticle-E. coli colloidal interaction in water and effect on E. coli survival.

Science.gov (United States)

Dror-Ehre, A; Mamane, H; Belenkova, T; Markovich, G; Adin, A

2009-11-15

Silver nanoparticles exhibit antibacterial properties via bacterial inactivation and growth inhibition. The mechanism is not yet completely understood. This work was aimed at elucidating the effect of silver nanoparticles on inactivation of Escherichia coli, by studying particle-particle interactions in aqueous suspensions. Stable, molecularly capped, positively or negatively charged silver nanoparticles were mixed at 1 to 60microgmL(-1) with suspended E. coli cells to examine their effect on inactivation of the bacteria. Gold nanoparticles with the same surfactant were used as a control, being of similar size but made up of a presumably inert metal. Log reduction of 5log(10) and complete inactivation were obtained with the silver nanoparticles while the gold nanoparticles did not show any inactivation ability. The effect of molecularly capped nanoparticles on E. coli survival was dependent on particle number. Log reduction of E. coli was associated with the ratio between the number of nanoparticles and the initial bacterial cell count. Electrostatic attraction or repulsion mechanisms in silver nanoparticle-E. coli cell interactions did not contribute to the inactivation process.

Inactivation of Escherichia coli in soil by solarization

International Nuclear Information System (INIS)

Wu, S.; Nishihara, M.; Kawasaki, Y.; Yokoyama, A.; Matsuura, K.; Koga, T.; Ueno, D.; Inoue, K.; Someya, T.

2009-01-01

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g −1 dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces. (author)

Calcium-phosphate biomineralization induced by alkaline phosphatase activity in Escherichia coli: localization, kinetics and potential signatures in the fossil record

Science.gov (United States)

Cosmidis, Julie; Benzerara, Karim; Guyot, François; Skouri-Panet, Fériel; Duprat, Elodie; Férard, Céline; Guigner, Jean-Michel; Babonneau, Florence; Coelho, Cristina

2015-12-01

Bacteria are thought to play an important role in the formation of calcium-phosphate minerals composing marine phosphorites, as supported by the common occurrence of fossil microbes in these rocks. Phosphatase enzymes may play a key role in this process. Indeed, they may increase the supersaturation with respect to Ca-phosphates by releasing orthophosphate ions following hydrolysis of organic phosphorus. However, several questions remain unanswered about the cellular-level mechanisms involved in this model, and its potential signatures in the mineral products. We studied Ca-phosphate precipitation by different strains of Escherichia coli which were genetically modified to differ in the abundance and cellular localization of the alkaline phosphatase (PHO A) produced. The mineral precipitated by either E. coli or purified PHO A was invariably identified as a carbonate-free non-stoichiometric hydroxyapatite. However, the bacterial precipitates could be discriminated from the ones formed by purified PHO A at the nano-scale. PHO A localization was shown to influence the pattern of Ca-phosphate nucleation and growth. Finally, the rate of calcification was proved to be consistent with the PHO A enzyme kinetics. Overall, this study provides mechanistic keys to better understand phosphogenesis in the environment, and experimental references to better interpret the microbial fossil record in phosphorites.

Asellus aquaticus as a potential carrier of Escherichia coli and other coliform bacteria into drinking water distribution systems

DEFF Research Database (Denmark)

Christensen, Sarah Christine Boesgaard; Arvin, Erik; Nissen, E.

2013-01-01

Individuals of the water louse, Asellus aquaticus, enter drinking water distribution systems in temperate parts of the world, where they establish breeding populations. Populations of A. aquaticus in surface water from 2 ponds were analysed for associated faecal indicator bacteria and the risk of A....... coli and 6 total coliforms A. aquaticus-1. During exposure to high concn. of coliforms, concn. reached 350 coliforms A. aquaticus-1. A. aquaticus associated E. coli were only detected as long as E. coli were present in the water and sediment. The calculated probability of exceeding drinking water...... for evaluating incidents with the presence of coliform indicators in drinking water by showing that intruding A. aquaticus are not important carriers of E. coli or other coliform bacteria even when emerging from faecally contaminated waters....

Changes in Escherichia coli to Cryptosporidium ratios for various fecal pollution sources and drinking water intakes.

Science.gov (United States)

Lalancette, Cindy; Papineau, Isabelle; Payment, Pierre; Dorner, Sarah; Servais, Pierre; Barbeau, Benoit; Di Giovanni, George D; Prévost, Michèle

2014-05-15

Assessing the presence of human pathogenic Cryptosporidium oocysts in surface water remains a significant water treatment and public health challenge. Most drinking water suppliers rely on fecal indicators, such as the well-established Escherichia coli (E. coli), to avoid costly Cryptosporidium assays. However, the use of E. coli has significant limitations in predicting the concentration, the removal and the transport of Cryptosporidium. This study presents a meta-analysis of E. coli to Cryptosporidium concentration paired ratios to compare their complex relationships in eight municipal wastewater sources, five agricultural fecal pollution sources and at 13 drinking water intakes (DWI) to a risk threshold based on US Environmental Protection Agency (USEPA) regulations. Ratios lower than the USEPA risk threshold suggested higher concentrations of oocysts in relation to E. coli concentrations, revealing an underestimed risk for Cryptosporidium based on E. coli measurements. In raw sewage (RS), high ratios proved E. coli (or fecal coliforms) concentrations were a conservative indicator of Cryptosporidium concentrations, which was also typically true for secondary treated wastewater (TWW). Removals of fecal indicator bacteria (FIB) and parasites were quantified in WWTPs and their differences are put forward as a plausible explanation of the sporadic ratio shift. Ratios measured from agricultural runoff surface water were typically lower than the USEPA risk threshold and within the range of risk misinterpretation. Indeed, heavy precipitation events in the agricultural watershed led to high oocyst concentrations but not to E. coli or enterococci concentrations. More importantly, ratios established in variously impacted DWI from 13 Canadian drinking water plants were found to be related to dominant fecal pollution sources, namely municipal sewage. In most cases, when DWIs were mainly influenced by municipal sewage, E. coli or fecal coliforms concentrations agreed with

Gold-Coated Iron Composite Nanospheres Targeted the Detection of Escherichia coli

Directory of Open Access Journals (Sweden)

İlker Dinçer

2013-03-01

Full Text Available We report the preparation and characterization of spherical core-shell structured Fe3O4–Au magnetic nanoparticles, modified with two component self-assembled monolayers (SAMs consisting of 3–mercaptophenylboronic acid (3–MBA and 1–decanethiol (1–DT. The rapid and room temperature synthesis of magnetic nanoparticles was achieved using the hydroxylamine reduction of HAuCl4 on the surface of ethylenediaminetetraacetic acid (EDTA-immobilized iron (magnetite Fe3O4 nanoparticles in the presence of an aqueous solution of hexadecyltrimetylammonium bromide (CTAB as a dispersant. The reduction of gold on the surface of Fe3O4 nanoparticles exhibits a uniform, highly stable, and narrow particle size distribution of Fe3O4–Au nanoparticles with an average diameter of 9 ± 2 nm. The saturation magnetization value for the resulting nanoparticles was found to be 15 emu/g at 298 K. Subsequent surface modification with SAMs against glucoside moieties on the surface of bacteria provided effective magnetic separation. Comparison of the bacteria capturing efficiency, by means of different molecular recognition agents 3–MBA, 1–DT and the mixed monolayer of 3–MBA and 1–DT was presented. The best capturing efficiency of E. coli was achieved with the mixed monolayer of 3–MBA and 1–DT-modified nanoparticles. Molecular specificity and selectivity were also demonstrated by comparing the surface-enhanced Raman scattering (SERS spectrum of E. coli-nanoparticle conjugates with bacterial growth media.

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation.

Science.gov (United States)

Nguyen-Chi, Mai; Phan, Quang Tien; Gonzalez, Catherine; Dubremetz, Jean-François; Levraud, Jean-Pierre; Lutfalla, Georges

2014-07-01

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation. © 2014. Published by The Company of Biologists Ltd.

Survival of pathogenic enterohemorrhagic Escherichia coli (EHEC) and control with calcium oxide in frozen meat products.

Science.gov (United States)

Ro, Eun Young; Ko, Young Mi; Yoon, Ki Sun

2015-08-01

This study investigated both the level of microbial contamination and the presence of enterohemorrhagic Escherichia coli (EHEC) in frozen meat products, followed by the evaluation of its survival over 180 days under frozen temperature. We also examined the effect of calcium oxide on the populations of EHEC, E. coli O157:H7 and EPEC under both 10 °C and -18 °C storage conditions. Afterward, the morphological changes occurring in EHEC cells in response to freezer storage temperature and calcium oxide (CaO) treatments were examined using transmission electron microscopy. Among the frozen meat products tested, the highest contamination levels of total aerobic counts, coliforms and E. coli were observed in pork cutlets. Examination showed that 20% of the frozen meat products contained virulence genes, including verotoxin (VT) 1 and 2. Over 180 days of frozen storage and after 3 freeze-thaw cycles, the population of EHEC did not change regardless of the type of products or initial inoculated concentration, indicating the strong survival ability of EHEC. Subsequent testing revealed that the growth of three pathogenic E. coli strains was completely inhibited in meat patties prepared with 1% CaO, stored at 10 °C. However, the addition of 2% CaO was necessary to control the survival of EHEC, E. coli O157:H7 and EPEC in meat patties stored at -18 °C. CaO reduced the population of E. coli O157:H7 more effectively than the other EHEC and EPEC strains at both 10 °C and -18 °C. Transmission electron microscopy analysis revealed that exposed EHEC cells were resistant to the freezer storage temperature, although some cells incurred injury and death after several freeze-thaw cycles. Most of the cells exposed to CaO were found to have died or lost their cellular integrity and membranes, indicating that CaO has the potential to be used as a powerful antimicrobial agent for manufacturing frozen meat products. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effectiveness of lytic bacteriophages in reducing E. coli O157:H7 populations introduced through cross-contamination on fresh cut lettuce

Science.gov (United States)

Previous research has shown that lytic bacteriophages (phages) can kill E. coli O157:H7 on produce surfaces. The role of lytic bacteriophages in preventing cross contamination of produce has not been evaluated. A cocktail of three lytic phages specific for E. coli O157:H7 (EcoShield) at 10^8 PFU/m...

Fate of Escherichia coli O157: H7 in agricultural soils amended with different organic fertilizers.

Science.gov (United States)

Yao, Zhiyuan; Yang, Li; Wang, Haizhen; Wu, Jianjun; Xu, Jianming

2015-10-15

Five organic fertilizers (vermicompost, pig manure, chicken manure, peat and oil residue) were applied to agricultural soils to study their effects on the survival of Escherichia coli O157:H7 (E. coli O157:H7). Results showed that E. coli O157:H7 survival changed greatly after organic fertilizers application, with shorter td values (survival time needed to reach the detection limit of 100 CFU g(-1)) (12.57±6.57 days) in soils amended with chicken manure and the longest (25.65±7.12 days) in soils amended with pig manure. Soil pH, EC and free Fe/Al (hydro) oxides were significant explanatory factors for E. coli O157:H7 survival in the original soils. Soil constituents (minerals and organic matter) and changes in their surface charges with pH increased the effect of soil pH on E. coli O157:H7 survival. However, electrical conductivity played a more important role in regulating E. coli O157:H7 survival in fertilizer-amended soils. This study highlighted the importance of choosing appropriate organic fertilizers in the preharvest environment to reduce food-borne bacterial contamination. Copyright © 2015 Elsevier B.V. All rights reserved.

Evolutionary Dynamics of Small RNAs in 27 Escherichia coli and Shigella Genomes

Science.gov (United States)

Skippington, Elizabeth; Ragan, Mark A.

2012-01-01

Small RNAs (sRNAs) are widespread in bacteria and play critical roles in regulating physiological processes. They are best characterized in Escherichia coli K-12 MG1655, where 83 sRNAs constitute nearly 2% of the gene complement. Most sRNAs act by base pairing with a target mRNA, modulating its translation and/or stability; many of these RNAs share only limited complementarity to their mRNA target, and require the chaperone Hfq to facilitate base pairing. Little is known about the evolutionary dynamics of bacterial sRNAs. Here, we apply phylogenetic and network analyses to investigate the evolutionary processes and principles that govern sRNA gene distribution in 27 E. coli and Shigella genomes. We identify core (encoded in all 27 genomes) and variable sRNAs; more than two-thirds of the E. coli K-12 MG1655 sRNAs are core, whereas the others show patterns of presence and absence that are principally due to genetic loss, not duplication or lateral genetic transfer. We present evidence that variable sRNAs are less tightly integrated into cellular genetic regulatory networks than are the core sRNAs, and that Hfq facilitates posttranscriptional cross talk between the E. coli–Shigella core and variable genomes. Finally, we present evidence that more than 80% of genes targeted by Hfq-associated core sRNAs have been transferred within the E. coli–Shigella clade, and that most of these genes have been transferred intact. These results suggest that Hfq and sRNAs help integrate laterally acquired genes into established regulatory networks. PMID:22223756

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Science.gov (United States)

Jones, Randall T.; Koeltzow, Donald E.; Stocker, B. A. D.

1972-01-01

Escherichia coli K-12 ϰ971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv+ hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his+ (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F′ factor (FS400) carrying the rfb–his region of S. typhimurium to the same two ilv+ hybrids gave similar results. LPS extracted from two ilv+,his+, factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his+ hybrids obtained from ϰ971 itself by similar HfrK9 and F′FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli ϰ971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli ϰ971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli ϰ971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his+ recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Ω8. This suggests that, although the parental E. coli K-12 strain ϰ971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units. PMID:4559827

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

Science.gov (United States)

Serra, Diego O.; Richter, Anja M.

2013-01-01

Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of “nature and nurture” in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used “domesticated” E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by “repairing” a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this “de-domesticated” strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that—together with the cell-encasing curli fiber network and geometrical constraints in a growing colony—explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and “tissue-like” bacterial biofilms. PMID:24097954

Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.

Science.gov (United States)

Levin, M S; Locke, B; Yang, N C; Li, E; Gordon, J I

1988-11-25

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) are 132-residue cytosolic proteins which have 56% amino acid sequence identity and bind all-trans-retinol as their endogenous ligand. They belong to a family of cytoplasmic proteins which have evolved to bind distinct hydrophobic ligands. Their patterns of tissue-specific and developmental regulation are distinct. We have compared the ligand binding properties of rat apo-CRBP and apo-CRBP II that have been expressed in Escherichia coli. Several observations indicate that the E. coli-derived apoproteins are structurally similar to the native rat proteins: they co-migrate on isoelectric focusing gels; and when complexed with all-trans-retinol, their absorption and excitation/emission spectra are nearly identical to those of the authentic rat holoproteins. Comparative lifetime and acrylamide quenching studies suggest that there are differences in the conformations of apo-CRBP and apo-CRBP II. The interaction of E. coli-derived apo-CRBP and apo-CRBP II with a variety of retinoids was analyzed using spectroscopic techniques. Both apoproteins formed high affinity complexes with all-trans-retinol (K'd approximately 10 nM). In direct binding assays, all-trans-retinal bound to both apoproteins (K'd approximately 50 nM for CRBP; K'd approximately 90 nM for CRBP II). However, all-trans-retinal could displace all-trans-retinol bound to CRBP II but not to CRBP. These observations suggests that there is a specific yet distinct interaction between these two proteins and all-trans-retinal. Apo-CRBP and apo-CRBP II did not demonstrate significant binding to either retinoic acid or methyl retinoate, an uncharged derivative of all-trans-retinoic acid. This indicates that the carboxymethyl group of methyl retinoate cannot be sterically accommodated in their binding pockets and that failure to bind retinoic acid probably is not simply due to the negative charge of its C-15 carboxylate group

Contamination of Ethiopian paper currency notes from various food handlers with E. coli.

Science.gov (United States)

Hiko, Adem; Abdata, Kasahun; Muktar, Yimer; Woyesa, Mezene; Mohammed, Abdela

2016-01-01

Contamination rate of Ethiopian paper currency notes handled by various food handlers with Escherichia coli and antimicrobial susceptibility of the isolates was assessed. A total of 384 Ethiopian Birr (ETB) notes were randomly sampled from meat handlers at butchers, bread and the related food handlers at cafeteria, fruit and vegetables handlers at supermarket, and milk sellers both at open market and dairy station. Fifty control new currencies were also sampled from Commercial Bank of Ethiopia. Both surfaces of the currency were swabbed using wet sterile cotton. The swab was overnight incubated in buffered peptone water. A loop full was streaked on eosin methylene blue agar and followed by biochemical test on presumptive E. coli colonies. Randomly selected isolates were exposed to chloramphenicol (C-30 µg), neomycin (N-30 µg), oxytetracycline (OT-30 µg), polymyxin-B (PB-300 IU) and trimethoprim-sulfamethoxazole (SXT-1.25/23.75/µg) susceptibility using disc diffusion techniques. E. coli was not isolated from currency used as control. A total of 288 (75 %) currency notes were found carrying E. coli. E. coli prevalence was ranges from 67.2 % at open market milk sellers to 87.2 % at dairy station milk sellers; from 64.8 % on ETB 100 to 82.9 % on ETB 1. Differences were not observed in E. coli prevalence on currency notes from among almost all food handlers (P > 0.05). Susceptibility of tested isolates to each chloramphenicol, oxytetracycline and trimethoprim-sulfamethoxazole was 100 %, and to polymyxin-B was 97.3 %. High resistance (83.7 %) was observed to neomycin. The finding indicates, contaminated food can be a source of E. coli for further contamination of currency which again transfer through various foods ready for consumption.

Transcriptomic analysis displays the effect of (-)-roemerine on the motility and nutrient uptake in Escherichia coli.

Science.gov (United States)

Ayyildiz, Dilara; Arga, Kazim Yalcin; Avci, Fatma Gizem; Altinisik, Fatma Ece; Gurer, Caglayan; Gulsoy Toplan, Gizem; Kazan, Dilek; Wozny, Katharina; Brügger, Britta; Mertoglu, Bulent; Sariyar Akbulut, Berna

2017-08-01

Among the different families of plant alkaloids, (-)-roemerine, an aporphine type, was recently shown to possess significant antibacterial activity in Escherichia coli. Based on the increasing demand for antibacterials with novel mechanisms of action, the present work investigates the potential of the plant-derived alkaloid (-)-roemerine as an antibacterial in E. coli cells using microarray technology. Analysis of the genome-wide transcriptional reprogramming in cells after 60 min treatment with 100 μg/mL (-)-roemerine showed significant changes in the expression of 241 genes (p value 2). Expression of selected genes was confirmed by qPCR. Differentially expressed genes were classified into functional categories to map biological processes and molecular pathways involved. Cellular activities with roles in carbohydrate transport and metabolism, energy production and conversion, lipid transport and metabolism, amino acid transport and metabolism, two-component signaling systems, and cell motility (in particular, the flagellar organization and motility) were among metabolic processes altered in the presence of (-)-roemerine. The down-regulation of the outer membrane proteins probably led to a decrease in carbohydrate uptake rate, which in turn results in nutrient limitation. Consequently, energy metabolism is slowed down. Interestingly, the majority of the expressional alterations were found in the flagellar system. This suggested reduction in motility and loss in the ability to form biofilms, thus affecting protection of E. coli against host cell defense mechanisms. In summary, our findings suggest that the antimicrobial action of (-)-roemerine in E. coli is linked to disturbances in motility and nutrient uptake.

Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

DEFF Research Database (Denmark)

Winberg, Line Kjær; Nielsen, Claus Henrik; Jacobsen, Søren

2017-01-01

Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes. These fea......Objectives: To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes...

Graphene Field-Effect Transistors for the Sensitive and Selective Detection of Escherichia coli Using Pyrene-Tagged DNA Aptamer.

Science.gov (United States)

Wu, Guangfu; Dai, Ziwen; Tang, Xin; Lin, Zihong; Lo, Pik Kwan; Meyyappan, M; Lai, King Wai Chiu

2017-10-01

This study reports biosensing using graphene field-effect transistors with the aid of pyrene-tagged DNA aptamers, which exhibit excellent selectivity, affinity, and stability for Escherichia coli (E. coli) detection. The aptamer is employed as the sensing probe due to its advantages such as high stability and high affinity toward small molecules and even whole cells. The change of the carrier density in the probe-modified graphene due to the attachment of E. coli is discussed theoretically for the first time and also verified experimentally. The conformational change of the aptamer due to the binding of E. coli brings the negatively charged E. coli close to the graphene surface, increasing the hole carrier density efficiently in graphene and achieving electrical detection. The binding of negatively charged E. coli induces holes in graphene, which are pumped into the graphene channel from the contact electrodes. The carrier mobility, which correlates the gate voltage to the electrical signal of the APG-FETs, is analyzed and optimized here. The excellent sensing performance such as low detection limit, high sensitivity, outstanding selectivity and stability of the graphene biosensor for E. coli detection paves the way to develop graphene biosensors for bacterial detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative genomics and transcriptomics of Escherichia coli isolates carrying virulence factors of both enteropathogenic and enterotoxigenic E. coli.

Science.gov (United States)

Hazen, Tracy H; Michalski, Jane; Luo, Qingwei; Shetty, Amol C; Daugherty, Sean C; Fleckenstein, James M; Rasko, David A

2017-06-14

Escherichia coli that are capable of causing human disease are often classified into pathogenic variants (pathovars) based on their virulence gene content. However, disease-associated hybrid E. coli, containing unique combinations of multiple canonical virulence factors have also been described. Such was the case of the E. coli O104:H4 outbreak in 2011, which caused significant morbidity and mortality. Among the pathovars of diarrheagenic E. coli that cause significant human disease are the enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC). In the current study we use comparative genomics, transcriptomics, and functional studies to characterize isolates that contain virulence factors of both EPEC and ETEC. Based on phylogenomic analysis, these hybrid isolates are more genomically-related to EPEC, but appear to have acquired ETEC virulence genes. Global transcriptional analysis using RNA sequencing, demonstrated that the EPEC and ETEC virulence genes of these hybrid isolates were differentially-expressed under virulence-inducing laboratory conditions, similar to reference isolates. Immunoblot assays further verified that the virulence gene products were produced and that the T3SS effector EspB of EPEC, and heat-labile toxin of ETEC were secreted. These findings document the existence and virulence potential of an E. coli pathovar hybrid that blurs the distinction between E. coli pathovars.

Binding of collagens to an enterotoxigenic strain of Escherichia coli

International Nuclear Information System (INIS)

Visai, L.; Speziale, P.; Bozzini, S.

1990-01-01

An enterotoxigenic strain of Escherichia coli, B34289c, has been shown to bind the N-terminal region of fibronectin with high affinity. We now report that this strain also binds collagen. The binding of 125I-labeled type II collagen to bacteria was time dependent and reversible. Bacteria expressed a limited number of collagen receptors (2.2 x 10(4) per cell) and bound collagen with a Kd of 20 nM. All collagen types tested (I to V) as well as all tested cyanogen bromide-generated peptides [alpha 1(I)CB2, alpha 1(I)CB3, alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB4] were recognized by bacterial receptors, as demonstrated by the ability of these proteins to inhibit the binding of 125I-labeled collagen to bacteria. Of several unlabeled proteins tested in competition experiments, fibronectin and its N-terminal region strongly inhibited binding of the radiolabeled collagen to E. coli cells. Conversely, collagen competed with an 125I-labeled 28-kilodalton fibronectin fragment for bacterial binding. Collagen bound to bacteria could be displaced by excess amounts of either unlabeled fibronectin or its N-terminal fragment. Similarly, collagen could displace 125I-labeled N-terminal peptide of fibronectin bound to the bacterial cell surface. Bacteria grown at 41 degrees C or in the presence of glucose did not express collagen or fibronectin receptors. These results indicate the presence of specific binding sites for collagen on the surface of E. coli cells and furthermore that the collagen and fibronectin binding sites are located in close proximity, possibly on the same structure

Thioredoxin from Escherichia coli

International Nuclear Information System (INIS)

Holmgren, A.; Ohlsson, I.; Grankvist, M.L.

1978-01-01

A competition radioimmunoassay for Escherichia coli thioredoxin using 125 I-labeled thioredoxin-S 2 and a double antibody technique was developed. The method permits determination of picomole amounts of thioredoxin in crude cell extracts and was used to study the localization of thioredoxin cell fractions. E. coli B was calculated to have approximately 10,000 copies of thioredoxin per cell mainly located in the soluble fraction after separation of the membrane and soluble fractions by gentle lysis and centrifugation. E. coli B tsnC mutants which are defective in the replication of phage T7 DNA in vivo and in vitro were examined for their content of thioredoxin. E. coli B tsnC 7004 contained no detectable level of thioredoxin in cell-free extracts examined under a variety of conditions. The results strongly suggest that tsnC 7004 is a nonsense or deletion mutant. Two other E. coli tsnC mutants, 7007 and 7008, contained detectable levels of thioredoxin in crude extracts as measured by thioredoxin reductase and gave similar immunoprecipitation reactions as the parent strain B/1. By radioimmunoassay incompletely cross-reacting material was present in both strains. These results show that tsnC 7007 and 7008 belong to a type of thioredoxin mutants with missence mutations in the thioredoxin gene affecting the function of thioredoxin as subunit in phage T7 DNA polymerase

Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

Energy Technology Data Exchange (ETDEWEB)

Svintradze, David V. [Virginia Commonwealth University, Richmond, VA 23298-0566 (United States); Virginia Commonwealth University, Richmond, VA 23219-1540 (United States); Peterson, Darrell L. [Virginia Commonwealth University, Richmond, VA 23219-1540 (United States); Virginia Commonwealth University, Richmond, VA 23298-0614 (United States); Collazo-Santiago, Evys A.; Lewis, Janina P. [Virginia Commonwealth University, Richmond, VA 23298-0566 (United States); Wright, H. Tonie, E-mail: xrdproc@vcu.edu [Virginia Commonwealth University, Richmond, VA 23219-1540 (United States); Virginia Commonwealth University, Richmond, VA 23298-0614 (United States); Virginia Commonwealth University, Richmond, VA 23298-0566 (United States)

2013-10-01

Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each.

Structures of the Porphyromonas gingivalis OxyR regulatory domain explain differences in expression of the OxyR regulon in Escherichia coli and P. gingivalis

International Nuclear Information System (INIS)

Svintradze, David V.; Peterson, Darrell L.; Collazo-Santiago, Evys A.; Lewis, Janina P.; Wright, H. Tonie

2013-01-01

Differences in OxyR regulated expression of oxidative stress genes between Escherichia coli and Porphyromonas gingivalis are explained by very minor differences in structure and amino-acid sequence of the respective oxidized and reduced OxyR regulatory domains. These differences affect OxyR quaternary structures and are predicted from model building of full length OxyR–DNA complexes to confer distinct modes of DNA binding on this transcriptional regulator. OxyR transcriptionally regulates Escherichia coli oxidative stress response genes through a reversibly reducible cysteine disulfide biosensor of cellular redox status. Structural changes induced by redox changes in these cysteines are conformationally transmitted to the dimer subunit interfaces, which alters dimer and tetramer interactions with DNA. In contrast to E. coli OxyR regulatory-domain structures, crystal structures of Porphyromonas gingivalis OxyR regulatory domains show minimal differences in dimer configuration on changes in cysteine disulfide redox status. This locked configuration of the P. gingivalis OxyR regulatory-domain dimer closely resembles the oxidized (activating) form of the E. coli OxyR regulatory-domain dimer. It correlates with the observed constitutive activation of some oxidative stress genes in P. gingivalis and is attributable to a single amino-acid insertion in P. gingivalis OxyR relative to E. coli OxyR. Modelling of full-length P. gingivalis, E. coli and Neisseria meningitidis OxyR–DNA complexes predicts different modes of DNA binding for the reduced and oxidized forms of each

Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR.

Science.gov (United States)

Liu, Yarui; Mustapha, Azlin

2014-01-17

Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 μM PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Cellular automata and statistical mechanical models

International Nuclear Information System (INIS)

Rujan, P.

1987-01-01

The authors elaborate on the analogy between the transfer matrix of usual lattice models and the master equation describing the time development of cellular automata. Transient and stationary properties of probabilistic automata are linked to surface and bulk properties, respectively, of restricted statistical mechanical systems. It is demonstrated that methods of statistical physics can be successfully used to describe the dynamic and the stationary behavior of such automata. Some exact results are derived, including duality transformations, exact mappings, disorder, and linear solutions. Many examples are worked out in detail to demonstrate how to use statistical physics in order to construct cellular automata with desired properties. This approach is considered to be a first step toward the design of fully parallel, probabilistic systems whose computational abilities rely on the cooperative behavior of their components

Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

Science.gov (United States)

Srinivasan, Ramanujam; Mishra, Mithilesh; Murata-Hori, Maki; Balasubramanian, Mohan K

2007-02-06

Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

Coating of silicone with mannoside-PAMAM dendrimers to enhance formation of non-pathogenic Escherichia coli biofilms against colonization of uropathogens.

Science.gov (United States)

Zhu, Zhiling; Yu, Fei; Chen, Haoqing; Wang, Jun; Lopez, Analette I; Chen, Quan; Li, Siheng; Long, Yuyu; Darouiche, Rabih O; Hull, Richard A; Zhang, Lijuan; Cai, Chengzhi

2017-12-01

Bacterial interference using non-pathogenic Escherichia coli 83972 is a novel strategy for preventing catheter-associated urinary tract infection (CAUTI). Crucial to the success of this strategy is to establish a high coverage and stable biofilm of the non-pathogenic bacteria on the catheter surface. However, this non-pathogenic strain is sluggish to form biofilms on silicone as the most widely used material for urinary catheters. We have addressed this issue by modifying the silicone catheter surfaces with mannosides that promote the biofilm formation, but the stability of the non-pathogenic biofilms challenged by uropathogens over long-term remains a concern. Herein, we report our study on the stability of the non-pathogenic biofilms grown on propynylphenyl mannoside-modified silicone. The result shows that 94% non-pathogenic bacteria were retained on the modified silicone under >0.5 Pa shear stress. After being challenged by three multidrug-resistant uropathogenic isolates in artificial urine for 11 days, large amounts (>4 × 10 6  CFU cm -2 ) of the non-pathogenic bacteria remained on the surfaces. These non-pathogenic biofilms reduced the colonization of the uropathogens by >3.2-log. In bacterial interference, the non-pathogenic Escherichia coli strains are sluggish to form biofilms on the catheter surfaces, due to rapid removal by urine flow. We have demonstrated a solution to this bottleneck by pre-functionalization of mannosides on the silicone surfaces to promote E. coli biofilm formation. A pre-conjugated high affinity propynylphenyl mannoside ligand tethered to the nanometric amino-terminated poly(amido amine) (PAMAM) dendrimer is used for binding to a major E. coli adhesin FimH. It greatly improves the efficiency for the catheter modification, the non-pathogenic biofilm coverage, as well as the (long-term) stability for prevention of uropathogen infections. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157:H7 by Propidium Monoazide Real-Time PCR

Directory of Open Access Journals (Sweden)

Hui Liu

2014-01-01

Full Text Available Conventional methods to determine the efficacy of bacteriophage (phage for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA, a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01 phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7.

Removal of bacteria Legionella pneumophila, Escherichia coli, and Bacillus subtilis by (super)cavitation.

Science.gov (United States)

Šarc, Andrej; Kosel, Janez; Stopar, David; Oder, Martina; Dular, Matevž

2018-04-01

In sufficient concentrations, the pathogenic bacteria L. pneumophila can cause a respiratory illness that is known as the "Legionnaires" disease. Moreover, toxic Shiga strains of bacteria E. coli can cause life-threatening hemolytic-uremic syndrome. Because of the recent restrictions imposed on the usage of chlorine, outbreaks of these two bacterial species have become more common. In this study we have developed a novel rotation generator and its effectiveness against bacteria Legionella pneumophila and Escherichia coli was tested for various types of hydrodynamic cavitation (attached steady cavitation, developed unsteady cavitation and supercavitation). The results show that the supercavitation was the only effective form of cavitation. It enabled more than 3 logs reductions for both bacterial species and was also effective against a more persistent Gram positive bacteria, B. subtilis. The deactivation mechanism is at present unknown. It is proposed that when bacterial cells enter a supercavitation cavity, an immediate pressure drop occurs and this results in bursting of the cellular membrane. The new rotation generator that induced supercavitation proved to be economically and microbiologically far more effective than the classical Venturi section (super)cavitation. Copyright © 2017 Elsevier B.V. All rights reserved.

Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface.

Science.gov (United States)

Fantappiè, Laura; Irene, Carmela; De Santis, Micaela; Armini, Alessandro; Gagliardi, Assunta; Tomasi, Michele; Parri, Matteo; Cafardi, Valeria; Bonomi, Serena; Ganfini, Luisa; Zerbini, Francesca; Zanella, Ilaria; Carnemolla, Chiara; Bini, Luca; Grandi, Alberto; Grandi, Guido

2017-07-01

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Parameters and characteristics governing cellular internalization and trans-barrier trafficking of nanostructures

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Murugan K

2015-03-01

Full Text Available Karmani Murugan, Yahya E Choonara, Pradeep Kumar, Divya Bijukumar, Lisa C du Toit, Viness Pillay Wits Advanced Drug Delivery Platform Research Unit, Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa Abstract: Cellular internalization and trans-barrier transport of nanoparticles can be manipulated on the basis of the physicochemical and mechanical characteristics of nanoparticles. Research has shown that these factors significantly influence the uptake of nanoparticles. Dictating these characteristics allows for the control of the rate and extent of cellular uptake, as well as delivering the drug-loaded nanosystem intra-cellularly, which is imperative for drugs that require a specific cellular level to exert their effects. Additionally, physicochemical characteristics of the nanoparticles should be optimal for the nanosystem to bypass the natural restricting phenomena of the body and act therapeutically at the targeted site. The factors at the focal point of emerging smart nanomedicines include nanoparticle size, surface charge, shape, hydrophobicity, surface chemistry, and even protein and ligand conjugates. Hence, this review discusses the mechanism of internalization of nanoparticles and ideal nanoparticle characteristics that allow them to evade the biological barriers in order to achieve optimal cellular uptake in different organ systems. Identifying these parameters assists with the progression of nanomedicine as an outstanding vector of pharmaceuticals. Keywords: nanoparticles, transport mechanisms, cellular uptake, size, shape, charge

The F4 fimbrial antigen of Escherichia coli and its receptors.

NARCIS (Netherlands)

Van den Broeck, W; Cox, E; Oudega, B.; Goddeeris, B

2000-01-01

F4 or K88 fimbriae are long filamentous polymeric surface proteins of enterotoxigenic Escherichia coli (ETEC), consisting of so-called major (FaeG) and minor (FaeF, FaeH, FaeC, and probably FaeI) subunits. Several serotypes of F4 have been described, namely F4ab, F4ac, and F4ad. The F4 fimbriae

Hierarchical Targeting Strategy for Enhanced Tumor Tissue Accumulation/Retention and Cellular Internalization.

Science.gov (United States)

Wang, Sheng; Huang, Peng; Chen, Xiaoyuan

2016-09-01

Targeted delivery of therapeutic agents is an important way to improve the therapeutic index and reduce side effects. To design nanoparticles for targeted delivery, both enhanced tumor tissue accumulation/retention and enhanced cellular internalization should be considered simultaneously. So far, there have been very few nanoparticles with immutable structures that can achieve this goal efficiently. Hierarchical targeting, a novel targeting strategy based on stimuli responsiveness, shows good potential to enhance both tumor tissue accumulation/retention and cellular internalization. Here, the recent design and development of hierarchical targeting nanoplatforms, based on changeable particle sizes, switchable surface charges and activatable surface ligands, will be introduced. In general, the targeting moieties in these nanoplatforms are not activated during blood circulation for efficient tumor tissue accumulation, but re-activated by certain internal or external stimuli in the tumor microenvironment for enhanced cellular internalization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical detection of specific DNA and respiratory activity of Escherichia coli

International Nuclear Information System (INIS)

Yamanaka, Keiichiro; Ikeuchi, Tomohiko; Saito, Masato; Nagatani, Naoki; Tamiya, Eiichi

2012-01-01

We present two rapid and simplified detection methods for Escherichia coli involving the use of a hand-held potentiostat and a disposable screen-printed carbon electrode. E. coli is one of the indicator organisms used to access for food safety. Commonly, microbiological culture techniques take more than one day to yield results and therefore, a simple, cost-effective, in situ detection system is required for testing food safety. This report describes two complementary techniques for high- and low-sensitivity detection of E. coli. High-sensitivity detection relies upon quantification of DNA amplification by using polymerase chain reaction (PCR), while the simplified, low-sensitivity detection can be obtained through measurement of oxygen consumption due to respiration; importantly, both techniques utilize the same type of electrode. The former entails mixing the PCR mixture with Hoechst, an electro-active DNA intercalator, and then, measuring the oxidation current. Binding of Hoechst molecules to the amplified DNA causes the peak current to decrease because of the slow diffusion of the Hoechst-amplified DNA complex to the electrode surface. The results showed that the oxidation peak current of Hoechst decreased depending on the number of E. coli cells added to the PCR mixture as the template for amplification, and the sensitivity of the method was as low as a single bacterium. Oxygen consumption was detected by direct measurement of the cell-containing culture medium. This method required only 10 μL to be applied on the screen-printed electrode, and the reduction in oxygen current was clearly observed within 30 min when a minimum of 1 × 10 5 cells were present. These results were obtained without purifying the culture, and the samples were applied onto the electrode without any surface modifications. The techniques describes in this report are versatile, because they require the same type of electrode, have simplistic nature, use a hand-held potentiostat, and have

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

Science.gov (United States)

Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T; Kralj, Joel M

2017-08-29

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules.

Science.gov (United States)

Kenny, Brendan; Ellis, Sarah; Leard, Alan D; Warawa, Jonathan; Mellor, Harry; Jepson, Mark A

2002-05-01

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction. We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

Protein N-myristoylation in Escherichia coli: Reconstitution of a eukaryotic protein modification in bacteria

International Nuclear Information System (INIS)

Duronio, R.J.; Jackson-Machelski, E.; Heuckeroth, R.O.; Gordon, J.I.; Olins, P.O.; Devine, C.S.; Yonemoto, W.; Slice, L.W.; Taylor, S.S.

1990-01-01

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH 2 -terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. The authors have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH 2 -terminal 39 amino acids. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed

Hemolytic Porcine Intestinal Escherichia coli without Virulence-Associated Genes Typical of Intestinal Pathogenic E. coli ▿ †

Science.gov (United States)

Schierack, Peter; Weinreich, Joerg; Ewers, Christa; Tachu, Babila; Nicholson, Bryon; Barth, Stefanie

2011-01-01

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli. PMID:21965399

Distribution, Numbers, and Diversity of ESBL-Producing E. coli in the Poultry Farm Environment.

Directory of Open Access Journals (Sweden)

Hetty Blaak

Full Text Available This study aimed to discern the contribution of poultry farms to the contamination of the environment with ESBL-producing Escherichia coli and therewith, potentially to the spread of these bacteria to humans and other animals. ESBL-producing E. coli were detected at all investigated laying hen farms (n = 5 and broiler farms (n = 3 in 65% (46/71 and 81% (57/70 of poultry faeces samples, respectively. They were detected in rinse water and run-off water (21/26; 81%, other farm animals (11/14; 79%, dust (21/35; 60%, surface water adjacent to farms (20/35; 57%, soil (48/87; 55%, on flies (11/73; 15%, and in barn air (2/33; 6%. The highest prevalence and concentrations in the outdoor environment were observed in soil of free-range areas at laying hen farms (100% of samples positive, geometric mean concentration 2.4×10(4 cfu/kg, and surface waters adjacent to broiler farms during, or shortly after, cleaning between production rounds (91% of samples positive, geometric mean concentration 1.9×10(2 cfu/l. The diversity of ESBL-producing E. coli variants with respect to sequence type, phylogenetic group, ESBL-genotype and antibiotic resistance profile was high, especially on broiler farms where on average 16 different variants were detected, and the average Simpson's Indices of diversity (SID; 1-D were 0.93 and 0.94 among flock and environmental isolates respectively. At laying hen farms on average nine variants were detected, with SIDs of 0.63 (flock isolates and 0.77 (environmental isolates. Sixty percent of environmental isolates were identical to flock isolates at the same farm. The highest proportions of 'flock variants' were observed in dust (94%, run-off gullies (82%, and barn air (67%, followed by surface water (57%, soil (56%, flies (50% and other farm animals (35%.The introduction of ESBL-producing E. coli from poultry farms to the environment may pose a health risk if these bacteria reach places where people may become exposed.

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex

DEFF Research Database (Denmark)

Kruse, Thomas; Bork-Jensen, Jette; Gerdes, Kenn

2005-01-01

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B...... carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and Mre....... subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became...

ESBL-Producing Escherichia coli

DEFF Research Database (Denmark)

Hertz, Frederik Boetius

Urinary tract infection (UTI) is one the most common bacterial infections and is regularly treated in primary health care. The most common cause of UTI is extraintestinal pathogenic Escherichia coli (ExPEC) already present in the intestinal microflora, often as the dominating strain. Resistance...... in E.coli is increasing and especially isolates producing Extended-Spectrum Beta-Lactamases (ESBL) have been reported worldwide. Treatment of UTI is usually initiated by the general practitioners and a significant proportion of clinical isolates are now resistant to first line antibiotics. The global...... to investigate (i) antibiotics involved in selection of ESBL-producing E.coli, in an experimental mouse model in vivo, (ii) risk factors for UTI with ESBL-producing E.coli and (iii) to describe the phylogenetic composition of E.coli populations with different resistance patterns. We found that different...

AFM studies of environmental effects on nanomechanical properties and cellular structure of human hair

International Nuclear Information System (INIS)

Bhushan, Bharat; Chen, Nianhuan

2006-01-01

Characterization of cellular structure and physical and mechanical properties of hair are essential to develop better cosmetic products and advance biological and cosmetic science. Although the morphology of the cellular structure of human hair has been traditionally investigated using scanning electron microscopy and transmission electron microscopy, these techniques provide limited capability to in situ study of the physical and mechanical properties of human hair in various environments. Atomic force microscopy (AFM) overcomes these problems and can be used for characterization in ambient conditions without requiring specific sample preparations and surface treatment. In this study, film thickness, adhesive forces and effective Young's modulus of various hair surfaces were measured at different environments (humidity and temperature) using force calibration plot technique with an AFM. Torsional resonance mode phase contrast images were also taken in order to characterize the morphology and cellular structure changes of human hair at different humidity. The correlation between the nanomechanical properties and the cellular structure of hair is discussed

On-line prediction of the feeding phase in high-cell density cultivation of rE. coli using constructive neural networks.

Science.gov (United States)

Nicoletti, M C; Bertini, J R; Tanizaki, M M; Zangirolami, T C; Gonçalves, V M; Horta, A C L; Giordano, R C

2013-07-01

Streptococcus pneumoniae (pneumococcus) is a bacterium responsible for a wide spectrum of illnesses. The surface of the bacterium consists of three distinctive membranes: plasmatic, cellular and the polysaccharide (PS) capsule. PS capsules may mediate several biological processes, particularly invasive infections of human beings. Prevention against pneumococcal related illnesses can be provided by vaccines. There is a sound investment worldwide in the investigation of a proteic antigen as a possible alternative to pneumococcal vaccines based exclusively on PS. A few proteins which are part of the membrane of the pneumococcus seem to have antigen potential to be part of a vaccine, particularly the PspA. A vital aspect in the production of the intended conjugate pneumococcal vaccine is the efficient production (in industrial scale) of both, the chosen PS serotypes as well as the PspA protein. Growing recombinant Escherichia coli (rE. coli) in high-cell density cultures (HCDC) under a fed-batch regime requires a refined continuous control over various process variables where the on-line prediction of the feeding phase is of particular relevance and one of the focuses of this paper. The viability of an on-line monitoring software system, based on constructive neural networks (CoNN), for automatically detecting the time to start the fed-phase of a HCDC of rE. coli that contains a plasmid used for PspA expression is investigated. The paper describes the data and methodology used for training five different types of CoNNs, four of them suitable for classification tasks and one suitable for regression tasks, aiming at comparatively investigate both approaches. Results of software simulations implementing five CoNN algorithms as well as conventional neural networks (FFNN), decision trees (DT) and support vector machines (SVM) are also presented and discussed. A modified CasCor algorithm, implementing a data softening process, has shown to be an efficient candidate to be

Hygienic-sanitary quality of vegetables and evaluation of treatments for the elimination of indigenous E. coli and E. coli O157:H7 from the surface of leaves of lettuce (Lactuca sativa L.)

OpenAIRE

Santos, Ytana Oliveira; Almeida, Rogeria Comastri de Castro; Guimarães, Alaise Gil; Almeida, Paulo Fernando de

2010-01-01

p.1083-1089 The purpose of this study is to evaluate the hygienic-sanitary quality of vegetables and irrigation water and assess the effectiveness of lemon juice and vinegar in reducing E. coli strains inoculated on lettuce. One hundred and forty samples of vegetables and 45 samples of irrigation water were investigated for thermotolerant coliforms and Salmonella spp. In order to verify the effectiveness of natural household sanitizers in reducing E. coli in inoculated lettuce, four treatm...

21 CFR 866.3255 - Escherichia coli serological reagents.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...

Production, properties, and applications of hydrocolloid cellular solids.

Science.gov (United States)

Nussinovitch, Amos

2005-02-01

Many common synthetic and edible materials are, in fact, cellular solids. When classifying the structure of cellular solids, a few variables, such as open vs. closed cells, flexible vs. brittle cell walls, cell-size distribution, cell-wall thickness, cell shape, the uniformity of the structure of the cellular solid and the different scales of length are taken into account. Compressive stress-strain relationships of most cellular solids can be easily identified according to their characteristic sigmoid shape, reflecting three deformation mechanisms: (i) elastic distortion under small strains, (ii) collapse and/or fracture of the cell walls, and (iii) densification. Various techniques are used to produce hydrocolloid (gum) cellular solids. The products of these include (i) sponges, obtained when the drying gel contains the occasionally produced gas bubbles; (ii) sponges produced by the immobilization of microorganisms; (iii) solid foams produced by drying foamed solutions or gels containing oils, and (iv) hydrocolloid sponges produced by enzymatic reactions. The porosity of the manufactured cellular solid is subject to change and depends on its composition and the processing technique. The porosity is controlled by a range of methods and the resulting surface structures can be investigated by microscopy and analyzed using fractal methods. Models used to describe stress-strain behaviors of hydrocolloid cellular solids as well as multilayered products and composites are discussed in detail in this manuscript. Hydrocolloid cellular solids have numerous purposes, simple and complex, ranging from dried texturized fruits to carriers of vitamins and other essential micronutrients. They can also be used to control the acoustic response of specific dry food products, and have a great potential for future use in countless different fields, from novel foods and packaging to medicine and medical care, daily commodities, farming and agriculture, and the environmental, chemical

Effect of surface modification by nitrogen ion implantation on the electrochemical and cellular behaviors of super-elastic NiTi shape memory alloy.

Science.gov (United States)

Maleki-Ghaleh, H; Khalil-Allafi, J; Sadeghpour-Motlagh, M; Shakeri, M S; Masoudfar, S; Farrokhi, A; Beygi Khosrowshahi, Y; Nadernezhad, A; Siadati, M H; Javidi, M; Shakiba, M; Aghaie, E

2014-12-01

The aim of this investigation was to enhance the biological behavior of NiTi shape memory alloy while preserving its super-elastic behavior in order to facilitate its compatibility for application in human body. The surfaces of NiTi samples were bombarded by three different nitrogen doses. Small-angle X-ray diffraction was employed for evaluating the generated phases on the bombarded surfaces. The electrochemical behaviors of the bare and surface-modified NiTi samples were studied in simulated body fluid (SBF) using electrochemical impedance and potentio-dynamic polarization tests. Ni ion release during a 2-month period of service in the SBF environment was evaluated using atomic absorption spectrometry. The cellular behavior of nitrogen-modified samples was studied using fibroblast cells. Furthermore, the effect of surface modification on super-elasticity was investigated by tensile test. The results showed the improvement of both corrosion and biological behaviors of the modified NiTi samples. However, no significant change in the super-elasticity was observed. Samples modified at 1.4E18 ion cm(-2) showed the highest corrosion resistance and the lowest Ni ion release.

Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

Science.gov (United States)

Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

2017-01-01

The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

Directory of Open Access Journals (Sweden)

Rita E. Serda

2011-01-01

Full Text Available Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.

Asymptomatic bacteriuria Escherichia coli strains

DEFF Research Database (Denmark)

Hancock, Viktoria; Nielsen, E.M.; Klemm, Per

2006-01-01

Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast...... to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete...

The DinB•RecA complex of Escherichia coli mediates an efficient and high-fidelity response to ubiquitous alkylation lesions.

Science.gov (United States)

Cafarelli, Tiziana M; Rands, Thomas J; Godoy, Veronica G

2014-03-01

Alkylation DNA lesions are ubiquitous, and result from normal cellular metabolism as well as from treatment with methylating agents and chemotherapeutics. DNA damage tolerance by translesion synthesis DNA polymerases has an important role in cellular resistance to alkylating agents. However, it is not yet known whether Escherichia coli (E. coli) DNA Pol IV (DinB) alkylation lesion bypass efficiency and fidelity in vitro are similar to those inferred by genetic analyses. We hypothesized that DinB-mediated bypass of 3-deaza-3-methyladenine, a stable analog of 3-methyladenine, the primary replication fork-stalling alkylation lesion, would be of high fidelity. We performed here the first kinetic analyses of E. coli DinB•RecA binary complexes. Whether alone or in a binary complex, DinB inserted the correct deoxyribonucleoside triphosphate (dNTP) opposite either lesion-containing or undamaged template; the incorporation of other dNTPs was largely inefficient. DinB prefers undamaged DNA, but the DinB•RecA binary complex increases its catalytic efficiency on lesion-containing template, perhaps as part of a regulatory mechanism to better respond to alkylation damage. Notably, we find that a DinB derivative with enhanced affinity for RecA, either alone or in a binary complex, is less efficient and has a lower fidelity than DinB or DinB•RecA. This finding contrasts our previous genetic analyses. Therefore, mutagenesis resulting from alkylation lesions is likely limited in cells by the activity of DinB•RecA. These two highly conserved proteins play an important role in maintaining genomic stability when cells are faced with ubiquitous DNA damage. Kinetic analyses are important to gain insights into the mechanism(s) regulating TLS DNA polymerases. Copyright © 2013 Wiley Periodicals, Inc.

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

Science.gov (United States)

Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

2016-01-22

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

International Nuclear Information System (INIS)

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-01-01

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis

Escherichia coli EDL933 Requires Gluconeogenic Nutrients To Successfully Colonize the Intestines of Streptomycin-Treated Mice Precolonized with E. coli Nissle 1917

Science.gov (United States)

Schinner, Silvia A. C.; Mokszycki, Matthew E.; Adediran, Jimmy; Leatham-Jensen, Mary; Conway, Tyrrell

2015-01-01

Escherichia coli MG1655, a K-12 strain, uses glycolytic nutrients exclusively to colonize the intestines of streptomycin-treated mice when it is the only E. coli strain present or when it is confronted with E. coli EDL933, an O157:H7 strain. In contrast, E. coli EDL933 uses glycolytic nutrients exclusively when it is the only E. coli strain in the intestine but switches in part to gluconeogenic nutrients when it colonizes mice precolonized with E. coli MG1655 (R. L. Miranda et al., Infect Immun 72:1666–1676, 2004, http://dx.doi.org/10.1128/IAI.72.3.1666-1676.2004). Recently, J. W. Njoroge et al. (mBio 3:e00280-12, 2012, http://dx.doi.org/10.1128/mBio.00280-12) reported that E. coli 86-24, an O157:H7 strain, activates the expression of virulence genes under gluconeogenic conditions, suggesting that colonization of the intestine with a probiotic E. coli strain that outcompetes O157:H7 strains for gluconeogenic nutrients could render them nonpathogenic. Here we report that E. coli Nissle 1917, a probiotic strain, uses both glycolytic and gluconeogenic nutrients to colonize the mouse intestine between 1 and 5 days postfeeding, appears to stop using gluconeogenic nutrients thereafter in a large, long-term colonization niche, but continues to use them in a smaller niche to compete with invading E. coli EDL933. Evidence is also presented suggesting that invading E. coli EDL933 uses both glycolytic and gluconeogenic nutrients and needs the ability to perform gluconeogenesis in order to colonize mice precolonized with E. coli Nissle 1917. The data presented here therefore rule out the possibility that E. coli Nissle 1917 can starve the O157:H7 E. coli strain EDL933 of gluconeogenic nutrients, even though E. coli Nissle 1917 uses such nutrients to compete with E. coli EDL933 in the mouse intestine. PMID:25733524