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Sample records for coli produces tailor-made

  1. Overproduction of Thermus sp. Strain T2 β-Galactosidase in Escherichia coli and Preparation by Using Tailor-Made Metal Chelate Supports

    PubMed Central

    Pessela, Benevides C. C.; Vian, Alejandro; Mateo, César; Fernández-Lafuente, Roberto; García, José L.; Guisán, José M.; Carrascosa, Alfonso V.

    2003-01-01

    A novel thermostable chimeric β-galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β-galactosidase from Thermus sp. strain T2 to facilitate its overexpression in Escherichia coli and its purification by immobilized metal-ion affinity chromatography (IMAC). The poly-His tag fusion did not affect the activation, kinetic parameters, and stability of the β-galactosidase. Copper-iminodiacetic acid (Cu-IDA) supports enabled the most rapid adsorption of the His-tagged enzyme, favoring multisubunit interactions, but caused deleterious effects on the enzyme stability. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-IDA or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made IMAC supports. PMID:12676671

  2. Tailor-made synthesis of hydrogels

    SciTech Connect

    Kopecek, J.; Yeh, P.Y.; Kopechkova, P.; Ulbrich, K.

    1993-12-31

    The tailor-made synthesis of hydrogels by crosslinking copolymerization, by crosslinking of polymer precursors, and by polymer-polymer reaction will be analyzed. During the synthesis side-reactions occur resulting in the presence of cycles, unreacted pendant groups, and entanglements in the three dimensional network. The extent of these side-reactions depends on the structure of the crosslinking agent; amount and character of solvent; diffusion control of termination, crosslinking, and eventually propagation; difference in the hydrophilicity of monomer and crosslinking agent, and conversion. Synthesis of biodegradable hydrogels containing degradable sequences in the crosslinks will be used as an example to demonstrate that biorecognition by enzymes depends on the detailed structure of the network.

  3. Recombinant lectins: an array of tailor-made glycan-interaction biosynthetic tools.

    PubMed

    Oliveira, Carla; Teixeira, José A; Domingues, Lucília

    2013-03-01

    Lectins are a heterogeneous group of proteins found in plants, animals and microorganisms, which possess at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharides. The range of lectins and respective biological activities is unsurprising given the immense diversity and complexity of glycan structures and the multiple modes of interaction with proteins. Recombinant DNA technology has been traditionally used for cloning and characterizing newly discovered lectins. It has also been employed as a means of producing pure and sequence-defined lectins for different biotechnological applications. This review focuses on the production of recombinant lectins in heterologous organisms, and highlighting the Escherichia coli and Pichia pastoris expression systems, which are the most employed. The choice of expression host depends on the lectin. Non-glycosylated recombinant lectins are produced in E. coli and post-translational modified recombinant lectins are produced in eukaryotic organisms, namely P. pastoris and non-microbial hosts such as mammalian cells. Emphasis is given to the applications of the recombinant lectins especially (a) in cancer diagnosis and/or therapeutics, (b) as anti-microbial, anti-viral, and anti-insect molecules or (c) in microarrays for glycome profiling. Most reported applications are from recombinant plant lectins. These applications benefit from the tailor-made design associated with recombinant production and will aid in unraveling the complex biological mechanisms of glycan-interactions, bringing recombinant lectins to the forefront of glycobiology. In conclusion, recombinant lectins are developing into valuable biosynthetic tools for biomedical research.

  4. Tailor-made polyamide membranes for water desalination.

    PubMed

    Choi, Wansuk; Gu, Joung-Eun; Park, Sang-Hee; Kim, Seyong; Bang, Joona; Baek, Kyung-Youl; Park, Byoungnam; Lee, Jong Suk; Chan, Edwin P; Lee, Jung-Hyun

    2015-01-27

    Independent control of the extrinsic and intrinsic properties of the polyamide (PA) selective layer is essential for designing thin-film composite (TFC) membranes with performance characteristics required for water purification applications besides seawater desalination. Current commercial TFC membranes fabricated via the well-established interfacial polymerization (IP) approach yield materials that are far from ideal because their layer thickness, surface roughness, polymer chemistry, and network structure cannot be separately tailored. In this work, tailor-made PA-based desalination membranes based on molecular layer-by-layer (mLbL) assembly are presented. The mLbL technique enables the construction of an ultrathin and highly cross-linked PA selective layer in a precisely and independently controlled manner. The mLbL-assembled TFC membranes exhibit significant enhancements in performance compared to their IP-assembled counterparts. A maximum sodium chloride rejection of 98.2% is achieved along with over 2.5 times higher water flux than the IP-assembled counterpart. More importantly, this work demonstrates the broad applicability of mLbL in fabricating a variety of PA-based TFC membranes with nanoscale control of the selective layer thickness and roughness independent of the specific polyamide chemistry.

  5. Gasifier feed - Tailor-made from Illinois coals

    SciTech Connect

    Ehrlinger, H.P. III ); Lytle, J.; Frost, R.R.; Lizzio, A.; Kohlenberger, L.; Brewer, K. DESTEC Energy Williams Technology, Illinois Coal Association )

    1992-01-01

    The main purpose of this project is to produce a feedstock from preparation plant fines from an illinois coal that is ideal for a slurry fed, slagging, entrained-flow coal gasifier. The high sulfur content and high Btu value of Illinois coals are particularly advantageous in such a gasifier; preliminary calculations indicate that the increased cost of removing sulfur from the gas from a high sulfur coal is more than offset by the increased revenue from the sale of the elemental sulfur; additionally the high Btu Illinois coal concentrates more energy into the slurry of a given coal to water ratio. The Btu is higher not only because of the higher Btu value of the coal but also because Illinois coal requires less water to produce a pumpable slurry than western coal, i.e., as little as 30--35% water may be used for Illinois coal as compared to approximately 45% for most western coals.

  6. Candida bombicola as a platform organism for the production of tailor-made biomolecules.

    PubMed

    Roelants, Sophie L K W; Saerens, Karen M J; Derycke, Thibaut; Li, Bing; Lin, Yao-Cheng; Van de Peer, Yves; De Maeseneire, Sofie L; Van Bogaert, Inge N A; Soetaert, Wim

    2013-09-01

    The yeast Candida bombicola is capable of producing high amounts (400 g/L) of the biosurfactant sophorolipids. The genetic makeup of this industrially important yeast has recently been uncovered and molecular manipulation techniques have been developed. Hence, all tools for the development of new bioprocesses with C. bombicola are now available. As a proof of concept, the production of two totally different molecules was aimed for: the bioplastic polyhydroxyalkanoate (PHA) and a new-to-nature cellobioselipid-biosurfactant. Integration of the new functionalities at genomic loci necessary for sophorolipid production safeguards the new biomolecules from sophorolipid contamination, while taking advantage of the regulation of the sophorolipid gene cluster. A maximum yield of 2.0% wt/dwt PHA was obtained; furthermore, this is the first time cellobioselipid synthesis by a non-natural producer is reported. We here provided proof of concept that C. bombicola can be transformed into a platform organism for the production of tailor-made biomolecules.

  7. Nanoscale tailor-made membranes for precise and rapid molecular sieve separation.

    PubMed

    Wang, Jing; Zhu, Junyong; Zhang, Yatao; Liu, Jindun; Van der Bruggen, Bart

    2017-03-02

    The precise and rapid separation of different molecules from aqueous, organic solutions and gas mixtures is critical to many technologies in the context of resource-saving and sustainable development. The strength of membrane-based technologies is well recognized and they are extensively applied as cost-effective, highly efficient separation techniques. Currently, empirical-based approaches, lacking an accurate nanoscale control, are used to prepare the most advanced membranes. In contrast, nanoscale control renders the membrane molecular specificity (sub-2 nm) necessary for efficient and rapid molecular separation. Therefore, as a growing trend in membrane technology, the field of nanoscale tailor-made membranes is highlighted in this review. An in-depth analysis of the latest advances in tailor-made membranes for precise and rapid molecule sieving is given, along with an outlook to future perspectives of such membranes. Special attention is paid to the established processing strategies, as well as the application of molecular dynamics (MD) simulation in nanoporous membrane design. This review will provide useful guidelines for future research in the development of nanoscale tailor-made membranes with a precise and rapid molecular sieve separation property.

  8. Development of a tailor-made bis(oxazolidine)pyridine-metal catalyst for the [3+2] cycloaddition of azomethine imines with propiolates.

    PubMed

    Arai, Takayoshi; Ogino, Yuta; Sato, Toru

    2013-09-14

    A series of bis(oxazolidine)pyridine ligands (the PyBodines) were newly designed and synthesized for the development of a highly efficient, tailor-made catalyst for the [3+2] cycloaddition of azomethine imines with propiolates. The PyBodine(l-Ala)-Cu(OAc)2 catalyst was selected on the basis of solid-phase catalysis/CD-HTS and exhibited high efficiency, producing the (R)-adducts with excellent enantioselectivity.

  9. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

  10. The effect of tailor-made additives on crystal growth of methyl paraben: Experiments and modelling

    NASA Astrophysics Data System (ADS)

    Cai, Zhihui; Liu, Yong; Song, Yang; Guan, Guoqiang; Jiang, Yanbin

    2017-03-01

    In this study, methyl paraben (MP) was selected as the model component, and acetaminophen (APAP), p-methyl acetanilide (PMAA) and acetanilide (ACET), which share the similar molecular structure as MP, were selected as the three tailor-made additives to study the effect of tailor-made additives on the crystal growth of MP. HPLC results indicated that the MP crystals induced by the three additives contained MP only. Photographs of the single crystals prepared indicated that the morphology of the MP crystals was greatly changed by the additives, but PXRD and single crystal diffraction results illustrated that the MP crystals were the same polymorph only with different crystal habits, and no new crystal form was found compared with other references. To investigate the effect of the additives on the crystal growth, the interaction between additives and facets was discussed in detail using the DFT methods and MD simulations. The results showed that APAP, PMAA and ACET would be selectively adsorbed on the growth surfaces of the crystal facets, which induced the change in MP crystal habits.

  11. Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues

    PubMed Central

    Karnaouri, Anthi; Matsakas, Leonidas; Topakas, Evangelos; Rova, Ulrika; Christakopoulos, Paul

    2016-01-01

    Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5, and MtEG7), in the presence of other four “accessory” enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a nine-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch, and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that

  12. Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues.

    PubMed

    Karnaouri, Anthi; Matsakas, Leonidas; Topakas, Evangelos; Rova, Ulrika; Christakopoulos, Paul

    2016-01-01

    Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5, and MtEG7), in the presence of other four "accessory" enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a nine-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch, and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that tailor-made

  13. Exploitation of desilylation chemistry in tailor-made functionalization on diverse surfaces

    NASA Astrophysics Data System (ADS)

    Fu, Yongchun; Chen, Songjie; Kuzume, Akiyoshi; Rudnev, Alexander; Huang, Cancan; Kaliginedi, Veerabhadrarao; Baghernejad, Masoud; Hong, Wenjing; Wandlowski, Thomas; Decurtins, Silvio; Liu, Shi-Xia

    2015-03-01

    Interface engineering to attain a uniform and compact self-assembled monolayer at atomically flat surfaces plays a crucial role in the bottom-up fabrication of organic molecular devices. Here we report a promising and operationally simple approach for modification/functionalization not only at ultraflat single-crystal metal surfaces, M(111) (M=Au, Pt, Pd, Rh and Ir) but also at the highly oriented pyrolytic graphite surface, upon efficient in situ cleavage of trimethylsilyl end groups of the molecules. The obtained self-assembled monolayers are ultrastable within a wide potential window. The carbon-surface bonding on various substrates is confirmed by shell-isolated nanoparticle-enhanced Raman spectroscopy. Application of this strategy in tuning surface wettability is also demonstrated. The most valuable finding is that a combination of the desilylation with the click chemistry represents an efficient method for covalent and tailor-made functionalization of diverse surfaces.

  14. Exploitation of desilylation chemistry in tailor-made functionalization on diverse surfaces

    PubMed Central

    Fu, Yongchun; Chen, Songjie; Kuzume, Akiyoshi; Rudnev, Alexander; Huang, Cancan; Kaliginedi, Veerabhadrarao; Baghernejad, Masoud; Hong, Wenjing; Wandlowski, Thomas; Decurtins, Silvio; Liu, Shi-Xia

    2015-01-01

    Interface engineering to attain a uniform and compact self-assembled monolayer at atomically flat surfaces plays a crucial role in the bottom-up fabrication of organic molecular devices. Here we report a promising and operationally simple approach for modification/functionalization not only at ultraflat single-crystal metal surfaces, M(111) (M=Au, Pt, Pd, Rh and Ir) but also at the highly oriented pyrolytic graphite surface, upon efficient in situ cleavage of trimethylsilyl end groups of the molecules. The obtained self-assembled monolayers are ultrastable within a wide potential window. The carbon–surface bonding on various substrates is confirmed by shell-isolated nanoparticle-enhanced Raman spectroscopy. Application of this strategy in tuning surface wettability is also demonstrated. The most valuable finding is that a combination of the desilylation with the click chemistry represents an efficient method for covalent and tailor-made functionalization of diverse surfaces. PMID:25758661

  15. Tailor-made anion-exchange membranes for salinity gradient power generation using reverse electrodialysis.

    PubMed

    Guler, Enver; Zhang, Yali; Saakes, Michel; Nijmeijer, Kitty

    2012-11-01

    Reverse electrodialysis (RED) or blue energy is a non-polluting, sustainable technology for generating power from the mixing of solutions with different salinity, that is, seawater and river water. A concentrated salt solution (e.g., seawater) and a diluted salt solution (e.g., river water) are brought into contact through an alternating series of polymeric anion-exchange membranes (AEMs) and cation-exchange membranes (CEMs), which are either selective for anions or cations. Currently available ion-exchange membranes are not optimized for RED, whereas successful RED operation notably depends on the used ion-exchange membranes. We designed such ion-exchange membranes and for the first time we show the performance of tailor-made membranes in RED. More specifically, we focus on the development of AEMs because these are much more complex to prepare. Herein we propose a safe and more environmentally friendly method and use halogenated polyethers, such as polyepichlorohydrin (PECH) as the starting material. A tertiary diamine (1,4-diazabicyclo[2.2.2]octane, DABCO) was used to introduce the ion-exchange groups by amination and for simultaneous cross-linking of the polymer membrane. Area resistances of the series of membranes ranged from 0.82 to 2.05 Ω cm² and permselectivities from 87 to 90 %. For the first time we showed that tailor-made ion-exchange membranes can be applied in RED. Depending on the properties and especially membrane thickness, application of these membranes in RED resulted in a high power density of 1.27 W m⁻², which exceeds the power output obtained with the commercially available AMX membranes. This shows the potential of the design of ion-exchange membranes for a viable blue energy process.

  16. Dry particle coating of polymer particles for tailor-made product properties

    SciTech Connect

    Blümel, C. Schmidt, J. Dielesen, A. Sachs, M. Winzer, B. Peukert, W. Wirth, K.-E.

    2014-05-15

    Disperse polymer powders with tailor-made particle properties are of increasing interest in industrial applications such as Selective Laser Beam Melting processes (SLM). This study focuses on dry particle coating processes to improve the conductivity of the insulating polymer powder in order to assemble conductive devices. Therefore PP particles were coated with Carbon Black nanoparticles in a dry particle coating process. This process was investigated in dependence of process time and mass fraction of Carbon Black. The conductivity of the functionalized powders was measured by impedance spectroscopy. It was found that there is a dependence of process time, respectively coating ratio and conductivity. The powder shows higher conductivities with increasing number of guest particles per host particle surface area, i.e. there is a correlation between surface functionalization density and conductivity. The assembled composite particles open new possibilities for processing distinct polymers such as PP in SLM process. The fundamentals of the dry particle coating process of PP host particles with Carbon Black guest particles as well as the influence on the electrical conductivity will be discussed.

  17. Effect of trehalose coating on basic fibroblast growth factor release from tailor-made bone implants.

    PubMed

    Choi, Sungjin; Lee, Jongil; Igawa, Kazuyo; Suzuki, Shigeki; Mochizuki, Manabu; Nishimura, Ryohei; Chung, Ung-il; Sasaki, Nobuo

    2011-12-01

    Artificial bone implants are often incorporated with osteoinductive factors to facilitate early bone regeneration. Calcium phosphate, the main component in artificial bone implants, strongly binds these factors, and in a few cases, the incorporated proteins are not released from the implant under conditions of physiological pH, thereby leading to reduction in their osteoinductivity. In this study, we coated tailor-made bone implants with trehalose to facilitate the release of basic fibroblast growth factor (bFGF). In an in vitro study, mouse osteoblastic cells were separately cultured for 48 hr in a medium with a untreated implant (T-), trehalose-coated implant (T+), bFGF-incorporated implant (FT-), and bFGF-incorporated implant with trehalose coating (FT+). In the FT+ group, cell viability was significantly higher than that in the other groups (P<0.05). Scanning electron microscopy (SEM) and X-ray diffraction (XRD) revealed that trehalose effectively covered the surface of the artificial bone implant without affecting the crystallinity or the mechanical strength of the artificial bone implant. These results suggest that coating artificial bone implants with trehalose could limit the binding of bFGF to calcium phosphate.

  18. Enhanced redifferentiation of chondrocytes on microperiodic silk/gelatin scaffolds: toward tailor-made tissue engineering.

    PubMed

    Das, Sanskrita; Pati, Falguni; Chameettachal, Shibu; Pahwa, Shikha; Ray, Alok R; Dhara, Santanu; Ghosh, Sourabh

    2013-02-11

    Direct-write assembly allows rapid fabrication of complex three-dimensional (3D) architectures, such as scaffolds simulating anatomical shapes, avoiding the need for expensive lithographic masks. However, proper selection of polymeric ink composition and tailor-made viscoelastic properties are critically important for smooth deposition of ink and shape retention. Deposition of only silk solution leads to frequent clogging due to shear-induced β-sheet crystallization, whereas optimized viscoelastic property of silk-gelatin blends facilitate the flow of these blends through microcapillary nozzles of varying diameter. This study demonstrates that induction of controlled changes in scaffold surface chemistry, by optimizing silk-gelatin ratio, can govern cell proliferation and maintenance of chondrocyte morphology. Microperiodic silk-gelatin scaffolds can influence postexpansion redifferentiation of goat chondrocytes by enhancing Sox-9 gene expression, aggregation, and driving cartilage matrix production, as evidenced by upregulation of collagen type II and aggrecan expression. The strategy for optimizing redifferentiation of chondrocytes can offer valuable consideration in scaffold-based cartilage repair strategies.

  19. Multiple-antibiotic-resistant Helicobacter pylori infection eradicated with a tailor-made quadruple therapy.

    PubMed

    Nakajima, Shigemi; Inoue, Hisayuki; Inoue, Tetsuya; Maruoka, Yuri

    2012-04-01

    In 2008, a 44-year-old woman with mild epigastralgia diagnosed as having Helicobacter pylori-positive chronic gastritis without peptic ulcer underwent eradication therapy with lansoprazole (LPZ), amoxicillin (AMPC) and clarithromycin (CAM) for 7 days, but it failed, so treatment with rabeprazole, AMPC, and metronidazole (MNZ) for another 7 days was given, but it also failed. She was then prescribed a modified, 14-day sequential therapy of LPZ and AMPC with an increased dose of CAM followed by MNZ supplement, but the infection was still not eradicated. The H. pylori was cultured and examined for antibiotic susceptibility with the agar dilution method and was found to be resistant to CAM, MNZ, and levofloxacin, and non-sensitive to AMPC, namely multiple-antibiotic-resistant, although sensitive to minocycline. The CYP2C19 genotype of the patient was an extensive metabolizer (G681A: G/A, G636A: G/G). In 2010, she gave informed consent for a 14-day, tailor-made, modified classical (or modified high-dose PPI + AMPC) quadruple therapy comprising 30 mg LPZ, 500 mg AMPC and 500 mg bismuth subnitrate, qid, and 100 mg minocycline, bid. Two months later, her urea breath test was negative. Histology and bacterial culture were still negative 1 year after the therapy. She did not have any adverse events during or after the novel therapy, nor did she feel any further epigastralgia.

  20. Tailor-made asymmetric PVDF hollow fibers for soluble gas removal

    SciTech Connect

    Li, K.; Kong, J.F.; Wang, D.; Teo, W.K.

    1999-06-01

    Tailor-made polyvinylidene fluoride (PVDF) asymmetric hollow-fiber membranes and their membrane modules were employed for soluble gas removal, such as H{sub 2}S from waste gas streams. This study focused on the techniques of fabricating and characterizing the PVDF asymmetric hollow-fiber membranes and their membrane modules for removal of H{sub 2}S using an aqueous solution containing 10% NaOH. A laminar parabolic velocity profile was used to characterize the flow of the H{sub 2}S gas mixture in the hollow-fiber lumen. Effects of operating conditions and the morphological structures of the membranes on the membrane`s coefficient, k{sub AM}, were examined both theoretically and experimentally. The capabilities of the hollow-fiber membranes developed for removal of H{sub 2}S from waste gas streams were evaluated and compared with conventional symmetric hydrophobic hollow-fiber membranes, such as polypropylene. An analysis of H{sub 2}S transfer across the more developed PVDF membranes reveals that the membrane`s coefficient, k{sub AM}, evaluated from its structure parameters, such as the effective surface porosity and mean radius, agreed well with the experimental data obtained from absorption experiments.

  1. Material efficiency: from top-down steering to tailor-made governance.

    PubMed

    Cramer, Jacqueline

    2013-03-13

    Material efficiency is one of the major challenges facing our society in the twenty-first century. Research can help to understand how we can make the transition towards a material-efficient society. This study focuses on the role of the government in such transition processes. Use is made of literature in the field of public administration and innovation literature, particularly transition management. On the basis of three Dutch examples (plastics, e-waste and bio-energy), the complex system change towards a material-efficient society will be reflected upon. These case studies underline the need for a tailor-made governance approach instead of a top-down government approach to enhance material efficiency in practice. The role of the government is not restricted to formulating policies and then leaving it up to other actors to implement these policies. Instead, it is a continuous interplay between the different actors during the whole implementation process. As such, the government's role is to steer the development in the desired direction and orchestrate the process from beginning to end. In order to govern with a better compass, scientifically underpinned guiding principles and indicators are needed. This is a challenge for researchers both in public administration and in transition management.

  2. Tailor-Made Distribution of Nanoparticles in Blend Structure toward Outstanding Electromagnetic Interference Shielding.

    PubMed

    Biswas, Sourav; Kar, Goutam Prasanna; Bose, Suryasarathi

    2015-11-18

    Engineering blend structure with tailor-made distribution of nanoparticles is the prime requisite to obtain materials with extraordinary properties. Herein, a unique strategy of distributing nanoparticles in different phases of a blend structure has resulted in >99% blocking of incoming electromagnetic (EM) radiation. This is accomplished by designing a ternary polymer blend structure using polycarbonate (PC), poly(vinylidene fluoride) (PVDF), and poly(methyl methacrylate) (PMMA) to simultaneously improve the structural, electrical, and electromagnetic interference shielding (EMI). The blend structure was made conducting by preferentially localizing the multi-wall nanotubes (MWNTs) in the PVDF phase. By taking advantage of "π-π stacking" MWNTs was noncovalently modified with an imidazolium based ionic liquid (IL). Interestingly, the enhanced dispersion of IL-MWNTs in PVDF improved the electrical conductivity of the blends significantly. While one key requisite to attenuate EM radiation (i.e., electrical conductivity) was achieved using MWNTs, the magnetic properties of the blend structure was tuned by introducing barium ferrite (BaFe) nanoparticles, which can interact with the incoming EM radiation. By suitably modifying the surface of BaFe nanoparticles, we can tailor their localization under the macroscopic processing condition. The precise localization of BaFe nanoparticles in the PC phase, due to nucleophilic substitution reaction, and the MWNTs in the PVDF phase not only improved the conductivity but also facilitated in absorption of the incoming microwave radiation due to synergetic effect from MWNT and BaFe. The shielding effectiveness (SE) was measured in X and Ku band, and an enhanced SE of -37 dB was noted at 18 GHz frequency. PMMA, which acted as an interfacial modifier in PC/PVDF blends further, resulting in a significant enhancement in the mechanical properties besides retaining high SE. This study opens a new avenue in designing mechanically strong

  3. Infection by verocytotoxin-producing Escherichia coli.

    PubMed Central

    Karmali, M A

    1989-01-01

    Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated

  4. ESBL-producing E. coli in Austrian sewage sludge.

    PubMed

    Reinthaler, Franz Ferdinand; Feierl, Gebhard; Galler, Herbert; Haas, Doris; Leitner, Eva; Mascher, Franz; Melkes, Angelika; Posch, Josefa; Winter, Ingrid; Zarfel, Gernot; Marth, Egon

    2010-03-01

    The aim of this study was to investigate the degree of contamination of sewage sludge with ESBL-producing Escherichia coli strains and the effectiveness of different sewage sludge treatment methods. Monthly sewage sludge samples were collected between January and September 2009 in 5 different sewage treatment plants and tested for the presence of ESBL E. coli. In addition, the number of colony forming units (CFU) of E. coli and coliform bacteria before and after the different sludge treatment methods (aerobic/anaerobic digestion, lime stabilization, and thermal treatment) was investigated. Of the 72 sewage sludge samples investigated, ESBL-positive E. coli were found in 44 (61.1%) sewage sludge samples. The classification of beta-lactamase groups was carried out in 15 strains resulting in the detection of 2 different groups (CTX-M and TEM) of bla genes. All 15 of them had a CTX-M gene and 4 of these strains furthermore carried a TEM gene. With regard to the CFU of E. coli and coliform bacteria, thermal treatment and lime stabilization following dehydration sufficiently reduced pathogen concentrations. The plants using merely stabilization and dehydration showed an increase of E. coli and coliform bacteria and thus also an increase in ESBL-producing E. coli.

  5. Tailor-made biopolymers porous scaffold fabrication for tissue engineering: application of radiant energy in the form of microwave under vacuum.

    PubMed

    Jaya, S; Durance, T D

    2008-01-01

    Many methods are available for developing three-dimensional porous scaffolds using various polymeric materials for tissue-engineering applications. Each has its own advantages and disadvantages. Some of the available methods and their limitations were discussed briefly. This paper focuses on the scope of novel technology called radiant energy application under vacuum for the fabrication of three-dimensional porous scaffolds for tissue engineering applications. Radiant energy application in the form of microwave under vacuum has been shown to develop and maintain the porous structure in fruits and vegetables after dehydration, which produced the microstructure similar to the freeze dried materials. Same principle of applying radiant energy under vacuum was used on the biopolymeric gels to create tailor-made, porous scaffolds for biomedical purposes. It has many advantages over the other existing methods of scaffold fabrication. This paper also reviews the scaffolds design recently fabricated by the authors using radiant energy under vacuum.

  6. Synergy Effects in the Chemical Synthesis and Extensions of Multicomponent Reactions (MCRs)-The Low Energy Way to Ultra-Short Syntheses of Tailor-Made Molecules.

    PubMed

    Eckert, Heiner

    2017-02-25

    Several novel methods, catalysts and reagents have been developed to improve organic synthesis. Synergistic effects between reactions, reagents and catalysts can lead to minor heats of reaction and occur as an inherent result of multicomponent reactions (MCRs) and their extensions. They enable syntheses to be performed at a low energy level and the number of synthesis steps to be drastically reduced in comparison with 'classical' two-component reactions, fulfilling the rules of Green Chemistry. The very high potential for variability, diversity and complexity of MCRs additionally generates an extremely diverse range of products, thus bringing us closer to the aim of being able to produce tailor-made and extremely low-cost materials, drugs and compound libraries.

  7. Gasifier feed - Tailor-made from Illinois coals. Technical report, December 1, 1991--February 29, 1992

    SciTech Connect

    Ehrlinger, H.P. III; Lytle, J.; Frost, R.R.; Lizzio, A.; Kohlenberger, L.; Brewer, K. |||

    1992-08-01

    The main purpose of this project is to produce a feedstock from preparation plant fines from an illinois coal that is ideal for a slurry fed, slagging, entrained-flow coal gasifier. The high sulfur content and high Btu value of Illinois coals are particularly advantageous in such a gasifier; preliminary calculations indicate that the increased cost of removing sulfur from the gas from a high sulfur coal is more than offset by the increased revenue from the sale of the elemental sulfur; additionally the high Btu Illinois coal concentrates more energy into the slurry of a given coal to water ratio. The Btu is higher not only because of the higher Btu value of the coal but also because Illinois coal requires less water to produce a pumpable slurry than western coal, i.e., as little as 30--35% water may be used for Illinois coal as compared to approximately 45% for most western coals.

  8. Tailor-made rehabilitation approach using multiple types of hybrid assistive limb robots for acute stroke patients: A pilot study.

    PubMed

    Fukuda, Hiroyuki; Morishita, Takashi; Ogata, Toshiyasu; Saita, Kazuya; Hyakutake, Koichi; Watanabe, Junko; Shiota, Etsuji; Inoue, Tooru

    2016-01-01

    This article investigated the feasibility of a tailor-made neurorehabilitation approach using multiple types of hybrid assistive limb (HAL) robots for acute stroke patients. We investigated the clinical outcomes of patients who underwent rehabilitation using the HAL robots. The Brunnstrom stage, Barthel index (BI), and functional independence measure (FIM) were evaluated at baseline and when patients were transferred to a rehabilitation facility. Scores were compared between the multiple-robot rehabilitation and single-robot rehabilitation groups. Nine hemiplegic acute stroke patients (five men and four women; mean age 59.4 ± 12.5 years; four hemorrhagic stroke and five ischemic stroke) underwent rehabilitation using multiple types of HAL robots for 19.4 ± 12.5 days, and 14 patients (six men and eight women; mean age 63.2 ± 13.9 years; nine hemorrhagic stroke and five ischemic stroke) underwent rehabilitation using a single type of HAL robot for 14.9 ± 8.9 days. The multiple-robot rehabilitation group showed significantly better outcomes in the Brunnstrom stage of the upper extremity, BI, and FIM scores. To the best of the authors' knowledge, this is the first pilot study demonstrating the feasibility of rehabilitation using multiple exoskeleton robots. The tailor-made rehabilitation approach may be useful for the treatment of acute stroke.

  9. Playing and listening to tailor-made notched music: cortical plasticity induced by unimodal and multimodal training in tinnitus patients.

    PubMed

    Pape, Janna; Paraskevopoulos, Evangelos; Bruchmann, Maximilian; Wollbrink, Andreas; Rudack, Claudia; Pantev, Christo

    2014-01-01

    BACKGROUND. The generation and maintenance of tinnitus are assumed to be based on maladaptive functional cortical reorganization. Listening to modified music, which contains no energy in the range of the individual tinnitus frequency, can inhibit the corresponding neuronal activity in the auditory cortex. Music making has been shown to be a powerful stimulator for brain plasticity, inducing changes in multiple sensory systems. Using magnetoencephalographic (MEG) and behavioral measurements we evaluated the cortical plasticity effects of two months of (a) active listening to (unisensory) versus (b) learning to play (multisensory) tailor-made notched music in nonmusician tinnitus patients. Taking into account the fact that uni- and multisensory trainings induce different patterns of cortical plasticity we hypothesized that these two protocols will have different affects. RESULTS. Only the active listening (unisensory) group showed significant reduction of tinnitus related activity of the middle temporal cortex and an increase in the activity of a tinnitus-coping related posterior parietal area. CONCLUSIONS. These findings indicate that active listening to tailor-made notched music induces greater neuroplastic changes in the maladaptively reorganized cortical network of tinnitus patients while additional integration of other sensory modalities during training reduces these neuroplastic effects.

  10. Biosynthesis, characterization, and hemostasis potential of tailor-made poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Haloferax mediterranei.

    PubMed

    Han, Jing; Wu, Lin-Ping; Hou, Jing; Zhao, Dahe; Xiang, Hua

    2015-02-09

    We report the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymers (R-PHBV) or higher-order copolymers (O-PHBV) in Haloferax mediterranei, with adjustable 3-hydroxyvalerate (3HV) incorporation by cofeeding valerate with glucose. Their microchemical structure, molecular weight and its distribution, and thermal and mechanical properties were characterized by NMR, GPC, DSC, TGA, and universal testing machine, respectively. (13)C NMR studies showed that O-PHBV copolymers consisted of short segments of PHB and PHV covalently linked together with random PHBV segments. Consistently, two Tg were observed in the DSC curves of O-PHBV. The "blocky" feature of O-PHBV enhanced crystallinity percentages and improved Young's modulus. Notably, the film of one O-PHBV copolymer, O-PHBV-1, showed unique foveolar cluster-like surface morphology with high hydrophobicity and roughness, as characterized using static contact angle and SEM and AFM analyses. It also exhibited increased platelet adhesion and accelerated blood clotting. The excellent hemostatic properties endow this copolymer with great potential in wound healing.

  11. Presence of Shiga toxin-producing Escherichia coli, Enteroinvasive E. coli, Enteropathogenic E. coli, and Enterotoxigenic E. coli on tomatoes from public markets in Mexico.

    PubMed

    Gómez-Aldapa, Carlos A; Torres-Vitela, M Del Refugio; Acevedo-Sandoval, Otilio A; Rangel-Vargas, Esmeralda; Villarruel-López, Angélica; Castro-Rosas, Andjavier

    2013-09-01

    Diarrheagenic Escherichia coli pathotypes (DEP) are important foodborne pathogens in various countries, including Mexico. However, no data exist on the presence of DEP on fresh tomatoes (Solanum lycopericum) from Mexico. The frequency of fecal coliforms (FC), E. coli, and DEP were determined for two tomato varieties. One hundred samples of a saladette tomato variety and 100 samples of a red round tomato variety were collected from public markets in Pachuca, Mexico. Each tomato sample consisted of four whole tomatoes. For the 100 saladette samples, coliform bacterial, FC, E. coli, and DEP were identified in 100, 70, 60, and 10% of samples, respectively. For the 100 red round samples, coliform bacterial, FC, E. coli, and DEP were identified in 100, 75, 65, and 11% of samples, respectively. Identified DEP included Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). STEC were isolated from 6% of saladette samples and 5% of red round samples. ETEC were isolated from 3% of saladette samples and 4% of red round samples. EPEC were isolated from 2% of saladette samples and 3% of red round samples, and EIEC were isolated from 1% of saladette samples. Both STEC and ETEC were identified in two saladette samples and 1 red round sample. E. coli O157:H7 was not detected in any STEC-positive samples.

  12. Microcin 25, a novel antimicrobial peptide produced by Escherichia coli.

    PubMed Central

    Salomón, R A; Farías, R N

    1992-01-01

    Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel filtration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell division, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system. Images PMID:1429464

  13. "Click chemistry" in tailor-made polymethacrylates bearing reactive furfuryl functionality: a new class of self-healing polymeric material.

    PubMed

    Kavitha, A Amalin; Singha, Nikhil K

    2009-07-01

    This investigation reports the effective use of the Diels-Alder (DA) reaction, a "click reaction" in the preparation of thermally amendable and self-healing polymeric materials having reactive furfuryl functionality. In this case, the DA and retro-DA (rDA) reactions were carried out between the tailor-made homo- and copolymer of furfuryl methacrylate prepared by atom-transfer radical polymerization and a bismaleimide (BM). The kinetic studies of DA and rDA reactions were carried out using Fourier transform infrared spectroscopy. The DA polymers were insoluble in toluene at room temperature. When the DA polymers were heated at 100 degrees C in toluene, it was soluble. This is because of the cleavage between furfuryl functionality and BM. The chemical cross-link density was determined by the Flory-Rehner equation. The cross-linked polymer showed much greater adhesive strength at room temperature, but the adhesive strength was quite low at higher temperature. The self-healing capability was studied by using scanning electron microscopy analysis. The thermal and dynamic mechanical properties of the thermally amendable cross-linked materials were investigated by thermogravimetric analysis and dynamic mechanical analysis.

  14. Tailor-made cell patterning using a near-infrared-responsive composite gel composed of agarose and carbon nanotubes.

    PubMed

    Koga, Haruka; Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi; Nakazawa, Kohji

    2013-03-01

    Micropatterning is useful for regulating culture environments. We developed a highly efficient near-infrared-(NIR)-responsive gel and established a new technique that enables cell patterning by NIR irradiation. As a new culture substratum, we designed a tissue culture plate that was coated with a composite gel composed of agarose and carbon nanotubes (CNTs). A culture plate coated with agarose only showed no response to NIR irradiation. In contrast, NIR laser irradiation induced heat generation by CNTs; this permitted local solation of the CNT/agarose gel, and consequently, selective cell-adhesive regions were exposed on the tissue culture plate. The solation area was controlled by the NIR intensity, magnification of the object lens and CNT concentration in the gel. Furthermore, we formed circular patterns of HeLa cells and linear patterns of 3T3 cells on the same culture plate through selective and stepwise NIR irradiation of the CNT/agarose gel, and we also demonstrated that individual 3T3 cells migrated along a linear path formed on the CNT/agarose gel by NIR irradiation. These results indicate that our technique is useful for tailor-made cell patterning of stepwise and/or complex cell patterns, which has various biological applications such as stepwise co-culture and the study of cell migration.

  15. Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana

    PubMed Central

    Rasmussen, Mette Marie; Opintan, Japheth A.; Frimodt-Møller, Niels; Styrishave, Bjarne

    2015-01-01

    The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype blaCTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential

  16. Beta-Lactamase Producing Escherichia coli Isolates in Imported and Locally Produced Chicken Meat from Ghana.

    PubMed

    Rasmussen, Mette Marie; Opintan, Japheth A; Frimodt-Møller, Niels; Styrishave, Bjarne

    2015-01-01

    The use of antibiotics in food animals is of public health concern, because resistant zoonotic pathogens can be transmitted to humans. Furthermore, global trade with food may rapidly spread multi-resistant pathogens between countries and even continents. The purpose of the study was to investigate whether imported chicken meat and meat from locally reared chicken are potential sources for human exposure to multi resistant Escherichia coli isolates. 188 samples from imported and locally produced chicken meat were sampled and analyzed. 153 bacteria isolates were successfully cultured and identified as E. coli using MALDI-ToF. Of these 109 isolates were from meat whereas the remaining 44 were isolated from the cloaca of locally reared live chickens. Antimicrobial susceptibility test was done on the identified E. coli isolates. Additionally, beta-lactamases production (ESBL and/or AmpC) were phenotypically confirmed on all isolates showing resistance to cefpodoxime. Beta-lactamase producing (BLP) E. coli meat isolates were further genotyped. Antimicrobial resistance to four antibiotic markers with highest resistance was detected more frequently in isolates from local chickens compared to imported chickens (tetracycline 88.9% vs. 57.5%, sulphonamide 75.0% vs. 46.6%, ampicillin 69.4% vs. 61.6% and trimethoprim 66.7% vs. 38.4%). Beta-lactamase production was found in 29 E. coli meat isolates, with 56.9% of them being multiple drug resistant (≥ 3). The predominant phylogroup identified was B1 followed by A and D, with similar distribution among the isolates from meat of locally reared chickens and imported chickens. Beta-lactamase producing genotype blaCTX-M-15 (50%; 10/20) was the most frequently drug resistant gene detected. More BLP E. coli isolates were found in imported chicken meat compared to locally reared chickens, demonstrating that these isolates may be spreading through food trade. In conclusion, both imported and locally produced chicken meats are potential

  17. 76 FR 72331 - Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-23

    ... Toxin-Producing Escherichia coli in Certain Raw Beef Products AGENCY: Food Safety and Inspection Service... methods for controlling non-O157 Shiga toxin-producing Escherichia coli in raw, intact and non-intact beef... Escherichia coli in raw, intact and non-intact beef products and product components on or before December...

  18. Enhancing Inhibition-Induced Plasticity in Tinnitus – Spectral Energy Contrasts in Tailor-Made Notched Music Matter

    PubMed Central

    Stein, Alwina; Engell, Alva; Lau, Pia; Wunderlich, Robert; Junghoefer, Markus; Wollbrink, Andreas; Bruchmann, Maximilian; Rudack, Claudia; Pantev, Christo

    2015-01-01

    Chronic tinnitus seems to be caused by reduced inhibition among frequency selective neurons in the auditory cortex. One possibility to reduce tinnitus perception is to induce inhibition onto over-activated neurons representing the tinnitus frequency via tailor-made notched music (TMNM). Since lateral inhibition is modifiable by spectral energy contrasts, the question arises if the effects of inhibition-induced plasticity can be enhanced by introducing increased spectral energy contrasts (ISEC) in TMNM. Eighteen participants suffering from chronic tonal tinnitus, pseudo randomly assigned to either a classical TMNM or an ISEC-TMNM group, listened to notched music for three hours on three consecutive days. The music was filtered for both groups by introducing a notch filter centered at the individual tinnitus frequency. For the ISEC-TMNM group a frequency bandwidth of 3/8 octaves on each side of the notch was amplified, additionally, by about 20 dB. Before and after each music exposure, participants rated their subjectively perceived tinnitus loudness on a visual analog scale. During the magnetoencephalographic recordings, participants were stimulated with either a reference tone of 500 Hz or a test tone with a carrier frequency representing the individual tinnitus pitch. Perceived tinnitus loudness was significantly reduced after TMNM exposure, though TMNM type did not influence the loudness ratings. Tinnitus related neural activity in the N1m time window and in the so called tinnitus network comprising temporal, parietal and frontal regions was reduced after TMNM exposure. The ISEC-TMNM group revealed even enhanced inhibition-induced plasticity in a temporal and a frontal cortical area. Overall, inhibition of tinnitus related neural activity could be strengthened in people affected with tinnitus by increasing spectral energy contrast in TMNM, confirming the concepts of inhibition-induced plasticity via TMNM and spectral energy contrasts. PMID:25951605

  19. Enhancing inhibition-induced plasticity in tinnitus--spectral energy contrasts in tailor-made notched music matter.

    PubMed

    Stein, Alwina; Engell, Alva; Lau, Pia; Wunderlich, Robert; Junghoefer, Markus; Wollbrink, Andreas; Bruchmann, Maximilian; Rudack, Claudia; Pantev, Christo

    2015-01-01

    Chronic tinnitus seems to be caused by reduced inhibition among frequency selective neurons in the auditory cortex. One possibility to reduce tinnitus perception is to induce inhibition onto over-activated neurons representing the tinnitus frequency via tailor-made notched music (TMNM). Since lateral inhibition is modifiable by spectral energy contrasts, the question arises if the effects of inhibition-induced plasticity can be enhanced by introducing increased spectral energy contrasts (ISEC) in TMNM. Eighteen participants suffering from chronic tonal tinnitus, pseudo randomly assigned to either a classical TMNM or an ISEC-TMNM group, listened to notched music for three hours on three consecutive days. The music was filtered for both groups by introducing a notch filter centered at the individual tinnitus frequency. For the ISEC-TMNM group a frequency bandwidth of 3/8 octaves on each side of the notch was amplified, additionally, by about 20 dB. Before and after each music exposure, participants rated their subjectively perceived tinnitus loudness on a visual analog scale. During the magnetoencephalographic recordings, participants were stimulated with either a reference tone of 500 Hz or a test tone with a carrier frequency representing the individual tinnitus pitch. Perceived tinnitus loudness was significantly reduced after TMNM exposure, though TMNM type did not influence the loudness ratings. Tinnitus related neural activity in the N1m time window and in the so called tinnitus network comprising temporal, parietal and frontal regions was reduced after TMNM exposure. The ISEC-TMNM group revealed even enhanced inhibition-induced plasticity in a temporal and a frontal cortical area. Overall, inhibition of tinnitus related neural activity could be strengthened in people affected with tinnitus by increasing spectral energy contrast in TMNM, confirming the concepts of inhibition-induced plasticity via TMNM and spectral energy contrasts.

  20. Field evaluation of a tailor-made new passive sampler for the determination of NO2 levels in ambient air.

    PubMed

    Ozden, Ozlem; Dogeroglu, Tuncay

    2008-07-01

    This study describes the field evaluation of a tailor-made new glass passive sampler developed for the determination of NO(2), based on the collection on triethanolemine (TEA)-coated fibre filter paper. The sampler has been derived from a Palmes design. The overall uncertainty of the sampler was determined by using Griess-Saltzman ASTM D 1607 standard test method as a reference method. The agreement between the results of the passive sampler and the reference method was +/-7.90% with the correlation coefficient of 0.90. Method precision in terms of coefficient of variance (CV) for three simultaneously applied passive samplers was 8.80%. The uptake rate of NO(2) was found to be 2.49 ml/min in a very good agreement with the value calculated from theory (2.63 ml/min). Sampler detection limit was 1.99 microg/m(3) for an exposure period of 1 week and the sampler can be stored safely for a period of up to 6 weeks before exposure. A comparison of the sampler performance was conducted against a commercially available diffusion tube (Gradko diffusion tube). The results from the applied statistical paired t test indicated that there was no significant difference between the performances of two passive samplers (R (2) > 0.90). Also, another statistical comparison was carried out between the dark and transparent glass passive samplers. The results from the dark-colour sampler were higher than that from the transparent sampler (approximately 25%) during the summer season because of the possible photodegradation of NO(2)-TEA complex.

  1. Impact of Spectral Notch Width on Neurophysiological Plasticity and Clinical Effectiveness of the Tailor-Made Notched Music Training

    PubMed Central

    Wunderlich, Robert; Lau, Pia; Stein, Alwina; Engell, Alva; Wollbrink, Andreas; Rudack, Claudia; Pantev, Christo

    2015-01-01

    Tinnitus, the ringing in the ears that is unrelated to any external source, causes a significant loss in quality of life, involving sleep disturbance and depression for 1 to 3% of the general population. While in the first place tinnitus may be triggered by damage to the inner ear cells, the neural generators of subjective tinnitus are located in central regions of the nervous system. A loss of lateral inhibition, tonotopical reorganization and a gain-increase in response to the sensory deprivation result in hypersensitivity and hyperactivity in certain regions of the auditory cortex. In the tailor-made notched music training (TMNMT) patients listen to music from which the frequency spectrum of the tinnitus has been removed. This evokes strong lateral inhibition from neurons tuned to adjacent frequencies onto the neurons involved in the tinnitus percept. A reduction of tinnitus loudness and tinnitus-related neural activity was achieved with TMNMT in previous studies. As the effect of lateral inhibition depends on the bandwidth of the notch, in the current study we altered the notch width to find the most effective notch width for TMNMT. We compared 1-octave notch width with ½-octave and ¼-octave. Participants chose their favorite music for the training that included three month of two hours daily listening. The outcome was measured by means of standardized questionnaires and magnetoencephalography. We found a general reduction of tinnitus distress in all administered tinnitus questionnaires after the training. Additionally, tinnitus-related neural activity was reduced after the training. Nevertheless, notch width did not have an influence on the behavioral or neural effects of TMNMT. This could be due to a non-linear resolution of lateral inhibition in high frequencies. PMID:26406446

  2. Shiga Toxin-Producing Escherichia coli (STEC) in Fresh Produce--A Food Safety Dilemma.

    PubMed

    Feng, Peter

    2014-08-01

    Produce contains high levels of mixed microflora, including coliforms and Escherichia coli, but occasionally pathogens may also be present. Enterotoxigenic E. coli and Shigatoxin-producing E. coli (STEC) have been isolated from various produce types, especially spinach. The presence of STEC in produce is easily detected by PCR for the Shiga toxin (Stx) gene, stx, but this is insufficient for risk analysis. STEC comprises hundreds of serotypes that include known pathogenic serotypes and strains that do not appear to cause severe illness. Moreover, Stx without a binding factor like intimin (encoded by eae) is deemed to be insufficient to cause severe disease. Hence, risk analyses require testing for other virulence or serotype-specific genes. Multiplex PCR enables simultaneous testing of many targets, but, in a mixed flora sample, not all targets detected may be coming from the same cell. The need to isolate and confirm STEC in produce is critical, but it is time- and labor-intensive due to the complexity of the group. Studies showed that only a handful of STEC strains in produce have eae, and most belonged to recognized pathogenic serotypes so are of definite health risks. Several eae-negative strains belonged to serotypes O113:H21 and O91:H21 that historically have caused severe illness and may also be of concern. Most of the other STEC strains in produce, however, are only partially serotyped or are unremarkable serotypes carrying putative virulence factors, whose role in pathogenesis is uncertain, thus making it difficult to assess the health risks of these STEC strains.

  3. Public Health Microbiology of Shiga Toxin-Producing Escherichia coli.

    PubMed

    Caprioli, Alfredo; Scavia, Gaia; Morabito, Stefano

    2014-12-01

    Shiga toxin-producing Escherichia coli (STEC) strains are the only pathogenic group of E. coli that has a definite zoonotic origin, with ruminants and, in particular, cattle being recognized as the major reservoir. Most human STEC infections are food borne, but the routes of transmission include direct contact with animals and a variety of environment-related exposures. Therefore, STEC public health microbiology spans the fields of medical, veterinary, food, water, and environmental microbiology, requiring a "One Health" perspective and laboratory scientists with the ability to work effectively across disciplines. Public health microbiology laboratories play a central role in the surveillance of STEC infections, as well as in the preparedness for responding to outbreaks and in providing scientific evidence for the implementation of prevention and control measures. This article reviews (i) how the integration of surveillance of STEC infections and monitoring of these pathogens in animal reservoirs and potential food vehicles may contribute to their control; (ii) the role of reference laboratories, in both the public health and veterinary and food sectors; and (iii) the public health perspectives, including those related to regulatory issues in both the European Union and the United States.

  4. Characterization of CTX-M-14-producing Escherichia coli from food-producing animals.

    PubMed

    Liao, Xiao-Ping; Xia, Jing; Yang, Lei; Li, Liang; Sun, Jian; Liu, Ya-Hong; Jiang, Hong-Xia

    2015-01-01

    Bacterial resistance to the third-generation cephalosporin antibiotics has become a major concern for public health. This study was aimed to determine the characteristics and distribution of bla CTX-M-14, which encodes an extended-spectrum β-lactamase, in Escherichia coli isolated from Guangdong Province, China. A total of 979 E. coli isolates isolated from healthy or diseased food-producing animals including swine and avian were examined for bla CTX-M-14 and then the bla CTX-M-14 -positive isolates were detected by other resistance determinants [extended-spectrum β-lactamase genes, plasmid-mediated quinolone resistance, rmtB, and floR] and analyzed by phylogenetic grouping analysis, PCR-based plasmid replicon typing, multilocus sequence typing, and plasmid analysis. The genetic environments of bla CTX-M-14 were also determined by PCR. The results showed that fourteen CTX-M-14-producing E. coli were identified, belonging to groups A (7/14), B1 (4/14), and D (3/14). The most predominant resistance gene was bla TEM (n = 8), followed by floR (n = 7), oqxA (n = 3), aac(6')-1b-cr (n = 2), and rmtB (n = 1). Plasmids carrying bla CTX-M-14 were classified to IncK, IncHI2, IncHI1, IncN, IncFIB, IncF or IncI1, ranged from about 30 to 200 kb, and with insertion sequence of ISEcp1, IS26, or ORF513 located upstream and IS903 downstream of bla CTX-M-14. The result of multilocus sequence typing showed that 14 isolates had 11 STs, and the 11 STs belonged to five groups. Many of the identified sequence types are reported to be common in E. coli isolates associated with extraintestinal infections in humans, suggesting possible transmission of bla CTX-M-14 between animals and humans. The difference in the flanking sequences of bla CTX-M-14 between the 2009 isolates and the early ones suggests that the resistance gene context continues to evolve in E. coli of food producing animals.

  5. Characterization of CTX-M-14-producing Escherichia coli from food-producing animals

    PubMed Central

    Liao, Xiao-Ping; Xia, Jing; Yang, Lei; Li, Liang; Sun, Jian; Liu, Ya-Hong; Jiang, Hong-Xia

    2015-01-01

    Bacterial resistance to the third-generation cephalosporin antibiotics has become a major concern for public health. This study was aimed to determine the characteristics and distribution of blaCTX-M-14, which encodes an extended-spectrum β-lactamase, in Escherichia coli isolated from Guangdong Province, China. A total of 979 E. coli isolates isolated from healthy or diseased food-producing animals including swine and avian were examined for blaCTX-M-14 and then the blaCTX-M-14 -positive isolates were detected by other resistance determinants [extended-spectrum β-lactamase genes, plasmid-mediated quinolone resistance, rmtB, and floR] and analyzed by phylogenetic grouping analysis, PCR-based plasmid replicon typing, multilocus sequence typing, and plasmid analysis. The genetic environments of blaCTX-M-14 were also determined by PCR. The results showed that fourteen CTX-M-14-producing E. coli were identified, belonging to groups A (7/14), B1 (4/14), and D (3/14). The most predominant resistance gene was blaTEM (n = 8), followed by floR (n = 7), oqxA (n = 3), aac(6′)-1b-cr (n = 2), and rmtB (n = 1). Plasmids carrying blaCTX-M-14 were classified to IncK, IncHI2, IncHI1, IncN, IncFIB, IncF or IncI1, ranged from about 30 to 200 kb, and with insertion sequence of ISEcp1, IS26, or ORF513 located upstream and IS903 downstream of blaCTX-M-14. The result of multilocus sequence typing showed that 14 isolates had 11 STs, and the 11 STs belonged to five groups. Many of the identified sequence types are reported to be common in E. coli isolates associated with extraintestinal infections in humans, suggesting possible transmission of blaCTX-M-14 between animals and humans. The difference in the flanking sequences of blaCTX-M-14 between the 2009 isolates and the early ones suggests that the resistance gene context continues to evolve in E. coli of food producing animals. PMID:26528278

  6. Thermolabile antifreeze protein produced in Escherichia coli for structural analysis.

    PubMed

    Lin, Feng-Hsu; Sun, Tianjun; Fletcher, Garth L; Davies, Peter L

    2012-03-01

    The only hyperactive antifreeze protein (AFP) found to date in fishes is an extreme variant of the 3-kDa, alpha-helical, alanine-rich type I AFP, which is referred to here as type Ih. Purification of the 33-kDa homodimeric AFP Ih from a natural source was hampered by its low levels in fish plasma; by the need to remove the more abundant smaller isoforms; and by its extreme thermolability. Moreover, ice affinity as a purification tool was spoiled by the tendency of fish IgM antibodies to bind to ice in the presence of AFPs. In order to produce enough protein for crystallography we expressed AFP Ih as a recombinant protein in the Arctic Express® strain of Escherichia coli at 12 °C, just below the thermal denaturation temperature of 16-18 °C. His-tags were not useful because they compromised the activity and yield of AFP Ih. But in the absence of fish antibodies we were able to recover 10-mg quantities of the antifreeze protein using two cycles of ice affinity purification followed by anion-exchange chromatography to remove contaminating chaperones. The purified recombinant AFP Ih yielded diffraction-quality crystals with an extremely asymmetrical unit cell. By transferring the genes of the chaperones into a methionine auxotroph we were able to grow this host at low temperatures and produce sufficient selenomethionine-labeled AFP Ih for crystallography.

  7. Resistance of various shiga toxin-producing Escherichia coli to electrolyzed oxidizing water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The resistance of thirty two strains of Escherichia coli O157:H7 and six major serotypes of non-O157 Shiga toxin- producing E. coli (STEC) plus E. coli O104 was tested against Electrolyzed oxidizing (EO) water using two different methods; modified AOAC 955.16 sequential inoculation method and minim...

  8. Behavior of non-O157 Shiga toxin-producing Escherichia coli, enteroinvasive E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli strains on alfalfa sprouts.

    PubMed

    Gómez-Aldapa, Carlos A; Rangel-Vargas, Esmeralda; Torres-Vitela, M Del Refugio; Villarruel-López, Angélica; Castro-Rosas, Javier

    2013-08-01

    Data about the behavior of non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enteropathogenic E. coli (EPEC) on seeds and alfalfa sprouts are not available. The behavior of STEC, EIEC, ETEC, and EPEC was determined during germination and sprouting of alfalfa seeds at 20 ± 2°C and 30 ± 2°C and on alfalfa sprouts at 3 ± 2°C. When alfalfa seeds were inoculated with STEC, EIEC, ETEC, or EPEC strains, all these diarrheagenic E. coli pathotypes (DEPs) grew during germination and sprouting of seeds, reaching counts of approximately 5 and 6 log CFU/g after 1 day at 20 ± 2°C and 30 ± 2°C, respectively. However, when the sprouts were inoculated after 1 day of seed germination and stored at 20 ± 2°C or 30 ± 2°C, no growth was observed for any DEP during sprouting at 20 ± 2°C or 30 ± 2°C for 9 days. Refrigeration reduced significantly (P < 0.0.5) the number of viable DEPs on sprouts after 20 days in storage; nevertheless, these decreases have no practical significance for the safety of the sprouts.

  9. Shiga-Like-Toxin-Producing Escherichia coli in Retail Meats and Cattle in Thailand

    DTIC Science & Technology

    1990-04-01

    animals in Thailand. SLT-producing E . coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and...from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E . coli types that hybridized...with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E . coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the

  10. Comparison of non-O157 Shiga toxin-producing E. coli detection systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Category: methodology improvements Objective: To identify strengths and weaknesses of commercial Shiga toxin-producing E. coli detection systems and kits in a side by side fashion. Experimental Design: Three commercial Shiga toxin-producing E. coli detection tests (BAX, GDS, and GeneDisc) and two t...

  11. Expansion of Shiga Toxin-Producing Escherichia coli by Use of Bovine Antibiotic Growth Promoters.

    PubMed

    Kim, Jong-Chul; Chui, Linda; Wang, Yang; Shen, Jianzhong; Jeon, Byeonghwa

    2016-05-01

    Antibiotics are routinely used in food-producing animals to promote growth and prevent infectious diseases. We investigated the effects of bovine antibiotic growth promoters (bAGPs) on the propagation and spread of Shiga toxin (Stx)-encoding phages in Escherichia coli. Co-culture of E. coli O157:H7 and other E. coli isolated from cattle in the presence of sublethal concentrations of bAGPs significantly increased the emergence of non-O157, Stx-producing E. coli by triggering the SOS response system in E. coli O157:H7. The most substantial mediation of Stx phage transmission was induced by oxytetracyline and chlortetracycline, which are commonly used in agriculture. bAGPs may therefore contribute to the expansion of pathogenic Stx-producing E. coli.

  12. Expansion of Shiga Toxin–Producing Escherichia coli by Use of Bovine Antibiotic Growth Promoters

    PubMed Central

    Kim, Jong-Chul; Chui, Linda; Wang, Yang; Shen, Jianzhong

    2016-01-01

    Antibiotics are routinely used in food-producing animals to promote growth and prevent infectious diseases. We investigated the effects of bovine antibiotic growth promoters (bAGPs) on the propagation and spread of Shiga toxin (Stx)–encoding phages in Escherichia coli. Co-culture of E. coli O157:H7 and other E. coli isolated from cattle in the presence of sublethal concentrations of bAGPs significantly increased the emergence of non-O157, Stx-producing E. coli by triggering the SOS response system in E. coli O157:H7. The most substantial mediation of Stx phage transmission was induced by oxytetracyline and chlortetracycline, which are commonly used in agriculture. bAGPs may therefore contribute to the expansion of pathogenic Stx-producing E. coli. PMID:27088186

  13. First report of Escherichia coli co-producing NDM-1 and OXA-232.

    PubMed

    Both, Anna; Huang, Jiabin; Kaase, Martin; Hezel, Julia; Wertheimer, Daniel; Fenner, Ines; Günther, Thomas; Grundhoff, Adam; Büttner, Henning; Aepfelbacher, Martin; Rohde, Holger; Hentschke, Moritz

    2016-12-01

    Recently Gram-negative bacteria co-producing multiple carbapenemases have emerged in different parts of the world. We report the first isolation of an Escherichia coli strain co-producing the carbapenemases NDM-1 and OXA-232.

  14. Translocation and thermal inactivation of Shiga-toxin producing Escherichia coli in non-intact beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We compared translocation of genetically-marked strains of serotype O157:H7 Escherichia coli (ECOH) to non-O157:H7 Shiga-Toxin producing Escherichia coli (STEC) following blade tenderization of beef subprimals and the subsequent lethality of these pathogens following cooking of steaks prepared from ...

  15. Complete genome sequence and comparison of two Shiga toxin-producing Escherichia coli O104 isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) O104 strains have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. E. coli O104:H21 caused an outbreak associated with milk in the U.S. in 1994. In this study, next generation sequencing techno...

  16. Detection and isolation of shiga toxin-producing Escherichia coli (STEC) O104 from sprouts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E. coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) are food-borne pathogens responsible for severe outbreaks of hemorrhagic colitis, which can lead to hemolytic uremic syndrome and/or death. STEC strains belonging to serogroup O104 have been associated with sporadic cases of illness a...

  17. Characterization of fimbriae produced by enteropathogenic Escherichia coli.

    PubMed Central

    Girón, J A; Ho, A S; Schoolnik, G K

    1993-01-01

    Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC. Images PMID:7901197

  18. Properties of a Clostridium thermocellum Endoglucanase Produced in Escherichia coli

    PubMed Central

    Schwarz, Wolfgang H.; Gräbnitz, Folke; Staudenbauer, Walter L.

    1986-01-01

    A cellulase gene of Clostridium thermocellum was transferred to Escherichia coli by molecular cloning with bacteriophage lambda and plasmid vectors and shown to be indentical with the celA gene. The celA gene product was purified from extracts of plasmid-bearing E. coli cells by heat treatment and chromatography on DEAE-Trisacryl. It was characterized as a thermophilic endo-β-1,4-glucanase, the properties of which closely resemble those of endoglucanase A previously isolated from C. thermocellum supernatants. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the enzyme purified from E. coli exhibited two protein bands with molecular weights of 49,000 and 52,000. It had a temperature optimum at 75°C and was stable for several hours at 60°C. Endoglucanase activity was optimal between pH 5.5 and 6.5. The enzyme was insensitive against end product inhibition by glucose and cellobiose and remarkably resistant to the denaturing effects of detergents and organic solvents. It was capable of degrading, in addition to cellulosic substrates, glucans with alternating β-1,4 and β-1,3 linkages such as barley β-glucan and lichenan. PMID:16347088

  19. Tailor-Made Zinc-Finger Transcription Factors Activate FLO11 Gene Expression with Phenotypic Consequences in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Shieh, Jia-Ching; Cheng, Yu-Che; Su, Mao-Chang; Moore, Michael; Choo, Yen; Klug, Aaron

    2007-01-01

    Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity. PMID:17710146

  20. Occurrence and phenotypic properties of verotoxin producing Escherichia coli in sporadic cases of gastroenteritis.

    PubMed

    Burnens, A P; Boss, P; Orskov, F; Orskov, I; Schaad, U B; Müller, F; Heinzle, R; Nicolet, J

    1992-07-01

    Five verotoxin producing Escherichia coli strains were detected in 405 patients with infectious gastroenteritis and 3 such strains were detected in 11 patients with the hemolytic uremic syndrome in Switzerland. Production of verotoxin 2 was associated with the latter three strains. Four strains reacted with the probe for the virulence plasmid of Escherichia coli O157:H7, and six reacted with a recently described probe for the eae gene of enteropathogenic Escherichia coli. None of the strains was of serotype O157:H7. The methods available at present for detecting toxins or toxin genes will reliably detect all such verotoxin producing strains.

  1. Emergence of Carbapenemase-Producing Escherichia coli Isolated from Companion Animals in Algeria.

    PubMed

    Yousfi, Massilia; Touati, Abdelaziz; Mairi, Assia; Brasme, Lucien; Gharout-Sait, Alima; Guillard, Thomas; De Champs, Christophe

    2016-06-01

    The emergence and worldwide spread of carbapenemase-producing Enterobacteriaceae is of great concern to public health. The aim of this study was to investigate the occurrence of carbapenemase-producing Escherichia coli in companion animals in Algeria. Two hundred fecal samples were obtained from healthy and diseased dogs and cats in one veterinary office and private owners in Bejaia city, Algeria, during November 2014 to March 2015. Isolates were screened by polymerase chain reaction for the presence of carbapenemase, acquired plasmidic AmpC (pAmpC) and extended-spectrum beta-lactamase genes. Five carbapenemase-producing E. coli isolates were detected including four OXA-48-producing isolates and one isolate producing NDM-5. Coexpression of ESBL and pAmpC genes was observed in these isolates. Phylogenetic grouping revealed that these isolates belonged to A and D phylogroups. The results of this study show that carbapenemase-producing E. coli spread to the companion animals in Algeria.

  2. Tailor-made heart simulation predicts the effect of cardiac resynchronization therapy in a canine model of heart failure.

    PubMed

    Panthee, Nirmal; Okada, Jun-ichi; Washio, Takumi; Mochizuki, Youhei; Suzuki, Ryohei; Koyama, Hidekazu; Ono, Minoru; Hisada, Toshiaki; Sugiura, Seiryo

    2016-07-01

    Despite extensive studies on clinical indices for the selection of patient candidates for cardiac resynchronization therapy (CRT), approximately 30% of selected patients do not respond to this therapy. Herein, we examined whether CRT simulations based on individualized realistic three-dimensional heart models can predict the therapeutic effect of CRT in a canine model of heart failure with left bundle branch block. In four canine models of failing heart with dyssynchrony, individualized three-dimensional heart models reproducing the electromechanical activity of each animal were created based on the computer tomographic images. CRT simulations were performed for 25 patterns of three ventricular pacing lead positions. Lead positions producing the best and the worst therapeutic effects were selected in each model. The validity of predictions was tested in acute experiments in which hearts were paced from the sites identified by simulations. We found significant correlations between the experimentally observed improvement in ejection fraction (EF) and the predicted improvements in ejection fraction (P<0.01) or the maximum value of the derivative of left ventricular pressure (P<0.01). The optimal lead positions produced better outcomes compared with the worst positioning in all dogs studied, although there were significant variations in responses. Variations in ventricular wall thickness among the dogs may have contributed to these responses. Thus CRT simulations using the individualized three-dimensional heart models can predict acute hemodynamic improvement, and help determine the optimal positions of the pacing lead.

  3. Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

    PubMed Central

    Kudva, I T; Hatfield, P G; Hovde, C J

    1997-01-01

    The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens. PMID:9157149

  4. Use of Photopolymerization for Genotyping Shiga Toxin-Producing Escherichia coli Recovered from Produce Production Regions in California

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype STEC strains, the present study employed the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen ...

  5. Antibacterial Activity of Some Plant Extracts Against Extended- Spectrum Beta-Lactamase Producing Escherichia coli Isolates

    PubMed Central

    Saeidi, Saeide; Amini Boroujeni, Negar; Ahmadi, Hassan; Hassanshahian, Mehdi

    2015-01-01

    Background: The extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli isolates make many serious infections, especially urinary tract infections. Objectives: The purpose of this study was to determine the antibacterial activities of some natural plant extracts against ESBL-producing E. coli isolates, which harbor the TEM gene in urine samples of the patients who have urinary tract infections. Materials and Methods: Evaluation has to be exactly determined for both methods of disk diffusion test and polymerase chain reaction (PCR), separately. We evaluated 120 strains of E. coli isolates from the urine culture of the patients in Boo-Ali Hospital (Zahedan, south-eastern Iran) who were suffering from urinary tract infections. The ESBL-producing E. coli isolates were evaluated by disk diffusion test and PCR through TEM gene detection. The minimal inhibitory concentration (MIC) of commonly used antibiotics including ceftazidime, ceftriaxon, amikacin, gentamicin and ciprofloxacin along with the MIC of the alcoholic extract of different natural plants including Myrtus communis L (Myrtaceae), Amaranthus retraflexus (Amaranthaceae), Cyminum cuminum L (Apiaceae), Marrubium vulgare (Laminaceae) and Peganum. harmala (Zygrophyllaceae) against the ESBL-producing E. coli isolates, which harbor the TEM genes, were determined using the microdulition method. Results: Results of this study showed that in disk diffusion method, 80 samples of E. coli produced ESBLs. In PCR method, the TEM gene distribution in the isolated ESBL-producing organisms was 50 (41.6%). Amikacin was the most effective anti-bacterial agent and ciprofloxacin was the least effective against E. coli isolates. All the natural plant extracts mentioned above, especially P. harmala, were effective against the selected isolates of ESBL-producing E. coli. The most frequent ESBL rate producing E. coli isolates (32 out of 50) had MIC of 2.5 mg/mL in ethanol extract of P. harmala. Conclusions: The alcoholic

  6. Characterization of Escherichia coli and other Enterobacteriaceae in producer-distributor bulk milk.

    PubMed

    Ntuli, V; Njage, P M K; Buys, E M

    2016-12-01

    The current study was undertaken to characterize Escherichia coli and other Enterobacteriaceae in raw and pasteurized producer-distributor bulk milk (PDBM). A total of 258 samples were collected from purchase points in 8 provinces in South Africa. The samples were tested for antibiotic residues, phosphatase, total aerobic bacteria, coliforms, and E. coli counts. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for identification of isolates. Escherichia coli isolates were characterized for virulence factors, antimicrobial resistance, serotypes, and presumptive E. coli O157:H7. Antibiotic residues and alkaline phosphatase were detected in 2% of both raw and pasteurized PDBM (n=258) and 21% pasteurized PDBM (n=104) samples, respectively. A total of 729 isolates belonging to 21 genera and 59 species were identified. Escherichia coli, Enterobacter cloacae, Klebsiella oxytoca, and Raoultella ornithinolytica were the most abundant species. Spoilage Enterobacteriaceae species exceeded 50% of the total isolates. Escherichia coli was detected and isolated from 36% of the milk samples. Thirty-one E. coli isolates harbored virulence genes stx1/stx2 and 38% (n=121) were presumptive O157:H7. The prevalence of samples with presumptive shigatoxin producing E. coli was 10%. Antimicrobial-resistant E. coli isolates were detected in 70% of the milk samples with 36% of stx1/stx2 positive E. coli showing multi-drug resistance. Information obtained from the study will be used for modeling the public health risk posed by milkborne pathogens in PDBM, which in many cases is consumed by poor and vulnerable members of the population.

  7. Effects of a tailor-made exercise program on exercise adherence and health outcomes in patients with knee osteoarthritis: a mixed-methods pilot study

    PubMed Central

    Lee, Fung-Kam Iris; Lee, Tze-Fan Diana; So, Winnie Kwok-Wei

    2016-01-01

    Introduction Previous studies showed that exercise intervention was effective in symptoms control of knee osteoarthritis (OA) but poor intervention adherence reduced the exercise effect. It has been suspected that the design of exercise intervention mainly from the health care professionals’ perspective could not address the patients’ barriers to exercise. Therefore, a tailor-made exercise program which incorporated the patient’s perspective in the design was developed and ready for evaluation. Objectives This pilot study estimated the effects of a tailor-made exercise program on exercise adherence and health outcomes, and explored the participants’ perception and experience of the program. Methods The intervention of this study was a 4-week community-based group exercise program, which required the participants to attend a 1-hour session each week. Thirty-four older people with knee OA were recruited to the program. Mixed-methods study design was used to estimate the effects of this program and explore the participants’ perception and experience of the program. Exercise adherence and performance in return-demonstration of the exercise were assessed at 12 weeks after the program. Disease-specific health status (Western Ontario and McMaster Universities Osteoarthritis Index), general health status (12-item Short Form of the Medical Outcome Study Questionnaire), knee range of motion, muscle strength, and endurance of the lower extremities (Timed-Stands Test) were measured at the beginning of the program and 12 weeks after. Six participants were interviewed individually on the 12th week. Results Thirty-three participants (75.0±7.3 years) completed the one-group pretest and post-test study. The participants’ exercise adherence was 91.4%±14.54%, and their correct performance in return-demonstration was 76.7%±21.75%. Most of the participants’ health outcomes significantly improved at posttests except the 12-item Short Form of the Medical Outcome Study

  8. Incidence of extended spectrum beta lactamase producing Escherichia coli among patients, healthy individuals and in the environment.

    PubMed

    George, E A; Sankar, S; Jesudasan, M V; Sudandiradoss, C; Nandagopal, B

    2014-01-01

    We investigated the faecal carriage of extended spectrum β-lactamases (ESBL) producing Escherichia coli in different groups of human subjects and in the environment. A total of 363 E. coli strains were isolated from stool samples of patients (n = 77), healthy subjects (n = 170) and from different environmental samples (n = 116). A total of 124 ESBL producing E. coli strains were isolated in this study. The frequency of ESBL producing E. coli was found to be highest (60.3%) among the strains isolated from patients, followed by healthy individuals (38%) and the environment (10.5%). The environment was observed to have a very low number of ESBL producing E. coli.

  9. The application of tailor-made force fields and molecular dynamics for NMR crystallography: a case study of free base cocaine

    PubMed Central

    Neumann, Marcus A.

    2017-01-01

    Motional averaging has been proven to be significant in predicting the chemical shifts in ab initio solid-state NMR calculations, and the applicability of motional averaging with molecular dynamics has been shown to depend on the accuracy of the molecular mechanical force field. The performance of a fully automatically generated tailor-made force field (TMFF) for the dynamic aspects of NMR crystallography is evaluated and compared with existing benchmarks, including static dispersion-corrected density functional theory calculations and the COMPASS force field. The crystal structure of free base cocaine is used as an example. The results reveal that, even though the TMFF outperforms the COMPASS force field for representing the energies and conformations of predicted structures, it does not give significant improvement in the accuracy of NMR calculations. Further studies should direct more attention to anisotropic chemical shifts and development of the method of solid-state NMR calculations. PMID:28250956

  10. Structure and mode of action of microcin 7, an antibacterial peptide produced by Escherichia coli.

    PubMed Central

    Garcia-Bustos, J F; Pezzi, N; Mendez, E

    1985-01-01

    Microcin 7 is a small peptide produced and excreted to the culture medium by stationary-phase Escherichia coli cells harboring the pMccC7 plasmid (formerly named pRYC7). This peptide inhibited the growth of the enterobacteria phylogenetically closer to E. coli, apparently by blocking protein biosynthesis. The molecule was degraded with trypsin, and the resulting fragments were purified and sequenced. The results show that microcin 7 is a linear heptapeptide blocked at both ends. PMID:2861788

  11. Changes in bone regeneration by trehalose coating and basic fibroblast growth factor after implantation of tailor-made bone implants in dogs.

    PubMed

    Choi, Sungjin; Lee, Jongil; Igawa, Kazuyo; Liu, I-Li; Honnami, Muneki; Suzuki, Shigeki; Nishimura, Ryohei; Chung, Ung-Il; Sasaki, Nobuo; Mochizuki, Manabu

    2013-01-01

    In this study, we aimed to determine the effect of trehalose coating and the optimal dose of basic fibroblast growth factor (bFGF), an osteoinductive protein, loaded onto tailor-made bone implants for implant-induced bone formation in vivo. We fabricated tailor-made α-tricalcium phosphate bone implants (11 mm diameter with 2 parallel cylindrical holes). bFGF 0, 1, 10, 100 or 200 μg/implant was incorporated into implants with and without a trehalose coating, and these were subsequently implanted into dogs to correct temporal bone defects of the same size and shape. Four weeks after implantation, we analyzed the bone implants and surrounding tissues by using micro-computed tomography imaging and histological analyses, as well as gross evaluation. No significant difference in new bone formation was observed between implants with and without a trehalose coating at any of the bFGF doses. Bone implants with 100 and 200 μg bFGF showed significantly more new bone formation at the implant site and within the cylindrical holes of the implants than those without bFGF (P<0.05). However, heterotopic bone formation on the skull near the implant was observed in the group that received 200 μg bFGF. These results suggest that 100 μg bFGF is the optimal dose for this implant in dogs, and that the trehalose coating may not be necessary in vivo, probably due to the presence of blood proteins and electrolytes at the implant site.

  12. Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

    PubMed

    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.

  13. Metabolic engineering of Escherichia coli to produce gamma-aminobutyric acid using xylose.

    PubMed

    Zhao, Anqi; Hu, Xiaoqing; Wang, Xiaoyuan

    2017-02-11

    Biomass-derived xylose is an economically interesting substrate for the sustainable microbial production of value-added compounds. Escherichia coli could barely use xylose to directly produce gamma-aminobutyric acid. In this study, E. coli strains that could directly produce gamma-aminobutyric acid were developed through the deletion of eight genes sucA, puuE, gabT, gabP, xylA, xylB, waaC, and waaF, and the overexpression of two E. coli genes gadB and gdhA, as well as five Caulobacter crescent genes CcxylA, CcxylB, CcxylC, CcxylD, and CcxylX. Both E. coli strains W3110 and JM109 could directly produce gamma-aminobutyric acid from xylose after either overexpression of the seven genes or deletion of the eight genes. Overexpression of the seven genes of in the multiple deletion mutants further increased gamma-aminobutyric acid production. Among the 28 recombinant E. coli strains constructed in this study, the highest gamma-aminobutyric acid was produced by JWZ08/pWZt7-g3/pWZt7-xyl. JWZ08/pWZt7-g3/pWZt7-xyl could produce 3.95 g/L gamma-aminobutyric acid in flask cultivation, using xylose as the sole carbon source.

  14. Teacher Training, Tailor-Made

    ERIC Educational Resources Information Center

    Newman, Katherine

    2009-01-01

    Family Partnerships for Achievement is not a course typical of most master's programs in education. The course was designed with one overriding goal: to prepare teachers to be effective in the Boston Public Schools (BPS). This goal drives every aspect of the Boston Teacher Residency (BTR), a district-based program for teacher training and…

  15. A Tailor-Made City

    ERIC Educational Resources Information Center

    Kahama, Clement George

    1975-01-01

    Dodoma, future capital of Tanzania, is one of the first planned attempts at integrating man in his environment. The four principle elements of the Master Plan include: the residential communities, the national capital central spine, the system of open spaces and the transportation network. (BT)

  16. [Estimation of the transfer of ESBL-producing Escherichia coli to humans in Germany].

    PubMed

    Sharp, Hannah; Valentin, Lars; Fischer, Jennie; Guerra, Beatriz; Appel, Bernd; Käsbohrer, Annemarie

    2014-01-01

    In 2011 EFSA has evaluated the risk for the consumer caused by ESBL-/AmpC-producing bacteria in food of animal origin and in livestock animals. Human-to-human transfer in hospitals.and in the community was considered as the most relevant route of transmission for ESBL-producing E. coli. ESBL-/AmpC-producing E. coli are in Germany, as in many other Member States of the European Union, widely spread in food of animal origin and in livestock animals. In an assessment of the relevance of livestock animals as reservoir for ESBL-/AmpC-producing E. coli as well as for ESBL-coding resistance genes the heterogeneity of the resistance genes, plasmids and bacteria in animals, foods and humans needs to be considered. In this context, both, the clonal spread of bacteria as well as horizontal transfer of resistance genes, e. g. by plasmids, have to be analyzed. Whereas studies in The Netherlands identified poultry as the most relevant reservoir, the transfer of ESBL-gene carrying plasmids from pigs to the farmers was demonstrated in Denmark. First attempts to quantify the relevance of livestock animals as reservoir for ESBL-producing E. coli in Germany showed, that the proportions of the most frequent ESBL-resistance genes are quite different between animal and human derived E. coli isolates. If in addition properties of the bacterial cells, e.g. resistance to several antibiotic classes are considered, only a small proportion of human isolates showed the same patterns as animal isolates. The results achieved so far demonstrate that certain ESBL-types are prevalent in all livestock populations investigated. Currently, the majority of cases of colonizations with ESBL-producing E. coli among humans cannot be directly linked to livestock and food-producing animals as reservoirs. This reflects that transmission routes are more complex and other reservoirs and sources including human-human interactions have to be taken into consideration.

  17. Thermal inactivation of Escherichia coli 0157:H7 (ECOH) and non-0157 Shiga toxin-producing E.coli (STEC)in mechanically tenderized veal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We quantified thermal destruction of Shiga toxin-producing Escherichia coli O157:H7 (ECOH) and Shiga toxin-producing non-O157 E. coli (STEC) cells within mechanically tenderized veal cutlets following cooking on an electric skillet. For each of five trials, flattened veal cutlets (ca. 71.6 g; ca. 1/...

  18. Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

    PubMed Central

    Saeed, A M; Magnuson, N S; Sriranganathan, N; Burger, D; Cosand, W

    1984-01-01

    Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Images PMID:6376355

  19. Rapid, Multiplexed Characterization of Shiga Toxin-Producing Escherichia coli (STEC) Isolates Using Suspension Array Technology

    PubMed Central

    Carter, John M.; Lin, Andrew; Clotilde, Laurie; Lesho, Matthew

    2016-01-01

    Molecular methods have emerged as the most reliable techniques to detect and characterize pathogenic Escherichia coli. These molecular techniques include conventional single analyte and multiplex PCR, PCR followed by microarray detection, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. The choice of methods used depends upon the specific needs of the particular study. One versatile method involves detecting serogroup-specific markers by hybridization or binding to encoded microbeads in a suspension array. This molecular serotyping method has been developed and adopted for investigating E. coli outbreaks. The major advantages of this technique are the ability to simultaneously serotype E. coli and detect the presence of virulence and pathogenicity markers. Here, we describe the development of a family of multiplex molecular serotyping methods for Shiga toxin-producing E. coli, compare their performance to traditional serotyping methods, and discuss the cost-benefit balance of these methods in the context of various food safety objectives. PMID:27242670

  20. Metabolic evolution of Escherichia coli strains that produce organic acids

    DOEpatents

    Grabar, Tammy; Gong, Wei; Yocum, R Rogers

    2014-10-28

    This invention relates to the metabolic evolution of a microbial organism previously optimized for producing an organic acid in commercially significant quantities under fermentative conditions using a hexose sugar as sole source of carbon in a minimal mineral medium. As a result of this metabolic evolution, the microbial organism acquires the ability to use pentose sugars derived from cellulosic materials for its growth while retaining the original growth kinetics, the rate of organic acid production and the ability to use hexose sugars as a source of carbon. This invention also discloses the genetic change in the microorganism that confers the ability to use both the hexose and pentose sugars simultaneously in the production of commercially significant quantities of organic acids.

  1. Behavior of enteroaggregative Escherichia coli, non-O157-shiga toxin-producing E. coli, enteroinvasive E. coli, enteropathogenic E. coli and enterotoxigenic E. coli strains on mung bean seeds and sprout.

    PubMed

    Gómez-Aldapa, Carlos A; Rangel-Vargas, Esmeralda; Bautista-De León, Haydee; Vázquez-Barrios, Ma Estela; Gordillo-Martínez, Alberto J; Castro-Rosas, Javier

    2013-09-16

    The behavior of enteroaggregative Escherichia coli (EAEC), non-O157 shiga toxin-producing E. coli (non-O157-STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) on mung bean seeds at 25±2 °C and during germination and sprouting of mung bean seeds at 20±2 ° and 30±2 °C and on mung bean sprouts at 3±2 °C was determined. When mung bean seeds were inoculated with EAEC, non-O157 STEC, EIEC, EPEC or ETEC strains, all these diarrheagenic E. coli pathotypes (DEPs) survived at least 90 days on mung bean seeds at 25±2 °C. All DEPs grew during germination and sprouting of seeds, reaching counts of approximately 5 Log and 7 Log CFU/g after 2 days at 20±2 ° and 30±2 °C, respectively. However, when the sprouts were inoculated after 1 day of seeds germination and stored at 20±2 ° or 30±2 °C, no growth was observed for any DEPs during sprouting at 20±2 °C per 9 d; however, a significant increase in the concentration of DEPs of approximately 0.7 log CFU/g was observed during sprouting at 30±2 °C after 1 day of sprout contamination. Refrigeration reduced the number of viable DEPs strains on sprouts after 10 days in storage; nevertheless, these decreases have no practical significance in the safety of the sprouts.

  2. Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

    PubMed Central

    Balière, C.; Rincé, A.; Delannoy, S.; Fach, P.

    2016-01-01

    ABSTRACT Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx1a eae-β1 strain was associated with a large number of virulence-associated genes (n = 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx1a, stx2a, stx2c, and stx2d) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2. IMPORTANCE The species E. coli includes a wide variety of strains, some of which may be responsible for severe infections. This study, a molecular risk assessment study of E. coli strains isolated from the coastal environment, was conducted to evaluate the potential risk for shellfish consumers. This report describes the characterization of virulence gene profiles and stx/eae polymorphisms of E. coli

  3. Quantitative PCR measurements of Escherichia coli including shiga toxin-producing E. coli (STEC) in animal feces and environmental waters.

    PubMed

    Ahmed, W; Gyawali, P; Toze, S

    2015-03-03

    Quantitative PCR (qPCR) assays were used to determine the concentrations of E. coli including shiga toxin-producing E. coli (STEC) associated virulence genes (eaeA, stx1, stx2, and hlyA) in ten animal species (fecal sources) and environmental water samples in Southeast Queensland, Australia. The mean Log10 concentrations and standard deviations of E. coli 23S rRNA across fecal sources ranged from 1.3 ± 0.1 (horse) to 6.3 ± 0.4 (cattle wastewater) gene copies at a test concentration of 10 ng of DNA. The differences in mean concentrations of E. coli 23S rRNA gene copies among fecal source samples were significantly different from each other (P < 0.0001). Among the virulence genes, stx2 (25%, 95% CI, 17-33%) was most prevalent among fecal sources, followed by eaeA (19%, 95% CI, 12-27%), stx1 (11%, 95% CI, 5%-17%) and hlyA (8%, 95% CI, 3-13%). The Log10 concentrations of STEC virulence genes in cattle wastewater samples ranged from 3.8 to 5.0 gene copies at a test concentration of 10 ng of DNA. Of the 18 environmental water samples tested, three (17%) were positive for eaeA and two (11%) samples were also positive for the stx2 virulence genes. The data presented in this study will aid in the estimation of quantitative microbial risk assessment (QMRA) from fecal pollution of domestic and wild animals in drinking/recreational water catchments.

  4. Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing

    PubMed Central

    Leonard, Susan R.; Mammel, Mark K.; Lacher, David W.; Elkins, Christopher A.

    2016-01-01

    Consumption of fresh bagged spinach contaminated with Shiga toxin-producing Escherichia coli (STEC) has led to severe illness and death; however current culture-based methods to detect foodborne STEC are time consuming. Since not all STEC strains are considered pathogenic to humans, it is crucial to incorporate virulence characterization of STEC in the detection method. In this study, we assess the comprehensiveness of utilizing a shotgun metagenomics approach for detection and strain-level identification by spiking spinach with a variety of genomically disparate STEC strains at a low contamination level of 0.1 CFU/g. Molecular serotyping, virulence gene characterization, microbial community analysis, and E. coli core gene single nucleotide polymorphism (SNP) analysis were performed on metagenomic sequence data from enriched samples. It was determined from bacterial community analysis that E. coli, which was classified at the phylogroup level, was a major component of the population in most samples. However, in over half the samples, molecular serotyping revealed the presence of indigenous E. coli which also contributed to the percent abundance of E. coli. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 94% of the samples with a total number of reads per sample averaging 2.4 million. Variation in STEC abundance and/or detection was observed in replicate spiked samples, indicating an effect from the indigenous microbiota during enrichment. SNP analysis of the metagenomic data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate in the enriched sample. Also, for these samples, our analysis demonstrates that strain-level phylogenetic resolution is possible using shotgun metagenomic data for determining the genomic relatedness of a contaminating STEC strain to other closely related E

  5. Classification of shiga toxin-producing escherichia coli (STEC) serotypes with hyperspectral microscope imagery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. Since a conventional microbiological method for cell counting is laborious and time-consuming process, optica...

  6. Shiga toxin-producing Escherichia coli: importance, outbreaks, and characterization methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen known to cause human gastrointestinal illnesses with diverse clinical manifestations. The varying disease severity, such as the onset of the hemolytic uremic syndrome, has been associated with certain serotypes of STEC and with th...

  7. Secondary Shiga Toxin–Producing Escherichia coli Infection, Japan, 2010–2012

    PubMed Central

    Iyoda, Sunao; Iguchi, Atsushi; Ohnishi, Makoto

    2016-01-01

    To evaluate the potential public health risk caused by secondary Shiga toxin–producing Escherichia coli (STEC) infections in Japan, we investigated the prevalence and characteristics of STEC isolated from healthy adults during 2010–2012. Although prevalence among healthy adults was high, most STEC organisms displayed characteristics rarely found in isolates from symptomatic patients. PMID:27869602

  8. Evaluation of beef trim sampling methods for detection of Shiga toxin-producing Escherichia coli (STEC)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Presence of Shiga toxin-producing Escherichia coli (STEC) is a major concern in ground beef. Several methods for sampling beef trim prior to grinding are currently used in the beef industry. The purpose of this study was to determine the efficacy of the sampling methods for detecting STEC in beef ...

  9. Epidemiology of Shiga toxin-producing Escherichia coli (STEC) in finishing swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Every year, approximately 200,000 cases of illness are estimated to be associated with Shiga toxin-producing Escherichia coli (STEC) in the United States. STEC strains are one of the leading causes of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. Many STEC outbreaks are a...

  10. Thermal inactivation of non-0157:H7 Shigatoxin producing Escherichia coli(STEC) on catfish fillets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-O157:H7 Shiga toxin-producing Escherichia coli (non-O157 STEC) strains have emerged as foodborne pathogens caused numerous foodborne illness outbreaks worldwide. Seafood (fish) consumption has significantly increased in recent years and it could be more common for STEC outbreaks due to non-O15...

  11. A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotyping of Shiga toxin-producing Escherichia coli (STEC) has been contingent upon the availability of antisera. Here we describe a 7-plex microbead-based immunoassay to simultaneously serotype seven STEC (i.e., belonging to serogroups O26, O45, O103, O111, O121, O145, and O157) by the Luminex xMA...

  12. Effect of stress on non-O157 Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-O157 Shiga toxin-producing E. coli (non-O157 STEC) have emerged as important food-borne pathogens worldwide. Non-O157 STEC serogroups O26, O45, O103, O111, O121, and O145 have been declared as adulterants in beef by the USDA Food Safety and Inspection Service. While documentation is limited, tre...

  13. Development of an automated multiplexed immunomagnetic separation system for isolating Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years, non-O157 Shiga toxin-producing Escherichia coli(STEC) have become an emerging problem. Efforts have been devoted to facilitating and speeding their detection, however, their isolation from high background microbiota foods remains problematic. To solve this problem, immunomagnetic se...

  14. Shiga toxin-producing escherichia coli: detection, differentiation, and implications for food safety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    All unprocessed food products typically harbor microorganisms. Some foods and the components that go into food production may contain pathogenic microorganisms such as Shiga toxin-producing Escherichia coli (STECs). When consumed, these STECs can cause serious illness or even death. In 2011, an out...

  15. Thermal inactivation of Shiga toxin-producing Escherichia coli cells within veal cordon bleu

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated the fate of Shiga toxin-producing Escherichia coli (STEC) within mechanically tenderized veal cordon bleu steaks following cooking on a flat-surface, non-stick griddle. Pre-flattened veal cutlets (ca. 75 g; ca. 0.34 cm thick) were purchased from a local vendor and both faces were surfac...

  16. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin (Stx) producing E. coli (STEC) are a major family of foodborne pathogens of immense public health, zoonotic and economic significance in the US and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal...

  17. Shiga toxin-producing Escherichia coli in swine: the public health perspective

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens that are an important public health concern. STEC infection is associated with severe clinical diseases in humans, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS), which can lead to kidney failure ...

  18. Characterization of shiga toxin subtypes and virulence genes in Porcine shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a...

  19. Tailor-made ion-imprinted polymer based on functionalized graphene oxide for the preconcentration and determination of trace copper in food samples.

    PubMed

    Liu, Yan; Qiu, Jian; Liu, Zhanchao; Ni, Liang; Jiang, Yinhua; Gong, Chongying; Meng, Xiangguo; Liu, Fangfang; Zhong, Guoxing

    2016-04-01

    A tailor-made Cu(II) ion-imprinted polymer based on large-surface-area graphene oxide sheets has been synthesized for the preconcentration and determination of trace copper from food samples by solid-phase extraction. Attributed to the ultrahigh surface area and hydrophilicity of graphene oxide, the Cu(II) ion-imprinted polymer prepared by the surface ion-imprinting technique exhibited a high binding capacity and a fast adsorption rate under the optimized experimental conditions. In the static adsorption experiments, the maximum adsorption capacity of Cu(II) ion-imprinted polymer is 109.38 mg/g at 25°C, which is much higher than that of the nonimprinted polymer (32.12 mg/g). Meanwhile, the adsorption is very rapid and equilibrium is reached after approximately 30 min. The adsorption mechanism is found to follow Langmuir adsorption model and the pseudo-second-order adsorption process. The Cu(II) ion-imprinted polymer was used for extracting and detecting Cu(II) in food samples combined with graphite flame atomic adsorption spectrometry with high recoveries in the range of 97.6-103.3%. The relative standard deviation and limit of detection of the method were evaluated as 1.2% and 0.37 μg/L, respectively. The results showed that the novel absorbent can be utilized as an effective material for the selective enrichment and determination of Cu(II) from food samples.

  20. Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains

    PubMed Central

    Botkin, Douglas J.; Galli, Lucía; Sankarapani, Vinoth; Soler, Michael; Rivas, Marta; Torres, Alfredo G.

    2012-01-01

    Escherichia coli O157:H7 and other pathogenic E. coli strains are enteric pathogens associated with food safety threats and which remain a significant cause of morbidity and mortality worldwide. In the current study, we investigated whether enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC) strains can be rapidly and specifically differentiated with multiplex PCR (mPCR) utilizing selected biomarkers associated with each strain’s respective virulence genotype. Primers were designed to amplify multiple intimin (eae) and long polar fimbriae (lpfA) variants, the bundle-forming pilus gene bfpA, and the Shiga toxin-encoding genes stx1 and stx2. We demonstrated consistent amplification of genes specific to the prototype EHEC O157:H7 EDL933 (lpfA1-3, lpfA2-2, stx1, stx2, and eae-γ) and EPEC O127:H6 E2348/69 (eae-α, lpfA1-1, and bfpA) strains using the optimized mPCR protocol with purified genomic DNA (gDNA). A screen of gDNA from isolates in a diarrheagenic E. coli collection revealed that the mPCR assay was successful in predicting the correct pathotype of EPEC and EHEC clones grouped in the distinctive phylogenetic disease clusters EPEC1 and EHEC1, and was able to differentiate EHEC1 from EHEC2 clusters. The assay detection threshold was 2 × 104 CFU per PCR reaction for EHEC and EPEC. mPCR was also used to screen Argentinean clinical samples from hemolytic uremic syndrome and diarrheal patients, resulting in 91% sensitivity and 84% specificity when compared to established molecular diagnostic procedures. In conclusion, our mPCR methodology permitted differentiation of EPEC, STEC and EHEC strains from other pathogenic E. coli; therefore, the assay becomes an additional tool for rapid diagnosis of these organisms. PMID:22919600

  1. Molecular Characterization and Epidemiologic Study of NDM-1-Producing Extensively Drug-Resistant Escherichia coli.

    PubMed

    Du, Jia; Li, Bin; Cao, Jianming; Wu, Qing; Chen, Huale; Hou, Yuanbo; Zhang, En; Zhou, Tieli

    2016-07-06

    The emergence and dissemination of NDM-1 (New Delhi metallo-β-lactamase-1)-producing Enterobacteriaceae have resulted in a worldwide public health risk. This study described a high incidence and endemic spread of NDM-1-producing extensively drug-resistant Escherichia coli in a teaching hospital in Zhejiang province, China. We recovered six nonduplicated NDM-1-producing E. coli isolates from May 2014 to August 2014 with positive modified Hodge test and EDTA synergistic test. These isolates were highly resistant to β-lactam antimicrobials, aminoglycosides, and quinolones. PCR and DNA sequences analysis showed that all isolates carried the blaNDM-1, blaSHV-11, aac(6')-ib-cr, and qnrB. Several isolates also harbored blaCTX-M-1, blaCTX-M-9, rmtB, and qnrA. Southern blot confirmed that blaNDM-1 was located on the same ∼55 kb plasmid and conjugation experiments further proved the contransferable characteristic of blaNDM-1. The ompC sequences showed various mutations, which was related to multidrug resistance in E. coli. Pulsed-field gel electrophoresis identified four of six isolates that belonged to the same genotype. Multilocus sequence typing assigned them to ST2, except one strain that belonged to ST594. Our study demonstrated that the resistance-associated genes and the loss of the outer membrane proteins could account for high resistance of NDM-1-producing E. coli to multiple antimicrobial drugs. Both horizontal transfer of IncN and transmission of ST2 were responsible for the spread of drug resistance. These findings highlighted an urgent need to limit the further dissemination of NDM-1-producing E. coli in this region.

  2. Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var Sotto in Escherichia coli.

    PubMed

    Rady, M H

    1991-01-01

    During sporulation, Bacillus thuringiensis var. Sotto produces a parasporal crystalline protein which is toxic for the silk-worm, Bombyx mori and the cotton leaf worm, Spodoptera littoralis. The gene coding this crystal protein is present in a single plasmid. The plasmid DNA was isolated, purified and physically mapped using restriction endonuclease enzymes (R.E.). The gene coding the delta-endotoxin was inserted into Escherichia coli-Jm103, using cloning vector pUC8. Transformed E. coli cells were found to synthesize the delta-endotoxin as demonstrated by the pathogenicity of the transformed cells against 4th instar larvae of S. littoralis.

  3. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4.

    PubMed

    Rohde, Holger; Qin, Junjie; Cui, Yujun; Li, Dongfang; Loman, Nicholas J; Hentschke, Moritz; Chen, Wentong; Pu, Fei; Peng, Yangqing; Li, Junhua; Xi, Feng; Li, Shenghui; Li, Yin; Zhang, Zhaoxi; Yang, Xianwei; Zhao, Meiru; Wang, Peng; Guan, Yuanlin; Cen, Zhong; Zhao, Xiangna; Christner, Martin; Kobbe, Robin; Loos, Sebastian; Oh, Jun; Yang, Liang; Danchin, Antoine; Gao, George F; Song, Yajun; Li, Yingrui; Yang, Huanming; Wang, Jian; Xu, Jianguo; Pallen, Mark J; Wang, Jun; Aepfelbacher, Martin; Yang, Ruifu

    2011-08-25

    An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.

  4. An outbreak of shiga toxin-producing Escherichia coli infection associated with a school camp.

    PubMed

    McCall, Bradley J; Slinko, Vicki G; Smith, Helen V; Heel, Karen; Culleton, Terry H; Kelk, Virgil R; Stafford, Russell J

    2010-03-01

    In November 2008, a case of Shiga toxin-producing Escherichia coli (STEC) infection was reported to the Brisbane Southside Public Health Unit. The case had participated in a school camp. Subsequent investigations confirmed 5 other asymptomatic cases among camp attendees or visitors. Examination of the camp water supply identified that most water sources had high levels of E. coli and did not meet the Australian Drinking Water Guidelines with STEC isolated from 2 water sources. This outbreak highlights the emerging issue of asymptomatic carriage of STEC and the importance of thorough maintenance and attention to drinking water supplies in the rural and school camp setting.

  5. The majority of enteroaggregative Escherichia coli strains produce the E. coli common pilus when adhering to cultured epithelial cells.

    PubMed

    Avelino, Fabiola; Saldaña, Zeus; Islam, Sohidul; Monteiro-Neto, Valerio; Dall'Agnol, Monique; Eslava, Carlos A; Girón, Jorge A

    2010-11-01

    Enteroaggregative Escherichia coli (EAEC) have emerged as a significant worldwide cause of chronic diarrhea in the pediatric population and in HIV patients. The vast majority of EAEC strains do not produce the aggregative adherence fimbriae I-III (AAFs) so far reported and thus, what adherence factors are present in these strains remains unknown. Here, we investigated the prevalence of the chromosomal E. coli common pilus (ECP) genes and ECP production amongst 130 EAEC strains of diverse origin as well as the role of ECP in EAEC adherence. Through multiplex PCR analysis we found that 96% of EAEC strains contained the ecpA structural pilin gene whereas only 3.1% and 5.4% were positive for AAF fimbrial genes aggA or aafA, respectively. Among the ecpA(+) strains, 63% produced ECP when adhering to cultured epithelial cells. An ecpA mutant derived from prototypic strain 042 (AAF/II(+)) was not altered in adherence suggesting that the AAF/II, and not ECP, plays a major role in this strain. In contrast, strain 278-1 (AAF(-)) deleted of the ecpA gene was significantly reduced in adherence to cultured epithelial cells. In all, these data indicate a potential role of ECP in adherence for EAEC strains lacking the known AAFs and that in association with other adhesive determinants, ECP may contribute to their survival and persistence within the host and in the environment.

  6. Developing and optimizing bacteriophage treatment to control enterohemorrhagic Escherichia coli on fresh produce.

    PubMed

    Snyder, Abigail B; Perry, Jennifer J; Yousef, Ahmed E

    2016-11-07

    Bacteriophages are potentially useful in controlling foodborne pathogens on minimally processed products since phage application is a non-destructive treatment. The purpose of this study was to evaluate the efficacy of a newly isolated environmental bacteriophage against enterohemorrhagic Escherichia coli on fresh produce, and optimize the treatment with consideration for potential application. Seven anti E. coli O157:H7 EDL933 bacteriophages were isolated from various sources; the most promising was isolated from municipal wastewater. This isolate (designated as E. coli phage OSY-SP) was propagated with the host, in a growth medium, to a titer of 10(8) PFU/ml. Before inoculation into fresh produce, E. coli phage OSY-SP was incubated with the host bacterium, spent medium was filter-sterilized, and the resulting crude lysate was used as a source of phage inocula for preliminary experiments. For optimized testing, phage in the crude lysate was purified by ultra-centrifugation and resuspension in phosphate-buffered saline. Efficacy of phage treatments was determined as a function of fresh produce type (cut green pepper or spinach leaves), treatment time (2 or 5min rinsing), and temperature of holding treated produce (4°C, 25°, or a combination of both temperatures). Cut green pepper was treated with UV light, to eliminate background microbiota, then spot-inoculated with E. coli O157:H7 EDL933 on cut edges, and the inoculum was allowed to dry. Because of its susceptibility to damage, baby spinach leaves were not subjected to a decontamination treatment. These leaves were inoculated with the green fluorescent protein-labeled E. coli O157:H7 B6-914 to facilitate inoculum enumeration in the presence of background microbiota. Phage suspension was applied to the inoculated fresh produce that was subsequently held for three days under variable storage conditions. The optimized phage treatment decreased the populations of pathogenic E. coli by 2.4-3.0logCFU/g on cut green

  7. Multicentre investigation of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in German hospitals.

    PubMed

    Kaase, Martin; Schimanski, Sven; Schiller, Reinhold; Beyreiß, Bettina; Thürmer, Alexander; Steinmann, Jörg; Kempf, Volkhard A; Hess, Christina; Sobottka, Ingo; Fenner, Ines; Ziesing, Stefan; Burckhardt, Irene; von Müller, Lutz; Hamprecht, Axel; Tammer, Ina; Wantia, Nina; Becker, Karsten; Holzmann, Thomas; Furitsch, Martina; Volmer, Gabriele; Gatermann, Sören G

    2016-09-01

    Aim of this study was to determine the incidence and molecular epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Germany. E. coli and K. pneumoniae isolates from clinical samples which were non-susceptible to carbapenems were collected in laboratories serving 20 hospitals throughout Germany from November 2013 to April 2014. The isolates were tested for the presence of carbapenemases by PCR and phenotypic methods and typed by multilocus sequence typing. Risk factors including a previous hospitalization abroad were analysed. Carbapenemases were detected in 24 isolates from 22 patients out of 464,514 admissions. Carbapenemases included OXA-48 (n=14), KPC-2 (n=8) and NDM-1 (n=2). Except for two K. pneumoniae isolates with ST101, all OXA-48 producing strains belonged to different clones. In contrast, half of KPC-2 producing K. pneumoniae were of ST258 and both NDM-1 producing strains were of ST11. Compared to carbapenem-susceptible controls, patients with carbapenemase-producing strains differed by a significantly higher proportion of males, a higher proportion of isolates from wound samples and a more frequent previous stay abroad in univariate analysis. This multicentre study demonstrated an incidence of carbapenemase-producing E. coli and K. pneumoniae from clinical samples in Germany of 0.047 cases per 1000 admissions. OXA-48 was more frequent than KPC-2 and NDM-1 and showed a multiclonal background.

  8. Emission of ESBL/AmpC-producing Escherichia coli from pig fattening farms to surrounding areas.

    PubMed

    von Salviati, Christina; Laube, Henriette; Guerra, Beatriz; Roesler, Uwe; Friese, Anika

    2015-01-30

    The presence of ESBL/AmpC-producing Escherichia coli in livestock such as pigs has been known for some time. However, to date there is little information about the transmission of these resistant bacteria between pig farms and their surroundings. Thus, the aim of this study was to explore this topic by investigating seven German pig fattening farms. Samples from outside (including ground surfaces, ambient air, slurry and digestate from biogas plants) and, in parallel, from inside the pig barns (including pig feces, dust, barn air, flies and mice feces) were examined for ESBL/AmpC-producing E. coli and selected isolates were compared by pulsed-field gel electrophoresis (PFGE) analysis. 14/17 (82.4%) slurry samples and three of four samples of digestate from biogas plants tested positive for ESBL/AmpC-producing E. coli. In the vicinity of the pig barns these resistant bacteria were detected in 14/87 (16.1%) boot swabs taken from various ground surfaces and in 2/36 (6%) ambient air samples. Inside the pig barns, 6/63 (9.5%) barn air samples and a small proportion of flies and mice feces samples were ESBL/AmpC-positive. PFGE analysis proved fecal emission as well as a possible spread via flies, as identical ESBL-E. coli isolates were detected in slurry and on fertilized fields, as well as in flies and pooled feces from inside the barn and slurry. Contaminated slurry presented the major emission source for ESBL/AmpC-producing E. coli in the pig fattening farms, but a spread via the airborne route or via different vectors also seems possible.

  9. Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction.

    PubMed

    Iversen, Hildegunn; L' Abée-Lund, Trine M; Aspholm, Marina; Arnesen, Lotte P S; Lindbäck, Toril

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS) and nervous system complications. Shiga toxin 2 (Stx2) is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424). Among 38 commensal E. coli strains from healthy children below 5 years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced) produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60%) was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.

  10. Persistence of Escherichia coli O157:H7 in major leafy green producing soils.

    PubMed

    Ma, Jincai; Ibekwe, A Mark; Crowley, David E; Yang, Ching-Hong

    2012-11-06

    Persistence of Escherichia coli O157:H7 in 32 (16 organically managed and 16 conventionally managed) soils from California (CA) and Arizona (AZ) was investigated. Results showed that the longest survival (ttd, time needed to reach detection limit, 100 CFU g(-1) dry soil) of E. coli O157:H7 was observed in the soils from Salinas Valley, CA and in organically managed soils from AZ. Detrended correspondence analysis revealed that the survival profiles in organically managed soils in Yuma, AZ were different from the ones in conventionally managed soils from the same site. Principal component analysis and stepwise regression analysis showed that E. coli O157:H7 survival in soils was negatively correlated with salinity (EC) (P < 0.001), while positively correlated with assimilable organic carbon (AOC) and total nitrogen (TN) (P < 0.01). Pearson correlation analysis revealed that a greater ttd was associated with a larger δ (time needed for first decimal reduction in E. coli population). EC was negatively correlated and TN was positively correlated (P < 0.05) with δ, suggesting that EC and TN likely have a direct impact on ttd. On the other hand, AOC showed a close correlation with p (the shape parameter) that was not directly related to ttd, indicating that AOC might have an indirect effect in the overall survival of E. coli O157:H7 in soils. Our data showed that AOC and EC significantly affected the survival of E. coli O157:H7 in leafy green producing soils and the development of good agricultural practices (manure/composting/irrigation water source management) in the preharvest environment must be followed to minimize foodborne bacterial contamination on fresh produce.

  11. Immune response in diarrheal patients and asymptomatic carrier with CS6-producing enterotoxigenic Escherichia coli infection.

    PubMed

    Puiprom, Orapim; Chantaroj, Siriporn; Matsuda, Shigeaki; Sawanpanyalert, Pathom; Honda, Takeshi; Iida, Tetsuya; Taniguchi, Tooru

    2012-11-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the major causes of diarrhea in children and travelers in developing countries. ETEC colonization factors (CFs) are virulence determinants considered as protective antigens and major targets for vaccine development against ETEC infections. One of the most prevalent CFs, coli surface antigen 6 (CS6), a non-fimbrial polymeric protein consisting of two major subunits, CssA and CssB, is produced by approximately 25-35% of ETEC worldwide. We could isolate only CS6-producing ETEC strains from two diarrheal patients and one asymptomatic carrier, but we could not detect CssA- or CssB-specific antibodies in the feces and blood of two patients convalescing from natural ETEC infection and of an asymptomatic carrier using western blotting. Therefore, in order to protect against infection with CS6-producing ETEC, protective levels of CS6 immunity should be incorporated in any future vaccines against ETEC.

  12. Molecular characterization of multiresistant Escherichia coli producing or not extended-spectrum β-lactamases

    PubMed Central

    2013-01-01

    Background The prevalence and type of plasmids, resistance genes and integrons carried by two collections of multiresistant E. coli producing or not extended-spectrum β-lactamases have been compared. Rep-PCR was used to determine the clonal relationship of the organisms. Plasmids were classified according to their incompatibility. Class 1 and Class 2 integrons and antibiotic resistance genes were analysed by PCR and sequencing. Results Both collections of organisms contained a large diversity of unrelated strains with some clones distributed in both groups of isolates. Large plasmids were identified in the two groups of organisms. Plasmids with replicons repK and repColE were more frequent among ESBL-producing isolates, while repFIA, repFII and repA/C replicons were more frequent in isolates lacking ESBL. Conjugative plasmids with repK and repA/C replicons coded for CTX-M-14 and CMY-2 β-lactamases, respectively. No significant differences were observed in the distribution of class 1 and class 2 integrons among multiresistant E. coli producing or not ESBL, and dfrA17-ant(3″)-Ie was the cassette arrangement most commonly found. Conclusions In the concrete temporal and geographical context of this study, multiresistant E. coli producing ESBL or other mechanisms of resistance were largely clonally diverse and present some differences in the types of harboured plasmids. Still, some clones were found in both ESBL-producing and –lacking isolates. PMID:23586437

  13. Isolation of Shiga Toxin-Producing Escherichia coli from a South American Camelid (Lama guanicoe) with Diarrhea

    PubMed Central

    Mercado, E. C.; Rodríguez, S. M.; Elizondo, A. M.; Marcoppido, G.; Parreño, V.

    2004-01-01

    Shiga toxin-producing Escherichia coli belonging to serotype O26:H11 was isolated from a 2-month-old guanaco with severe watery diarrhea. E. coli colonies carried the stx1 and eae genes, showed localized adherence to HEp-2 cells, and produced enterohemolysin. A serological response to lipopolysaccharide O26 was observed at the onset of diarrhea. PMID:15472347

  14. Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

    PubMed Central

    Vogeleer, Philippe; Tremblay, Yannick D. N.; Jubelin, Grégory; Jacques, Mario

    2015-01-01

    Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment. PMID:26712549

  15. Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections.

    PubMed

    Vogeleer, Philippe; Tremblay, Yannick D N; Jubelin, Grégory; Jacques, Mario; Harel, Josée

    2015-12-28

    Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment.

  16. Escherichia coli Probiotic Strain ED1a in Pigs Has a Limited Impact on the Gut Carriage of Extended-Spectrum-β-Lactamase-Producing E. coli.

    PubMed

    Mourand, G; Paboeuf, F; Fleury, M A; Jouy, E; Bougeard, S; Denamur, E; Kempf, I

    2017-01-01

    Four trials were conducted to evaluate the impact of Escherichia coli probiotic strain ED1a administration to pigs on the gut carriage or survival in manure of extended-spectrum-β-lactamase-producing E. coli Groups of pigs were orally inoculated with strain E. coli M63 carrying the blaCTX-M-1 gene (n = 84) or used as a control (n = 26). In the first two trials, 24 of 40 E. coli M63-inoculated pigs were given E. coli ED1a orally for 6 days starting 8 days after oral inoculation. In the third trial, 10 E. coli M63-inoculated pigs were given either E. coli ED1a or probiotic E. coli Nissle 1917 for 5 days. In the fourth trial, E. coli ED1a was given to a sow and its 12 piglets, and these 12 piglets plus 12 piglets that had not received E. coli ED1a were then inoculated with E. coli M63. Fecal shedding of cefotaxime-resistant Enterobacteriaceae (CTX-RE) was studied by culture, and blaCTX-M-1 genes were quantified by PCR. The persistence of CTX-RE in manure samples from inoculated pigs or manure samples inoculated in vitro with E. coli M63 with or without probiotics was studied. The results showed that E. coli M63 and ED1a were good gut colonizers. The reduction in the level of fecal excretion of CTX-RE in E. coli ED1a-treated pigs compared to that in nontreated pigs was usually less than 1 log10 CFU and was mainly observed during the probiotic administration period. The results obtained with E. coli Nissle 1917 did not differ significantly from those obtained with E. coli ED1a. CTX-RE survival did not differ significantly in manure samples with or without probiotic treatment. In conclusion, under our experimental conditions, E. coli ED1a and E. coli Nissle 1917 could not durably prevent CTX-RE colonization of the pig gut.

  17. Escherichia coli O104:H4 Pathogenesis: an Enteroaggregative E. coli/Shiga Toxin-Producing E. coli Explosive Cocktail of High Virulence.

    PubMed

    Navarro-Garcia, Fernando

    2014-12-01

    A major outbreak caused by Escherichia coli of serotype O104:H4 spread throughout Europe in 2011. This large outbreak was caused by an unusual strain that is most similar to enteroaggregative E. coli (EAEC) of serotype O104:H4. A significant difference, however, is the presence of a prophage encoding the Shiga toxin, which is characteristic of enterohemorrhagic E. coli (EHEC) strains. This combination of genomic features, associating characteristics from both EAEC and EHEC, represents a new pathotype. The 2011 E. coli O104:H4 outbreak of hemorrhagic diarrhea in Germany is an example of the explosive cocktail of high virulence and resistance that can emerge in this species. A total of 46 deaths, 782 cases of hemolytic-uremic syndrome, and 3,128 cases of acute gastroenteritis were attributed to this new clone of EAEC/EHEC. In addition, recent identification in France of similar O104:H4 clones exhibiting the same virulence factors suggests that the EHEC O104:H4 pathogen has become endemically established in Europe after the end of the outbreak. EAEC strains of serotype O104:H4 contain a large set of virulence-associated genes regulated by the AggR transcription factor. They include, among other factors, the pAA plasmid genes encoding the aggregative adherence fimbriae, which anchor the bacterium to the intestinal mucosa (stacked-brick adherence pattern on epithelial cells). Furthermore, sequencing studies showed that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga toxin-producing EAEC O104:H4 strain that caused the German outbreak. This article discusses the role these virulence factors could have in EAEC/EHEC O104:H4 pathogenesis.

  18. Detection of Shiga toxin-producing Escherichia coli in meat marketed in Casablanca (Morocco).

    PubMed

    Badri, S; Fassouane, A; Filliol, I; Hassar, M; Cohen, N

    2011-03-01

    The contamination of meat and meat products with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC), obtained from markets in Casablanca, Morocco, was investigated. A total of 460 meat and meat products were sampled between March 2004 and July 2006 analysed and 176 strains of E. coli were isolated from these samples. The presence of the stx1, stx2, eae and ehxA genes, recognized as major virulence factors of STEC, was tested in E. coli isolates by polymerase chain reaction (PCR). STEC was detected in 4 (0.9%) samples. The result of serotyping by molecular method showed that two of these STEC isolates corresponded to the serotype O157:H7. The others Shiga toxin-producing E. coli non-O157 corresponded to O6:H21 and O76:H19. The presence of O157:H7 and non-O157 STEC in meat and meat products marketed in Casablanca, Morocco, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.

  19. Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli.

    PubMed

    Park, Si Jae; Kim, Eun Young; Noh, Won; Oh, Young Hoon; Kim, Hye Young; Song, Bong Keun; Cho, Kwang Myung; Hong, Soon Ho; Lee, Seung Hwan; Jegal, Jonggeon

    2013-07-01

    In this study, we developed recombinant Escherichia coli strains expressing Lactococcus lactis subsp. lactis Il1403 glutamate decarboxylase (GadB) for the production of GABA from glutamate monosodium salt (MSG). Syntheses of GABA from MSG were examined by employing recombinant E. coli XL1-Blue as a whole cell biocatalyst in buffer solution. By increasing the concentration of E. coli XL1-Blue expressing GadB from the OD₆₀₀ of 2-10, the concentration and conversion yield of GABA produced from 10 g/L of MSG could be increased from 4.3 to 4.8 g/L and from 70 to 78 %, respectively. Furthermore, E. coli XL1-Blue expressing GadB highly concentrated to the OD₆₀₀ of 100 produced 76.2 g/L of GABA from 200 g/L of MSG with 62.4 % of GABA yield. Finally, nylon 4 could be synthesized by the bulk polymerization using 2-pyrrolidone that was prepared from microbially synthesized GABA by the reaction with Al₂O₃ as catalyst in toluene with the yield of 96 %.

  20. Cytolethal distending toxin-producing Escherichia coli strains causing severe diarrhoea in young Mexican children

    PubMed Central

    Maldonado-Puga, Samantha; Huerta-Cantillo, Jazmin; Chavez-Dueñas, Lucia; Navarro-Garcia, Fernando

    2017-01-01

    Introduction. Cytolethal distending toxins (CDTs), encoded by cdt genes, have DNase activity leading to cellular and nuclear distension, resulting in irreversible cell cycle arrest and apoptosis of target cells. cdt-positive Escherichia coli strains have been isolated from children with diarrhoea. There is, however, scant information on the prevalence and clinical presentation of diarrhoeal disease caused by these strains. Furthermore, toxin production of cdt-positive strains is rarely confirmed. We report five young children with diarrhoea caused by CDT-producing E. coli in whom stools were negative for other bacterial or enteric pathogens. Case presentation. On admission to hospital, all children presented watery diarrhoea with high stool output (range 7–20 stools/24 h); five had fever of 38 °C or more and four presented vomiting. Dehydration was present in four patients, one of whom had hypovolaemic shock; one child also presented hyponatraemia and hypokalaemia. In two children, cdt-positive strains were classified as typical and atypical enteropathogenic E. coli, and the remaining three harboured cdt-positive strains that did not belong to any diarrhoeagenic pathogroup. One cdt-positive strain from each case was characterized by a CDT cytotoxic assay and a cdt type-specific PCR. All strains produced the characteristic cellular intoxication due to CDT. Two strains carried the cdt-I, one cdt-III, one cdt-IV, and one concurrently had cdt-I, cdt-II and cdt-III genes. Conclusion. Our results suggest that CDT-producing E. coli strains are an infrequent, albeit significant, cause of severe diarrhoeal illness in children. Future research should measure the true burden of cdt-positive E. coli diarrhoea among children. PMID:28348804

  1. Absence of direct association between coliforms and Escherichia coli in irrigation water and on produce.

    PubMed

    Won, Gayeon; Schlegel, Pamela J; Schrock, Jennifer M; LeJeune, Jeffrey T

    2013-06-01

    Irrigation water is considered a potential source of preharvest pathogen contamination of vegetables. Hence, several organizations have recommended microbiological standards for water used to irrigate edible plants. The purpose of this study was to determine the strength of association between microbial quality indicators (coliforms and Escherichia coli) in irrigation water and on irrigated vegetables. Data analyzed included original results from a cross-sectional study conducted in the Midwestern United States during summer 2009 and information presented in two previously published studies performed in France and Portugal to investigate microbial quality of irrigation water and watered produce. In the cross-sectional study, repetitive PCR (rep-PCR) was used to characterize genetic relatedness of E. coli isolates from water and vegetables. No significant correlations were found between fecal indicators on leafy greens (lettuce and parsley, n = 91) or fruit (tomatoes and green peppers, n = 22) and those found in irrigation water used in the cross-sectional study (P > 0.40) or in the previously published data sets (data set 1: lettuce and waste irrigation water, n = 15, P > 0.40; data set 2: lettuce and irrigation water, n = 32, P = 0.06). Rep-PCR banding patterns of E. coli strains were all distinguishable among the pairs of E. coli isolates recovered from produce and irrigation water on the same farm. From the available data, the concentration of indicator organisms based on a single measure of irrigation water quality was not associated with the presence of these indicators on produce. In the absence of additional information, the use of a single microbial water quality parameter as an indicator of produce safety is of limited value for predicting the safety of the produce.

  2. The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans

    PubMed Central

    Sack, R. Bradley

    2011-01-01

    Non-vibrio cholera has been recognized as a clinical entity for as long as cholera was known to be caused by Vibrio cholerae. Until 1968, the aetiologic agent of this syndrome was not known. Following a series of studies in patients with non-vibrio cholera it was found that these patients had large concentrations of Escherichia coli in the small bowel and stools which produced cholera toxin-like enterotoxins, and had fluid and electrolyte transport abnormalities in the small bowel similar to patients with documented cholera. Furthermore, these patients developed antibodies to the cholera-like enterotoxin. Later studies showed that these strains, when fed to volunteers produced a cholera-like disease and that two enterotoxins were found to be produced by these organisms: a heat-labile enterotoxin (LT) which is nearly identical to cholera toxin, and a heat-stable enterotoxin (ST), a small molecular weight polypeptide. E. coli that produced one or both of these enterotoxins were designated enterotoxigenic E. coli (ETEC). ETEC are now known not only to cause a severe cholera-like illness, but to be the most common bacterial cause of acute diarrhoea in children in the developing world, and to be the most common cause of travellers’ diarrhoea in persons who visit the developing world. PMID:21415491

  3. A simple and low-cost platform technology for producing pexiganan antimicrobial peptide in E. coli.

    PubMed

    Zhao, Chun-Xia; Dwyer, Mirjana Dimitrijev; Yu, Alice Lei; Wu, Yang; Fang, Sheng; Middelberg, Anton P J

    2015-05-01

    Antimicrobial peptides, as a new class of antibiotics, have generated tremendous interest as potential alternatives to classical antibiotics. However, the large-scale production of antimicrobial peptides remains a significant challenge. This paper reports a simple and low-cost chromatography-free platform technology for producing antimicrobial peptides in Escherichia coli (E. coli). A fusion protein comprising a variant of the helical biosurfactant protein DAMP4 and the known antimicrobial peptide pexiganan is designed by joining the two polypeptides, at the DNA level, via an acid-sensitive cleavage site. The resulting DAMP4(var)-pexiganan fusion protein expresses at high level and solubility in recombinant E. coli, and a simple heat-purification method was applied to disrupt cells and deliver high-purity DAMP4(var)-pexiganan protein. Simple acid cleavage successfully separated the DAMP4 variant protein and the antimicrobial peptide. Antimicrobial activity tests confirmed that the bio-produced antimicrobial peptide has the same antimicrobial activity as the equivalent product made by conventional chemical peptide synthesis. This simple and low-cost platform technology can be easily adapted to produce other valuable peptide products, and opens a new manufacturing approach for producing antimicrobial peptides at large scale using the tools and approaches of biochemical engineering.

  4. Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

    PubMed Central

    Kehl, K S; Havens, P; Behnke, C E; Acheson, D W

    1997-01-01

    An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children. PMID:9230380

  5. Shiga Toxin-Producing Escherichia coli O157, England and Wales, 1983-2012.

    PubMed

    Adams, Natalie L; Byrne, Lisa; Smith, Geraldine A; Elson, Richard; Harris, John P; Salmon, Roland; Smith, Robert; O'Brien, Sarah J; Adak, Goutam K; Jenkins, Claire

    2016-04-01

    We evaluated clinical Shiga toxin-producing Escherichia coli O157 infections in England and Wales during 1983-2012 to describe changes in microbiological and surveillance methods. A strain replacement event was captured; phage type (PT) 2 decreased to account for just 3% of cases by 2012, whereas PT8 and PT21/28 strains concurrently emerged, constituting almost two thirds of cases by 2012. Despite interventions to control and reduce transmission, incidence remained constant. However, sources of infection changed over time; outbreaks caused by contaminated meat and milk declined, suggesting that interventions aimed at reducing meat cross-contamination were effective. Petting farm and school and nursery outbreaks increased, suggesting the emergence of other modes of transmission and potentially contributing to the sustained incidence over time. Studies assessing interventions and consideration of policies and guidance should be undertaken to reduce Shiga toxin-producing E. coli O157 infections in England and Wales in line with the latest epidemiologic findings.

  6. Virulence characterization of Shiga-toxigenic Escherichia coli isolates from wholesale produce.

    PubMed

    Feng, Peter C H; Councell, Terry; Keys, Christine; Monday, Steven R

    2011-01-01

    The 13 Shiga-toxigenic Escherichia coli (STEC) strains isolated from wholesale spinach and lettuce consisted mostly of serotypes that have not been implicated in illness. Among these strains, however, were two O113:H21 that carried virulence genes common to this pathogenic serotype (stx(2), ehxA, saa, and subAB), suggesting that their presence in ready-to-eat produce may be of health concern.

  7. Multidrug Resistant CTX-M-Producing Escherichia coli: A Growing Threat among HIV Patients in India

    PubMed Central

    Padmavathy, Kesavaram; Padma, Krishnan; Rajasekaran, Sikhamani

    2016-01-01

    Extended Spectrum β-Lactamases (ESBLs) confer resistance to third-generation cephalosporins and CTX-M types have emerged as the most prominent ESBLs worldwide. This study was designed to determine the prevalence of CTX-M positive ESBL-producing urinary E. coli isolates from HIV patients and to establish the association of multidrug resistance, phylogeny, and virulence profile with CTX-M production. A total of 57 ESBL producers identified among 76 E. coli strains isolated from HIV patients from South India were screened for blaCTX-M, AmpC production, multidrug resistance, and nine virulence associated genes (VAGs), fimH, pap, afa/dra, sfa/foc, iutA, fyuA, iroN, usp, and kpsMII. The majority (70.2%) of the ESBL producers harbored blaCTX-M and were AmpC coproducers. Among the CTX-M producers, 47.5% were found to be UPEC, 10% harbored as many as 7 VAGs, and 45% possessed kpsMII. Multidrug resistance (CIPRSXTRGENR) was significantly more common among the CTX-M producers compared to the nonproducers (70% versus 41.2%). However, 71.4% of the multidrug resistant CTX-M producers exhibited susceptibility to nitrofurantoin thereby making it an effective alternative to cephalosporins/fluoroquinolones. The emergence of CTX-M-producing highly virulent, multidrug resistant uropathogenic E. coli is of significant public health concern in countries like India with a high burden of HIV/AIDS. PMID:27123344

  8. Sorbitol non-fermenting shiga toxin-producing Escherichia coli in cattle on smallholdings.

    PubMed

    Islam, M Z; Christensen, J P; Biswas, P K

    2015-01-01

    We investigated faecal samples collected from the rectum of 518 cattle on 371 randomly selected smallholdings in Bangladesh for the presence of sorbitol non-fermenting (SN-F) shiga toxin-producing Escherichia coli (STEC). The SN-F isolates were tested for the presence of rfb O157, stx1, stx2, eae and hlyA genes by polymerase chain reaction (PCR). Seven SN-F isolates lacking these genes were profiled by pulsed-field gel electrophoresis (PFGE) to verify their clonality. SN-F E. coli was identified in 44 [8·5%, 95% confidence interval (CI) 6·4-11·2] samples; of these, 28 (5·4%, 95% CI 3·8-7·7) had shiga toxin-producing strains, although only two carried the rfb O157 gene. Thirteen isolates carried the hlyA gene while 18 harboured the eae gene. Based on PFGE, six pulsotypes were observed among the seven isolates that had no virulence genes. To the best of our knowledge this is the first report on shiga toxin-producing E. coli from direct rectal faecal samples of cattle on smallholdings.

  9. (13)C-metabolic flux analysis for mevalonate-producing strain of Escherichia coli.

    PubMed

    Wada, Keisuke; Toya, Yoshihiro; Banno, Satomi; Yoshikawa, Katsunori; Matsuda, Fumio; Shimizu, Hiroshi

    2017-02-01

    Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives. An MVA-producing strain of Escherichia coli (engineered) was constructed by introducing mvaES genes from Enterococcus faecalis. The engineered strain produced 1.84 mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. The mass balance analysis revealed that the MVA yield of the engineered strain was close to the upper limit at the biomass yield. Since MVA is synthesized from acetyl-CoA using NADPH as a cofactor, the production of MVA affects central metabolism in terms of carbon utilization and NADPH requirements. The reason for this highly efficient MVA production was investigated based on (13)C-metabolic flux analysis. The estimated flux distributions revealed that the fluxes of acetate formation and the TCA cycle in the engineered strain were lower than those in the control strain. Although the oxidative pentose phosphate pathway is considered as the NADPH generating pathway in E. coli, no difference of the flux was observed between the control and engineered strains. The production/consumption balance of NADPH suggested that additional requirement of NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. Comparison between the measured flux distribution and the ideal values for MVA production proposes a strategy for further engineering to improve the MVA production in E. coli.

  10. [Morphologic changes in the intestine of swine after infection with verotoxin-producing Escherichia coli].

    PubMed

    Appel, G; Ewald, C; Heer, A; von Mickwitz, G; Aleksić, S; Rüssmann, H; Meyer, T; Karch, H

    1990-09-01

    Spontaneous morphologic lesions are described in 12 of 66 pigs submitted for necropsy. All 12 pigs were culture positive for Verotoxin-producing E. coli (VTEC). 10 of them were weaned pigs, one a suckling piglet and one a fattening hog. In 6 cases E. coli serovar 0139:H1 and in one case each the serovars 0139:H40; 0138:H-; 0125ac:H27 and 0154:H- were isolated. From the fecal samples of 2 animals E. coli ONT (O-group non typable):H- were cultured. Macroscopically there were cyanosis, edema of the eye lids, catarrhal enteritis and/or colitis as well as edema of the mesentery, swelling of the mesenteric lymph nodes and congestion of the lung to varying degrees. Histopathologic examination of 5 animals was carried out. In 3 animals atrophy and edema of the villi in the jejunum and ileum were discovered. In one animal an additional infection with corona virus was confirmed electron microscopically. Furthermore there was disseminated necrosis of lymphocytes in Peyer's patches of the small intestine and in secondary follicles of the mesenteric lymph nodes. In one of the animals a hemorrhagic-necrotising ileitis, occurred characterized by necrosis of villi and thrombosis of blood vessels in the mucosa. The highest number of VTEC with seven out of twelve animals was found in weaned pigs in association with the E. coli serovar 0139.

  11. Occurrence of generic Escherichia coli, E. coli O157 and Salmonella spp. in water and sediment from leafy green produce farms and streams on the Central California coast.

    PubMed

    Benjamin, Lisa; Atwill, Edward R; Jay-Russell, Michele; Cooley, Michael; Carychao, Diana; Gorski, Lisa; Mandrell, Robert E

    2013-07-01

    Irrigation with water of poor microbiological quality can elevate levels of bacteria on produce. This study aimed to identify climate and management variables associated with generic Escherichia coli in irrigation water on leafy green produce farms and to measure the prevalence of E. coli O157 and Salmonella spp. in irrigation and non-irrigation water sources on these farms. Water and sediment samples collected from various points along irrigation systems, as well as from streams and ponds on farms on the Central California coast between May 27th, 2008 and October 26th, 2010 were cultured for generic E. coli (MPN/100 mL or cfu 100 g) (n=436), E. coli O157 (n=437), and (n=163) Salmonella. Variables were based on grower's management practices, landscape features in proximity to samples (e.g., distance to roads and ranches/livestock), and climate data accessed from an online database. Negative binomial regression models were constructed to test associations between generic E. coli (MPN/100 mL) in water from farms and variables. Arithmetic mean concentration of E. coli for water, not including those from Moore swabs, and sediment samples, was 7.1×10(2) MPN/100 mL and 1.0×10(4) cfu/100 g, respectively. Matched by collection day, E. coli concentration in sediment (cfu/100 g) was typically 10- to 1000-fold higher than the overlying water (MPN/100 mL) for these irrigation systems. Generic E. coli concentration (MPN/100 mL) increased by 60.1% for each 1m/s increase in wind speed and decreased by 3% for each 10 m increase in the distance between the sample location and rangeland. Moore swabs detected a higher proportion of E. coli O157 (13.8%) positive water samples compared to grab samples (1.8%); 1.7% of sediment samples had detectable levels of this pathogen. Interestingly, season was not significantly associated with E. coli O157 presence in water or sediments from produce farms or water sources with public access. Salmonella was detected in 6% (6/96) water and 4.3% (3

  12. PL3 Amidase, a Tailor-made Lysin Constructed by Domain Shuffling with Potent Killing Activity against Pneumococci and Related Species

    PubMed Central

    Blázquez, Blas; Fresco-Taboada, Alba; Iglesias-Bexiga, Manuel; Menéndez, Margarita; García, Pedro

    2016-01-01

    that the structure/function-based domain shuffling approach is a successful method to construct tailor-made endolysins with higher bactericidal activities than their parental enzymes. PMID:27516758

  13. Diminazene aceturate: an antibacterial agent for Shiga-toxin-producing Escherichia coli O157:H7

    PubMed Central

    Wu, Si-Ying; Park, Gil-Yong; Kim, So-Hee; Hulme, John; An, Seong Soo A

    2016-01-01

    The aim of this study was to investigate the bacteriostatic and bactericidal effects of diminazene aceturate (DA) against five strains of pathogenic bacteria and two strains of nonpathogenic bacteria. The results showed that 5 μg/mL of DA suppressed the growth of pathogenic Escherichia coli by as much as 77% compared with the controls. Enterohemorrhagic E. coli EDL933 (an E. coli O157:H7 strain) was the most sensitive to DA with a minimum inhibitory concentration of 20 μg/mL. Additional investigations showed that DA induced the highest level of intracellular reactive oxygen species in EDL933. A positive correlation between the reactive oxygen species levels and DA concentration was demonstrated. DA (5 μg/mL) was also a potent uncoupler, inducing a stationary phase collapse (70%–75%) in both strains of E. coli O157:H7. Further investigation showed that the collapse was due to the NaCl:DA ratio in the broth and was potassium ion dependent. A protease screening assay was conducted to elucidate the underlying mechanism. It was found that at neutral pH, the hydrolysis of H-Asp-pNA increased by a factor of 2–3 in the presence of DA, implying that DA causes dysregulation of the proton motive force and a decrease in cellular pH. Finally, a commercial verotoxin test showed that DA did not significantly increase toxin production in EDL933 and was a suitable antibacterial agent for Shiga-toxin-producing E. coli. PMID:27789937

  14. Molecular Diversity and Plasmid Analysis of KPC-Producing Escherichia coli.

    PubMed

    Chavda, Kalyan D; Chen, Liang; Jacobs, Michael R; Bonomo, Robert A; Kreiswirth, Barry N

    2016-07-01

    The emergence and spread of Klebsiella pneumoniae carbapenemase (KPC) among Enterobacteriaceae presents a major public health threat to the world. Although not as common as in K. pneumoniae, KPC is also found in Escherichia coli strains. Here, we genetically characterized 9 carbapenem-resistant E. coli strains isolated from six hospitals in the United States and completely sequenced their blaKPC-harboring plasmids. The nine strains were isolated from different geographical locations and belonged to 8 different E. coli sequence types. Seven blaKPC-harboring plasmids belonged to four different known incompatibility groups (IncN, -FIA, -FIIK2, and -FIIK1) and ranged in size from ∼16 kb to ∼241 kb. In this analysis, we also identified two plasmids that have novel replicons: (i) pBK28610, which is similar to p34978-3 with an insertion of Tn4401b, and (ii) pBK31611, which does not have an apparent homologue in the GenBank database. Moreover, we report the emergence of a pKP048-like plasmid, pBK34397, in E. coli in the United States. Meanwhile, we also found examples of interspecies spread of blaKPC plasmids, as pBK34592 is identical to pBK30683, isolated from K. pneumoniae In addition, we discovered examples of acquisition (pBK32602 acquired an ∼46-kb fragment including a novel replication gene, along with Tn4401b and other resistance genes) and/or loss (pKpQIL-Ec has a 14.5-kb deletion compared to pKpQIL-10 and pBK33689) of DNA, demonstrating the plasticity of these plasmids and their rapid evolution in the clinic. Overall, our study shows that the spread of blaKPC-producing E. coli is largely due to horizontal transfer of blaKPC-harboring plasmids and related mobile elements into diverse genetic backgrounds.

  15. Molecular Diversity and Plasmid Analysis of KPC-Producing Escherichia coli

    PubMed Central

    Chavda, Kalyan D.; Chen, Liang; Jacobs, Michael R.; Bonomo, Robert A.

    2016-01-01

    The emergence and spread of Klebsiella pneumoniae carbapenemase (KPC) among Enterobacteriaceae presents a major public health threat to the world. Although not as common as in K. pneumoniae, KPC is also found in Escherichia coli strains. Here, we genetically characterized 9 carbapenem-resistant E. coli strains isolated from six hospitals in the United States and completely sequenced their blaKPC-harboring plasmids. The nine strains were isolated from different geographical locations and belonged to 8 different E. coli sequence types. Seven blaKPC-harboring plasmids belonged to four different known incompatibility groups (IncN, -FIA, -FIIK2, and -FIIK1) and ranged in size from ∼16 kb to ∼241 kb. In this analysis, we also identified two plasmids that have novel replicons: (i) pBK28610, which is similar to p34978-3 with an insertion of Tn4401b, and (ii) pBK31611, which does not have an apparent homologue in the GenBank database. Moreover, we report the emergence of a pKP048-like plasmid, pBK34397, in E. coli in the United States. Meanwhile, we also found examples of interspecies spread of blaKPC plasmids, as pBK34592 is identical to pBK30683, isolated from K. pneumoniae. In addition, we discovered examples of acquisition (pBK32602 acquired an ∼46-kb fragment including a novel replication gene, along with Tn4401b and other resistance genes) and/or loss (pKpQIL-Ec has a 14.5-kb deletion compared to pKpQIL-10 and pBK33689) of DNA, demonstrating the plasticity of these plasmids and their rapid evolution in the clinic. Overall, our study shows that the spread of blaKPC-producing E. coli is largely due to horizontal transfer of blaKPC-harboring plasmids and related mobile elements into diverse genetic backgrounds. PMID:27114279

  16. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

  17. Increasing incidence of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Belgian hospitals.

    PubMed

    De Laveleye, M; Huang, T D; Bogaerts, P; Berhin, C; Bauraing, C; Sacré, P; Noel, A; Glupczynski, Y

    2017-01-01

    Carbapenemase-producing Enterobacteriaceae are increasingly reported worldwide. The aim of the study was to determine the incidence and molecular epidemiology of carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae (CP-E/K) in Belgium. Eleven hospital-based laboratories collected carbapenem non-susceptible (CNS) isolates of E. coli and K. pneumoniae detected in clinical specimens from January 2013 to December 2014. All CNS strains were tested for carbapenemase production and typed by multilocus sequence typing (MLST) for a 6-month period as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE) structured survey. In addition, an equal number of carbapenem-susceptible isolates collected were preserved as a control group for risk factor analysis. The overall incidence rate of CP-E/K isolates in hospitals increased from 0.124 in 2013 to 0.223 per 1000 admissions in 2014. From November 2013 to April 2014, 30 CP K. pneumoniae [OXA-48 (n = 16), KPC (n = 13), OXA-427 (n = 1)] and five CP E. coli [OXA-48 (n = 3), NDM (n = 1), OXA-427 (n = 1)] isolates were detected in ten hospitals. The 16 OXA-48-producing K. pneumoniae strains were distributed into eight sequence types (STs), while the 13 KPC-producing K. pneumoniae clustered into three STs dominated by ST512 (n = 7) and ST101 (n = 5). Compared to controls, we observed among CP-E/K carriers significantly higher proportion of males, respiratory origins, previous hospitalization, nosocomial setting, and a significantly lower proportion of bloodstream infections. Our study confirms the rapid spread of CP-E/K in Belgian hospitals and the urgent need for a well-structured and coordinated national surveillance plan in order to limit their dissemination.

  18. Media composition and incubation temperature affect Congo red dye affinity of Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Escherichia coli biofilm formation is dependent on curli fimbriae and cellulose, and the expression of both varies among Shiga toxin-producing E. coli (STEC). Curli and cellulose expression are often identified by their affinity for Congo red dye (CR) but media composition and incubation...

  19. High genotypic and phenotypic similarity among Shiga toxin-producing Escherichia coli O111 environmental and outbreak strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E. coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing Escherichia coli (STEC), which are emerging foodborne pathogens that have caused numerous outbreaks and sporadic cases of enteric illness in industrialized countries. We have assembled a collection of en...

  20. Shiga toxin-producing Escherichia coli and rectoanal junction persistence in ruminants: a study of bacterial-epithelial interactions.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O157:H7 (O157) was the first Shiga toxin-producing E. coli serotype to be associated with bloody diarrhea or hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. It has since been implicated in several outbreaks in the U.S. and globally. Non-O157 STEC have not bee...

  1. Molecular insights into the unique phenotypes exhibited by super shed shiga toxin producing Escherichia coli O157

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin producing Escherichia coli (STEC) serovar O157:H7 is a major foodborne pathogen that can cause bloody diarrhea and life threatening hemolytic uremic syndrome in humans. Asymptomatic cattle are colonized with E. coli O157:H7 at the mucosal interface of the recto-anal junction (RAJ). Sup...

  2. The polymorphic aggregative phenotype of Shiga toxin-producing Escherichia coli O111 depends on rpoS and curli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli O111 is an emerging non-O157:H7 Shiga toxin-producing E. coli (STEC). We previously reported that outbreak and environmental, but not sporadic case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and ...

  3. Detection of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Market-Ready Chickens in Zambia

    PubMed Central

    Chishimba, K.; Hang'ombe, B. M.; Muzandu, K.; Mshana, S. E.; Matee, M. I.; Nakajima, C.; Suzuki, Y.

    2016-01-01

    The frequent administering of antibiotics in the treatment of poultry diseases may contribute to emergence of antimicrobial-resistant strains. The objective of this study was to detect the presence of extended-spectrum β-lactamase- (ESBL-) producing Escherichia coli in poultry in Zambia. A total of 384 poultry samples were collected and analyzed for ESBL-producing Escherichia coli. The cultured E. coli isolates were subjected to antimicrobial susceptibility tests and the polymerase chain reaction for detection of blaCTX-M, blaSHV, and blaTEM genes. Overall 20.1%, 77/384, (95% CI; 43.2–65.5%) of total samples analyzed contained ESBL-producing Escherichia coli. The antimicrobial sensitivity test revealed that 85.7% (66/77; CI: 75.7–92) of ESBL-producing E. coli isolates conferred resistance to beta-lactam and other antimicrobial agents. These results indicate that poultry is a potential reservoir for ESBL-producing Escherichia coli. The presence of ESBL-producing Escherichia coli in poultry destined for human consumption requires strengthening of the antibiotic administering policy. This is important as antibiotic administration in food animals is gaining momentum for improved animal productivity in developing countries such as Zambia. PMID:27190518

  4. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol.

    SciTech Connect

    Donnelly, M. I.; Millard, C. S.; Clark, D. P.; Chen, M. J.; Rathke, J. W.; Southern Illinois Univ.

    1998-04-01

    Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E.coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.

  5. AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals.

    PubMed

    Le Bouguénec, C; Bertin, Y

    1999-01-01

    AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenic Escherichia coli strains in domestic animals. F17-related fimbriae are mainly detected on bovine and ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosamine (GlcNAc) receptors present on bovine intestinal cells. Some F17 subtypes also bind to GlcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to the laminin contained in the basement of mammalian membranes. F17 is often associated with other virulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31A or AFA adhesins) on pathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fimbrial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the fact that: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between structural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently, determinants related to the afa gene clusters from human pathogenic E. coli associated with intestinal and extra-intestinal infections were identified in strains isolated from calves and piglets with diarrhoea and septicaemia. Two afa-related gene clusters, designated afa-7 and afa-8, that encode afimbrial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal afa gene clusters were plasmid and chromosome borne and were expressed by strains that produced other virulence factors such as CNF toxins, F17, PAP and CS31A adhesins. A high frequency of afa-8 and a low prevalence of afa-7 among bovine E. coli isolates were suggested by preliminary epidemiological studies. As with the human afa gene clusters, the animal ones encode an adhesive structure composed of two proteins: AfaE which

  6. Preharvest evaluation of coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers.

    PubMed

    Mukherjee, Avik; Speh, Dorinda; Dyck, Elizabeth; Diez-Gonzalez, Francisco

    2004-05-01

    Microbiological analyses of fresh fruits and vegetables produced by organic and conventional farmers in Minnesota were conducted to determine the coliform count and the prevalence of Escherichia coli, Salmonella, and E. coli O157:H7. A total of 476 and 129 produce samples were collected from 32 organic and 8 conventional farms, respectively. The samples included tomatoes, leafy greens, lettuce, green peppers, cabbage, cucumbers, broccoli, strawberries, apples, and seven other types of produce. The numbers of fruits and vegetables was influenced by their availability at participating farms and varied from 11 strawberry samples to 108 tomato samples. Among the organic farms, eight were certified by accredited agencies and the rest reported the use of organic practices. All organic farms used aged or composted animal manure as fertilizer. The average coliform counts in both organic and conventional produce were 2.9 log most probable number per g. The percentages of E. coli-positive samples in conventional and organic produce were 1.6 and 9.7%, respectively. However, the E. coli prevalence in certified organic produce was 4.3%, a level not statistically different from that in conventional samples. Organic lettuce had the largest prevalence of E. coli (22.4%) compared with other produce types. Organic samples from farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials. Serotype O157:H7 was not detected in any produce samples, but Salmonella was isolated from one organic lettuce and one organic green pepper. These results provide the first microbiological assessment of organic fruits and vegetables at the farm level.

  7. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

    PubMed Central

    Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

    2014-01-01

    Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

  8. Transmission of ESBL/AmpC-producing Escherichia coli from broiler chicken farms to surrounding areas.

    PubMed

    Laube, H; Friese, A; von Salviati, C; Guerra, B; Rösler, U

    2014-08-27

    Although previous studies have demonstrated high carriage of ESBL/AmpC-producing Escherichia coli in livestock, especially in broiler chickens, data on emission sources of these bacteria into the environment are still rare. Therefore, this study was designed to systematically investigate the occurrence of ESBL/AmpC-producing E. coli in slurry, air (inside animal houses), ambient air (outside animal houses) and on soil surfaces in the areas surrounding of seven ESBL/AmpC-positive broiler chicken fattening farms, including investigation of the possible spread of these bacteria via the faecal route and/or exhaust air into the environment. Seven German broiler fattening farms were each investigated at three points in time (3-36 h after restocking, 14-18 and 26-35 days after housing) during one fattening period. The occurrence of ESBL/AmpC genes in the investigated samples was confirmed by PCR, detecting blaCTX-M, blaSHV, blaTEM and blaCMY-genes, and, if necessary, by sequencing and/or the disc diffusion method. The results showed a wide spread of ESBL/AmpC-producing E. coli in broiler farms, as well as emissions into the surroundings. 12 out of 14 (86%) slurry samples were positive for ESBL/AmpC-producing E. coli. Additionally, 28.8% (n=23/80) of boot swabs taken from various surfaces in the areas surrounding of the farms as well as 7.5% (n=3/40) of the exhaust air samples turned out to be positive for these microorganisms. Moreover, a small proportion of air samples from inside the barns were ESBL/AmpC-positive. By comparing selected isolates using pulsed field gel electrophoresis, we proved that faecal and airborne transfer of ESBL/AmpC-producing microorganisms from broiler fattening farms to the surrounding areas is possible. Two isolates from farm G2 (slurry and boot swab 50 m downwind), two isolates from farm G3 (slurry and individual animal swab) as well as two isolates from farm G6 (air sample in the barn and air sample 50 m downwind) showed 100% similarity in

  9. Molecular characterization and phylogeny of Shiga toxin-producing E. coli (STEC) from imported beef meat in Malaysia.

    PubMed

    Abuelhassan, Nawal Nouridaim; Mutalib, Sahilah Abdul; Gimba, Fufa Ido; Yusoff, Wan Mohtar

    2016-09-01

    This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak

  10. Whole-Genome Characterization and Strain Comparison of VT2f-Producing Escherichia coli Causing Hemolytic Uremic Syndrome

    PubMed Central

    Michelacci, Valeria; Bondì, Roslen; Gigliucci, Federica; Franz, Eelco; Badouei, Mahdi Askari; Schlager, Sabine; Minelli, Fabio; Tozzoli, Rosangela; Caprioli, Alfredo; Morabito, Stefano

    2016-01-01

    Verotoxigenic Escherichia coli infections in humans cause disease ranging from uncomplicated intestinal illnesses to bloody diarrhea and systemic sequelae, such as hemolytic uremic syndrome (HUS). Previous research indicated that pigeons may be a reservoir for a population of verotoxigenic E. coli producing the VT2f variant. We used whole-genome sequencing to characterize a set of VT2f-producing E. coli strains from human patients with diarrhea or HUS and from healthy pigeons. We describe a phage conveying the vtx2f genes and provide evidence that the strains causing milder diarrheal disease may be transmitted to humans from pigeons. The strains causing HUS could derive from VT2f phage acquisition by E. coli strains with a virulence genes asset resembling that of typical HUS-associated verotoxigenic E. coli. PMID:27584691

  11. An in vitro combined antibiotic/antibody treatment eliminates toxicity from Shiga toxin-producing E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is a difficult endeavor. The utility of antibiotics as an STEC treatment is controversial since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase express...

  12. Individualized Tailor-Made Dietetic Intervention Program at Schools Enhances Eating Behaviors and Dietary Habits in Obese Hispanic Children of Low Socioeconomic Status

    PubMed Central

    Moreno-Sànchez, Diana; Gutierrez, Norma G.; Lamadrid-Zertuche, Ana C.; Hernandez-Torre, Martin M.

    2014-01-01

    Hispanic children and those from low-socioeconomic status are predisposed to unhealthy eating habits and obesity. Aim. to implement an individualized, face-to-face, parent supported, and school-partnership dietetic intervention to promote healthy eating habits and decrease body mass index. Prospective school year dietetic intervention of 101 obese, Hispanic, low-socioeconomic school-age children representative of Monterrey, Mexico, consisted of anthropometrics, dietetic assessment, energy-restriction tailor-made daily menus, and parental education every three weeks. Student's t-test was used for means comparison. A significant decrease was found in body mass index percentile (96.43 ± 3.32 to 93.42 ± 8.12/P = 0.00) and energy intake/day of −755.7 kcal/day (P = 0.00). Among other energy dense foods with significant decline in servings/day and servings/week were processed meats (3.13 ± 1.43 to 2.19 ± 1.04/P = 0.00 and 5.60 ± 1.75 to 4.37 ± 2.10/P = 0.00, resp.), saturated fat (1.47 ± 1.08 to 0.78 ± 0.79/P = 0.00 and 2.19 ± 2.18 to 1.1 ± 1.36/P = 0.00), sweetened beverages (2.79 ± 1.99 to 1.42 ± 1.21 and 6.21 ± 1.72 to 3.89 ± 2.80/P = 0.00), and desserts and refined-grain bakery (1.99 ± 1.54 to 1.32 ± 1.59 and 2.85 ± 2.54 to 1.57 ± 2.20/P = 0.00). There was a significant increase in servings/day and servings/week of water (2.98 ± 2.02 to 4.91 ± 2.37 and 6.62 ± 2.03 to 6.87 ± 0.91/P = 0.00, resp.) and nutrient dense foods such as fruits (1.31 ± 0.89 to 1.66 ± 0.96 and 3.34 ± 2.24 to 4.28 ± 2.43/P = 0.00) and fish and poultry (3.76 ± 2.15 to 4.54 ± 2.25/P = 0.00). This intervention created healthy eating habits and decreased body mass index in a high risk population. Trial registration number: NCT01925976. PMID:24592170

  13. Prevalence and characteristics of shiga toxin-producing Escherichia coli from healthy cattle in Japan.

    PubMed

    Kobayashi, H; Shimada, J; Nakazawa, M; Morozumi, T; Pohjanvirta, T; Pelkonen, S; Yamamoto, K

    2001-01-01

    The prevalence of Shiga toxin-producing Escherichia coli (STEC) in Japan was examined by using stool samples from 87 calves, 88 heifers, and 183 cows on 78 farms. As determined by screening with stx-PCR, the prevalence was 46% in calves, 66% in heifers, and 69% in cows; as determined by nested stx-PCR, the prevalence was 100% in all animal groups. Of the 962 isolates picked by colony stx hybridization, 92 isolates from 54 farms were characterized to determine their O serogroups, virulence factor genes, and antimicrobial resistance. Of these 92 isolates, 74 (80%) could be classified into O serogroups; 50% of these 74 isolates belonged to O serogroups O8, O26, O84, O113, and O116 and 1 isolate belonged to O serogroup O157. Locus of enterocyte effacement genes were detected in 24% of the isolates, and enterohemorrhagic E. coli (EHEC) hlyA genes were detected in 72% of the isolates. Neither the bundle-forming pilus gene nor the enteropathogenic E. coli adherence factor plasmid was found. STEC strains with characteristics typical of isolates from human EHEC infections, which were regarded as potential EHEC strains, were present on 11.5% of the farms.

  14. Inactivation of Escherichia coli ATCC 11775 in fresh produce using atmospheric pressure cold plasma

    NASA Astrophysics Data System (ADS)

    Bermudez-Aguirre, Daniela; Wemlinger, Erik; Barbosa-Canovas, Gustavo; Pedrow, Patrick; Garcia-Perez, Manuel

    2011-10-01

    Food-borne outbreaks are associated with the presence of pathogenic bacteria in food products such as fresh produce. One of the target microorganisms is Escherichia coli which exhibits resistance to being inactivated with conventional disinfection methods for vegetables. Atmospheric pressure cold plasma (APCP) was tested to disinfect three vegetables with challenge surfaces, lettuce, carrots and tomatoes. The produce was inoculated with the bacteria to reach an initial microbial concentration of 107 cfu/g. Vegetables were initially exposed to the APCP discharges from a needle array at 5.7 kV RMS in argon, processing times of 0.5, 3 and 5 min. Initial results indicate that microbial decontamination is effective on the lettuce (1.2 log reduction) when compared with other vegetables. To claim disinfection, a 3 log reduction or more is needed, which makes APCP treatment very promising technology for decontamination of produce. We propose that with method refinements full disinfection can be achieved using APCP.

  15. Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain.

    PubMed

    Huang, Qin; Lin, Yuheng; Yan, Yajun

    2013-12-01

    Caffeic acid is a plant-specific phenylpropanoic acid with multiple health-improving effects reported, and its therapeutic derivatives have also been studied throughout the last decade. To meet its market need and achieve high-level production, microbial production of caffeic acid approaches have been developed in metabolically engineered Escherichia coli. In our previous work, we have established the first artificial pathway that realized de novo production of caffeic acid using E. coli endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HP3H). In this work, we exploited the catalytic potential of 4HPA3H in the whole-cell bioconversion study and produced 3.82 g/L (461.12 mg/L/OD) caffeic acid from p-coumaric acid, a direct precursor. We further engineered a phenylalanine over-producer into a tyrosine over-producer and then introduced the artificial pathway. After adjusting the expression strategy and optimizing the inoculants timing, de novo production of caffeic acid reached 766.68 mg/L. Both results from the direct precursor and simple carbon sources represent the highest titers of caffeic acid from microbial production so far.

  16. Distribution, Numbers, and Diversity of ESBL-Producing E. coli in the Poultry Farm Environment

    PubMed Central

    Blaak, Hetty; van Hoek, Angela H. A. M.; Hamidjaja, Raditijo A.; van der Plaats, Rozemarijn Q. J.; Kerkhof-de Heer, Lianne; de Roda Husman, Ana Maria; Schets, Franciska M.

    2015-01-01

    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 2.4×104 cfu/kg), and surface waters adjacent to broiler farms during, or shortly after, cleaning between production rounds (91% of samples positive, geometric mean concentration 1.9×102 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. PMID

  17. Prevalence of Salmonella enterica and Shiga toxin-producing Escherichia coli in zoo animals from Chile

    PubMed Central

    Marchant, Paulina; Hidalgo-Hermoso, Ezequiel; Espinoza, Karen

    2016-01-01

    Salmonella (S.) enterica and Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens. Here, we report the prevalence of S. enterica and STEC in feces of 316 zoo animals belonging to 61 species from Chile. S. enterica and STEC strains were detected in 7.5% and 4.4% of animals, respectively. All Salmonella isolates corresponded to the serotype Enteritidis. To the best of our knowledge, this is the first report of S. Enteritidis in the culpeo fox (Lycalopex culpaeus), black-capped capuchin (Sapajus apella) and Peruvian pelican (Pelecanus thagus) and the first STEC report in Thomson's gazelle (Eudorcas thomsonii). PMID:27030195

  18. Epidemiological factors associated with ESBL- and non ESBL-producing E. coli causing urinary tract infection in general practice.

    PubMed

    Hertz, Frederik Boëtius; Schønning, Kristian; Rasmussen, Steen Christian; Littauer, Pia; Knudsen, Jenny Dahl; Løbner-Olesen, Anders; Frimodt-Møller, Niels

    2016-01-01

    The purpose of the study was to evaluate how use of antibiotics precedes the presence of ESBL-producing E.coli in general practice. The authors performed a triple-case-control study where three case groups were individually compared to a single control group of uninfected individuals. Urine samples were prospectively collected and retrospective statistical analyses were done. This study included 98 cases with urinary tract infection (UTI) caused by ESBL-producing E. coli, 174 with antibiotic-resistant (non-ESBL) E. coli, 177 with susceptible E. coli and 200 with culture negative urine samples. Case groups had significantly higher use of antibiotics than the control group within 30 days before infection (p < 0.0001). The ESBL group had significantly more hospital admissions than the other case groups (p < 0.05). Hospital admission was an independent risk factor for community onset UTI by ESBL-producing E. coli. Exposure to antibiotics was a risk factor for UTI with E. coli, while prior antibiotic usage was not an indisputable predictor for infection with ESBL-producing E.coli in general practice.

  19. Behavior of shiga toxin-producing Escherichia coli, enteroinvasive E. coli, enteropathogenic E. coli and enterotoxigenic E. coli strains on whole and sliced jalapeño and serrano peppers.

    PubMed

    Gómez-Aldapa, Carlos A; Rangel-Vargas, Esmeralda; Gordillo-Martínez, Alberto J; Castro-Rosas, Javier

    2014-06-01

    The behavior of enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and non-O157 shiga toxin-producing E. coli (non-O157-STEC) on whole and slices of jalapeño and serrano peppers as well as in blended sauce at 25 ± 2 °C and 3 ± 2 °C was investigated. Chili peppers were collected from markets of Pachuca city, Hidalgo, Mexico. On whole serrano and jalapeño stored at 25 ± 2 °C or 3 ± 2 °C, no growth was observed for EPEC, ETEC, EIEC and non-O157-STEC rifampicin resistant strains. After twelve days at 25 ± 2 °C, on serrano peppers all diarrheagenic E. coli pathotypes (DEP) strains had decreased by a total of approximately 3.7 log, whereas on jalapeño peppers the strains had decreased by approximately 2.8 log, and at 3 ± 2 °C they decreased to approximately 2.5 and 2.2 log respectively, on serrano and jalapeño. All E. coli pathotypes grew onto sliced chili peppers and in blended sauce: after 24 h at 25 ± 2 °C, all pathotypes had grown to approximately 3 and 4 log CFU on pepper slices and sauce, respectively. At 3 ± 2 °C the bacterial growth was inhibited.

  20. Characterization of extended-spectrum-beta-lactamase-producing Escherichia coli strains involved in maternal-fetal colonization: prevalence of E. coli ST131.

    PubMed

    Birgy, André; Mariani-Kurkdjian, Patricia; Bidet, Philippe; Doit, Catherine; Genel, Nathalie; Courroux, Céline; Arlet, Guillaume; Bingen, Edouard

    2013-06-01

    Maternal-fetal Escherichia coli infections, such as neonatal bacteremia and meningitis, are important causes of morbidity and mortality. From 2006 to 2010, we studied newborns and their mothers who were colonized with E. coli in a French hospital in order to document (i) the epidemiology and genetic characteristics of extended-spectrum-beta-lactamase (ESBL)-producing E. coli strains, (ii) the prevalence of associated virulence genes, (iii) the prevalence of clone sequence type 131 (ST131), and (iv) the genetic relationship among ESBL-producing strains. Among the 2,755 E. coli cultures recovered from vaginal or neonatal samples, 68 were ESBL producers (2.46%). We found a wide diversity of ESBL genes, with the majority being bla(CTX-M-14), bla(CTX-M-1), and bla(CTX-M-15), distributed among the 4 main phylogenetic groups. Genes encoding virulence factors were found in 90.7% of the isolates, with ≥ 2 virulence genes present in 76% of cases. The prevalence of ST131 among ESBL-producing E. coli isolates was 9.4% (6/64). Five of these 6 ST131 isolates possessed bla(CTX-M-15) enzymes (and also were resistant to quinolones), and one possessed bla(CTX-M-2) enzymes. Two possessed virulence genes, suggesting the presence of pathogenicity island IIJ96 (PAI IIJ96)-like domains. Pulsed-field gel electrophoresis (PFGE) revealed a high level of genomic diversity overall, except for 3 closely related isolates belonging to clonal group ST131. Repetitive PCR showed that the six ST131 isolates were closely related to ST131 control strains (>95% similarity). This study shows a high prevalence of ESBL-producing E. coli strains and clonal group ST131 in the French maternal-fetal population. These results suggest a widespread distribution of ESBL enzymes in the community and highlight the early transmission between mothers and neonates. These findings are worrisome, especially for this particularly vulnerable population.

  1. Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins.

    PubMed

    Desimone, Martín F; De Marzi, Mauricio C; Copello, Guillermo J; Fernández, Marisa M; Malchiodi, Emilio L; Diaz, Luis E

    2005-10-01

    The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.

  2. Selective recovery by different culture methods of Shiga toxin-producing Escherichia coli genotypes from a major produce production region in California(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The higher consumption of fresh fruits and vegetables in the United States has corresponded to an increase in the number of outbreaks. More than 45% of the leafy greens-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) O157:H7 have been linked to produce from California’s central...

  3. A new immunoassay for detecting all subtypes of Shiga toxins produced by Shiga toxin-producing E. coli in ground beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none o...

  4. Investigation of environmental factors on the prevalence of free bacteriophages against Shiga toxin-producing Escherichia coli strains in produce pre-harvest environment in Salinas, California

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Investigation of environmental factors on the prevalence of free bacteriophages against Shiga toxin-producing Escherichia coli strains in produce pre-harvest environment in Salinas, California Yen-Te Liaoa, Irwin Quintelab, Kimberly Nguyena, Alexandra Salvadora, Michael Cooleya, and Vivian C.H. Wu*a...

  5. Tailor-made Au@Ag core-shell nanoparticle 2D arrays on protein-coated graphene oxide with assembly enhanced antibacterial activity

    NASA Astrophysics Data System (ADS)

    Wang, Huiqiao; Liu, Jinbin; Wu, Xuan; Tong, Zhonghua; Deng, Zhaoxiang

    2013-05-01

    Water-dispersible two-dimensional (2D) assemblies of Au@Ag core-shell nanoparticles are obtained through a highly selective electroless silver deposition on pre-assembled gold nanoparticles on bovine serum albumin (BSA)-coated graphene oxide (BSA-GO). While neither BSA-GO nor AuNP-decorated BSA-GO shows any antibacterial ability, the silver-coated GO@Au nanosheets (namely GO@Au@Ag) exhibit an enhanced antibacterial activity against Gram-negative Escherichia coli (E. coli) bacteria, superior to unassembled Au@Ag nanoparticles and even ionic Ag. Such an improvement may be attributed to the increased local concentration of silver nanoparticles around a bacterium and a polyvalent interaction with the bacterial surface. In addition, the colloidal stability of this novel nano-antimicrobial against the formation of random nanoparticle aggregates guarantees a minimized activity loss of the Au@Ag nanoparticles. The antibacterial efficacy of GO@Au@Ag is less sensitive to the existence of Cl-, in comparison with silver ions, providing another advantage for wound dressing applications. Our research unambiguously reveals a strong and very specific interaction between the GO@Au@Ag nanoassembly and E. coli, which could be an important clue toward a rational design, synthesis and assembly of innovative and highly active antibacterial nanomaterials.

  6. An Automated Fluorescent PCR Method for Detection of Shiga Toxin-Producing Escherichia coli in Foods

    PubMed Central

    Chen, Shu; Xu, Renlin; Yee, Arlene; Wu, Kai Yuan; Wang, Chang-Ning; Read, Susan; De Grandis, Stephanie A.

    1998-01-01

    An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx1, stx2, and stxe. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples. PMID:9797267

  7. Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104 from sprouts.

    PubMed

    Baranzoni, Gian Marco; Fratamico, Pina M; Rubio, Fernando; Glaze, Thomas; Bagi, Lori K; Albonetti, Sabrina

    2014-03-03

    Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O104 have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. An outbreak that occurred in Europe in 2011 linked to fenugreek sprouts was caused by E. coli O104:H4 that had characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that encoded for Shiga toxin 2. In this study, methods were developed for detection of this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR assays for enteroaggregative STEC O104:H4 targeted the stx2, aggR, and wzx104 genes, and for STEC O104 targeted the stx1-2, ehxA, and wzx104 genes. After incubating artificially contaminated sprouts at 4 °C for 48 h and overnight enrichment in modified buffered peptone water with pyruvate supplemented with three antibiotics (mBPWp), the pathogens were detected in all samples inoculated at a level of ca. 100CFU/25 g. Several samples inoculated at lower concentrations of ca. 10CFU/25 g were negative by the PCR assays, and this could have been due to cells not surviving or not being able to recover after the stress treatment at 4 °C for 48 h. For isolation of the pathogens, immunomagnetic separation (IMS) using magnetic beads coated with antibodies against O104 were employed, and this was followed by plating the beads onto mRBA and CHROMagar STEC O104 for isolation of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7. Presumptive colonies were confirmed by agglutination using latex particles attached to antibodies against serogroup O104 and by the multiplex PCR assays. The methodologies described in this study for detection of enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104, and they enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well.

  8. [The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants].

    PubMed

    Doroshenko, V G; Lobanov, A O; Fedorina, E A

    2013-01-01

    Strain MG 1655+hisGr hisL'-Delta, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG 1655+, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisGr (E271 K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisGr through the deleting attenuator region (hisL'-Delta). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

  9. Tigecycline treatment of infection caused by KPC-producing Escherichia coli in a pediatric patient

    PubMed Central

    2013-01-01

    Tigecycline shows great antimicrobial activity against both Gram-positive and Gram-negative bacteria, and has been considered to be an appropriate choice in controlling infection caused by multi-drug resistant (MDR) pathogens, such as carbapenemase-producing Enterobacteriaceae (CPE). Although many clinical trials evaluate the efficacy and safety of tigecycline on adults, rare reports recommend tigecycline to treat pediatric patient. In this study, we presented a clinical case with tigecycline as an anti-infectious agent on a 14-year-old child who was suffering from infection of intraperitoneal abscess caused by Klebsiella pneumoniae carbapenemases (KPC)-producing Escherichia coli with extreme drug resistant profile. By accessing the clinical outcome and efficacy of the patient, and the side effects of tigecycline, our research explored the documented experience of tigecycline on controlling infection caused by CPE isolate in children. PMID:23941473

  10. 60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage.

    PubMed

    Yamamoto, Tatsuo; Kojio, Seiichi; Taneike, Ikue; Nakagawa, Saori; Iwakura, Nobuhiro; Wakisaka-Saito, Noriko

    2003-05-16

    Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.

  11. Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhoea and controls in Teresina/PI, Brazil.

    PubMed

    Nunes, Maria do Rosário Conceição Moura; Magalhães, Paula Prazeres; Macêdo, Antônio da Silva; Franco, Roger Teixeira; Penna, Francisco José; Mendes, Edilberto Nogueira

    2012-01-01

    This 3.5-year prospective study was conducted to ascertain the level of attaching and effacing Escherichia coli (AEEC) associated diarrhoea in children from Teresina, a northeastern state of Brazil. Passed faecal specimens from 400 patients (250 with and 150 without diarrhoea) up to 60 months of age attending from 2004 to 2007 at two public hospitals were investigated. Conventional microbiology methods and PCR were employed. Escherichia coli was isolated from 390 children, 240 of them with diarrhoea. A total of 117 AEEC strains were cultivated from specimens from 63 children, 37 with and 26 without diarrhoea. No association between AEEC and diarrhoea was observed. Atypical enteropathogenic E. coli (a-EPEC) (79.4%) was more commonly found than typical EPEC (t-EPEC). Association between EPEC and EPEC subtypes and diarrhoea was not detected. Mixed infection by t-EPEC and a-EPEC and infection by Shiga toxin-producing E. coli (STEC) were rare. Enteropathogenic E. coli was more common in males and in children aged less than 12 months. Correlation between serotyping and PCR results was 0.19. High resistance rates of AEEC to ampicillin, cephalotin, and trimethoprim-sulfamethoxazole were found. In conclusion, EPEC is very common in children with diarrhoea and controls in the population we studied, with a-EPEC predominating. This diarrhoeagenic E. coli (DEC) pathotype is more common in infant males and is resistant to drugs frequently used in clinical practice.

  12. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    DOE PAGES

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; ...

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using amore » Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections.« less

  13. Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-01-01

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. The present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles associated with human infections. PMID:27148249

  14. Clonal spread and interspecies transmission of clinically relevant ESBL-producing Escherichia coli of ST410--another successful pandemic clone?

    PubMed

    Schaufler, Katharina; Semmler, Torsten; Wieler, Lothar H; Wöhrmann, Michael; Baddam, Ramani; Ahmed, Niyaz; Müller, Kerstin; Kola, Axel; Fruth, Angelika; Ewers, Christa; Guenther, Sebastian

    2016-01-01

    Clinically relevant extended-spectrum beta-lactamase (ESBL)-producing multi-resistant Escherichia coli have been on the rise for years. Initially restricted to mostly a clinical context, recent findings prove their prevalence in extraclinical settings independent of the original occurrence of antimicrobial resistance in the environment. To get further insights into the complex ecology of potentially clinically relevant ESBL-producing E. coli, 24 isolates from wild birds in Berlin, Germany, and 40 ESBL-producing human clinical E. coli isolates were comparatively analyzed. Isolates of ST410 occurred in both sample groups (six). In addition, three ESBL-producing E. coli isolates of ST410 from environmental dog feces and one clinical dog isolate were included. All 10 isolates were clonally analyzed showing almost identical macrorestriction patterns. They were chosen for whole-genome sequencing revealing that the whole-genome content of these 10 E. coli isolates showed a very high genetic similarity, differing by low numbers of single nucleotide polymorphisms only. This study gives initial evidence for a recent interspecies transmission of a new successful clone of ST410 E. coli between wildlife, humans, companion animals and the environment. The results underline the zoonotic potential of clinically relevant multi-resistant bacteria found in the environment as well as the mandatory nature of the 'One Health' approach.

  15. Study of antimicrobial resistance due to extended spectrum beta-lactamase-producing Escherichia coli in healthy broilers of Jabalpur

    PubMed Central

    Shrivastav, Arpita; Sharma, R. K.; Sahni, Y. P.; Shrivastav, Neeraj; Gautam, Vidhi; Jain, Sachin

    2016-01-01

    Aim: To study the prevalence of antimicrobial resistance due to extended spectrum beta-lactamase (ESBL)-producing Escherichia coli in samples collected from the ceca of healthy broilers of poultry sale outlets (PSOs) Jabalpur. Materials and Methods: A total of 400 cecal swab samples were taken randomly from freshly slaughtered poultry of 39 PSOs located at four different zones or areas of Jabalpur and were screened for the presence of ESBL-producing E. coli using standard methods. Further they were characterized phenotypically by standard methods. Results: All the 400 samples were screened for E. coli producing ESBL enzyme. Among the samples positive for E. coli 135 were positive for ESBL E. coli giving an overall prevalence of 33.5%. Conclusion: This study related to the prevalence of ESBL-producing E. coli in healthy broilers in Jabalpur is indicative of antibiotic resistance prevalent in the healthy birds which are used for human consumption as well. It also signifies resistance prevalent against beta-lactam antibiotics including third and fourth generations of cephalosporins. PMID:27956778

  16. 2′-Deoxyribonolactone lesion produces G→A transitions in Escherichia coli

    PubMed Central

    Faure, Virginie; Constant, Jean-François; Dumy, Pascal; Saparbaev, Murat

    2004-01-01

    2′-Deoxyribonolactone (dL) is a C1′-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and γ-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZα gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 × 10–5, similar to that of the classic abasic site 2′-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo. PMID:15159441

  17. Functional and phylogenetic analysis of ureD in Shiga toxin-producing Escherichia coli.

    PubMed

    Steyert, Susan R; Rasko, David A; Kaper, James B

    2011-02-01

    Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

  18. Occurrence of Escherichia coli, Campylobcter, Salmonella and Shiga-Toxin Producing E. coli in Norwegian Primary Strawberry Production.

    PubMed

    Johannessen, Gro S; Eckner, Karl F; Heiberg, Nina; Monshaugen, Marte; Begum, Mumtaz; Økland, Marianne; Høgåsen, Helga R

    2015-06-17

    The aim of this study was to investigate the bacteriological quality of strawberries at harvest and to study risk factors such as irrigation water, soil and picker's hand cleanliness. Four farms were visited during the harvest season in 2012. Samples of strawberries, irrigation water, soil and hand swabs were collected and analyzed for E. coli, Campylobacter, Salmonella and STEC Although fecal indicators and pathogens were found in environmental samples, only one of 80 samples of strawberries was positive for E. coli (1.0 log10 cfu/g) and pathogens were not detected in any of the strawberry samples. The water samples from all irrigation sources were contaminated with E. coli in numbers ranging from 0 to 3.3 log10 cfu/g. Campylobacter (8/16 samples) and Salmonella (1/16 samples) were isolated from samples with high numbers of E. coli. The water samples collected from a lake had lower numbers of E. coli than the samples from rivers and a stream. The present study indicated continuous background contamination in the primary production environment. Although the background contamination was not reflected on the strawberries tested here, the results must be interpreted with caution due to the limited number of samples.

  19. Occurrence of Escherichia coli, Campylobcter, Salmonella and Shiga-Toxin Producing E. coli in Norwegian Primary Strawberry Production

    PubMed Central

    Johannessen, Gro S.; Eckner, Karl F.; Heiberg, Nina; Monshaugen, Marte; Begum, Mumtaz; Økland, Marianne; Høgåsen, Helga R.

    2015-01-01

    The aim of this study was to investigate the bacteriological quality of strawberries at harvest and to study risk factors such as irrigation water, soil and picker’s hand cleanliness. Four farms were visited during the harvest season in 2012. Samples of strawberries, irrigation water, soil and hand swabs were collected and analyzed for E. coli, Campylobacter, Salmonella and STEC Although fecal indicators and pathogens were found in environmental samples, only one of 80 samples of strawberries was positive for E. coli (1.0 log10 cfu/g) and pathogens were not detected in any of the strawberry samples. The water samples from all irrigation sources were contaminated with E. coli in numbers ranging from 0 to 3.3 log10 cfu/g. Campylobacter (8/16 samples) and Salmonella (1/16 samples) were isolated from samples with high numbers of E. coli. The water samples collected from a lake had lower numbers of E. coli than the samples from rivers and a stream. The present study indicated continuous background contamination in the primary production environment. Although the background contamination was not reflected on the strawberries tested here, the results must be interpreted with caution due to the limited number of samples. PMID:26090606

  20. Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli.

    PubMed

    Kueh, Chare Li; Yong, Chean Yeah; Masoomi Dezfooli, Seyedehsara; Bhassu, Subha; Tan, Soon Guan; Tan, Wen Siang

    2016-11-14

    Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  1. Method for the detection of priority Shiga toxin-producing Escherichia coli in beef trim.

    PubMed

    Huszczynski, George; Gauthier, Martine; Mohajer, Sam; Gill, Alexander; Blais, Burton

    2013-10-01

    A method has been developed for the detection in beef trim of priority Shiga toxin-producing E. coli (STEC) strains, defined as E. coli possessing the virulence factors stx1 and/or stx2 and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae (STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

  2. Characterization of Shiga toxin – producing Escherichia coli infections in beef feeder calves and the effectiveness of a prebiotic in alleviating Shiga toxin - producing Escherichia coli infections

    PubMed Central

    2013-01-01

    Background In the less-sensitive mouse model, Shiga toxin-producing Escherichia coli (STEC) challenges result in shedding that reflect the amount of infection and the expression of virulence factors such as Shiga toxins (Stx). The purpose of this study was to characterize the contribution of STEC diversity and Stx expression to shedding in beef feeder calves and to evaluate the effectiveness of a prebiotic, Celmanax®, to alleviate STEC shedding. Fecal samples were collected from calves at entry and after 35 days in the feedlot in spring and summer. STECs were evaluated using selective media, biochemical profile, serotyping and Stx detection. Statistical analysis was performed using repeated measures ANOVA and logistic regression. Results At entry, non-O157 STEC were dominant in shedding calves. In spring, 21%, 14% and 14% of calves acquired O157, non-O157 and mixed STEC infections, respectively. In contrast, 45%, 48% and 46% of calves in summer acquired O157, non-O157 and mixed STEC infections, respectively. Treatment with a prebiotic, Celmanax®, in spring significantly reduced 50% of the O157 STEC infections, 50% of the non-O157 STEC infections and 36% of the STEC co-infections (P = 0.037). In summer, there was no significant effect of the prebiotic on STEC infections. The amount of shedding at entry was significantly related to the number and type of STECs present and Stx expression (r2 = 0.82). The same relationship was found for shedding at day 35 (r2 = 0.85), but it was also related to the number and type of STECs present at entry. Stx - producing STEC infections resulted in 100 to 1000 × higher shedding in calves compared with Stx-negative STECs. Conclusions STEC infections in beef feeder calves reflect the number and type of STECs involved in the infection and STEC expression of Stx. Application of Celmanax® reduced O157 and non-O157 STEC shedding by calves but further research is required to determine appropriate dosages to manage STEC

  3. Draft Genome Sequence of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strain CCUG 62462, Isolated from a Urine Sample

    PubMed Central

    Johnning, Anna; Jakobsson, Hedvig E.; Boulund, Fredrik; Salvà-Serra, Francisco; Åhrén, Christina; Kristiansson, Erik

    2016-01-01

    The draft genome sequence has been determined for an extended-spectrum-β-lactamase (ESBL)-producing (blaCTX-M-15) Escherichia coli strain (CCUG 62462), composed of 119 contigs and a total size of 5.27 Mb. This E. coli is serotype O25b and sequence type 131, a pandemic clonal group, causing worldwide antimicrobial-resistant infections. PMID:27979938

  4. Effects of stresses on the growth and Cytotoxicity of Shiga-Toxin producing Escherichia coli in ground beef and spinach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to examine the effect of stresses on the growth and cytotoxicity of pathogenic Escherichia coli in beef and spinach. A mixture of three strains of Shiga toxin-producing E. coli (STEC) O157:H7 or four strains of non-O157 STEC O26:H11, O103:H1, O104:H4, and O145:NM wa...

  5. Escherichia coli Sequence Type 131 H30 Is the Main Driver of Emerging Extended-Spectrum-β-Lactamase-Producing E. coli at a Tertiary Care Center.

    PubMed

    Johnson, James R; Johnston, Brian; Thuras, Paul; Launer, Bryn; Sokurenko, Evgeni V; Miller, Loren G

    2016-01-01

    The H30 strain of Escherichia coli sequence type 131 (ST131-H30) is a recently emerged, globally disseminated lineage associated with fluoroquinolone resistance and, via its H30Rx subclone, the CTX-M-15 extended-spectrum beta-lactamase (ESBL). Here, we studied the clonal background and resistance characteristics of 109 consecutive recent E. coli clinical isolates (2015) and 41 historical ESBL-producing E. coli blood isolates (2004 to 2011) from a public tertiary care center in California with a rising prevalence of ESBL-producing E. coli isolates. Among the 2015 isolates, ST131, which was represented mainly by ST131-H30, was the most common clonal lineage (23% overall). ST131-H30 accounted for 47% (8/17) of ESBL-producing, 47% (14/30) of fluoroquinolone-resistant, and 33% (11/33) of multidrug-resistant isolates. ST131-H30 also accounted for 53% (8/14) of dually fluoroquinolone-resistant, ESBL-producing isolates, with the remaining 47% comprised of diverse clonal groups that contributed a single isolate each. ST131-H30Rx, with CTX-M-15, was the major ESBL producer (6/8) among ST131-H30 isolates. ST131-H30 and H30Rx also dominated (46% and 37%, respectively) among the historical ESBL-producing isolates (2004 to 2011), without significant temporal shifts in relative prevalence. Thus, this medical center's recently emerging ESBL-producing E. coli strains, although multiclonal, are dominated by ST131-H30 and H30Rx, which are the only clonally expanded fluoroquinolone-resistant, ESBL-producing lineages. Measures to rapidly and effectively detect, treat, and control these highly successful lineages are needed. IMPORTANCE The ever-rising prevalence of resistance to first-line antibiotics among clinical Escherichia coli isolates leads to worse clinical outcomes and higher health care costs, thereby creating a need to discover its basis so that effective interventions can be developed. We found that the H30 subset within E. coli sequence type 131 (ST131-H30) is

  6. Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli.

    PubMed

    Ravenscroft, Neil; Haeuptle, Micha A; Kowarik, Michael; Fernandez, Fabiana S; Carranza, Paula; Brunner, Andreas; Steffen, Michael; Wetter, Michael; Keller, Sacha; Ruch, Corina; Wacker, Michael

    2016-01-01

    Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.

  7. Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

    NASA Astrophysics Data System (ADS)

    Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

    2017-02-01

    Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population.

  8. Bet-hedging in bacteriocin producing Escherichia coli populations: the single cell perspective

    PubMed Central

    Bayramoglu, Bihter; Toubiana, David; van Vliet, Simon; Inglis, R. Fredrik; Shnerb, Nadav; Gillor, Osnat

    2017-01-01

    Production of public goods in biological systems is often a collaborative effort that may be detrimental to the producers. It is therefore sustainable only if a small fraction of the population shoulders the cost while the majority reap the benefits. We modelled this scenario using Escherichia coli populations producing colicins, an antibiotic that kills producer cells’ close relatives. Colicin expression is a costly trait, and it has been proposed that only a small fraction of the population actively expresses the antibiotic. Colicinogenic populations were followed at the single-cell level using time-lapse microscopy, and showed two distinct, albeit dynamic, subpopulations: the majority silenced colicin expression, while a small fraction of elongated, slow-growing cells formed colicin-expressing hotspots, placing a significant burden on expressers. Moreover, monitoring lineages of individual colicinogenic cells showed stochastic switching between expressers and non-expressers. Hence, colicin expressers may be engaged in risk-reducing strategies—or bet-hedging—as they balance the cost of colicin production with the need to repel competitors. To test the bet-hedging strategy in colicin-mediated interactions, competitions between colicin-sensitive and producer cells were simulated using a numerical model, demonstrating a finely balanced expression range that is essential to sustaining the colicinogenic population. PMID:28165017

  9. Growth of Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing Escherichia coli , and Salmonella in Water and Hydroponic Fertilizer Solutions.

    PubMed

    Shaw, Angela; Helterbran, Kara; Evans, Michael R; Currey, Christopher

    2016-12-01

    The desire for local, fresh produce year round is driving the growth of hydroponic growing systems in the United States. Many food crops, such as leafy greens and culinary herbs, grown within hydroponics systems have their root systems submerged in recirculating nutrient-dense fertilizer solutions from planting through harvest. If a foodborne pathogen were introduced into this water system, the risk of contamination to the entire crop would be high. Hence, this study was designed to determine whether Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli , and Salmonella were able to survive and reproduce in two common hydroponic fertilizer solutions and in water or whether the bacteria would be killed or suppressed by the fertilizer solutions. All the pathogens grew by 1 to 6 log CFU/ml over a 24-h period, depending on the solution. E. coli O157:H7 reached higher levels in the fertilizer solution with plants (3.12 log CFU/ml), whereas non-O157 Shiga toxin-producing E. coli and Salmonella reached higher levels in the fertilizer solution without plants (1.36 to 3.77 log CFU/ml). The foodborne pathogens evaluated here survived for 24 h in the fertilizer solution, and populations grew more rapidly in these solutions than in plain water. Therefore, human pathogens entering the fertilizer solution tanks in hydroponic systems would be expected to rapidly propagate and spread throughout the system and potentially contaminate the entire crop.

  10. Prevalence and characteristics of ESBL-producing E. coli in Dutch recreational waters influenced by wastewater treatment plants.

    PubMed

    Blaak, Hetty; de Kruijf, Patrick; Hamidjaja, Raditijo A; van Hoek, Angela H A M; de Roda Husman, Ana Maria; Schets, Franciska M

    2014-07-16

    Outside health care settings, people may acquire ESBL-producing bacteria through different exposure routes, including contact with human or animal carriers or consumption of contaminated food. However, contact with faecally contaminated surface water may also represent a possible exposure route. The current study investigated the prevalence and characteristics of ESBL-producing Escherichia coli in four Dutch recreational waters and the possible role of nearby waste water treatment plants (WWTP) as contamination source. Isolates from recreational waters were compared with isolates from WWTP effluents, from surface water upstream of the WWTPs, at WWTP discharge points, and in connecting water bodies not influenced by the studied WWTPs. ESBL-producing E. coli were detected in all four recreational waters, with an average concentration of 1.3 colony forming units/100ml, and in 62% of all samples. In surface waters not influenced by the studied WWTPs, ESBL-producing E. coli were detected in similar concentrations, indicating the existence of additional ESBL-E. coli contamination sources. Isolates with identical ESBL-genes, phylogenetic background, antibiotic resistance profiles, and sequence type, were obtained from effluent and different surface water sites in the same watershed, on the same day; occasionally this included isolates from recreational waters. Recreational waters were identified as a potential exposure source of ESBL-producing E. coli. WWTPs were shown to contribute to the presence of these bacteria in surface waters, but other (yet unidentified) sources likely co-contribute.

  11. Extended-Spectrum Beta-Lactamases Producing E. coli in Wildlife, yet Another Form of Environmental Pollution?

    PubMed Central

    Guenther, Sebastian; Ewers, Christa; Wieler, Lothar H.

    2011-01-01

    Wildlife is normally not exposed to clinically used antimicrobial agents but can acquire antimicrobial resistant bacteria through contact with humans, domesticated animals and the environment, where water polluted with feces seems to be the most important vector. Escherichia coli, an ubiquitous commensal bacterial species colonizing the intestinal tract of mammals and birds, is also found in the environment. Extended-spectrum beta-lactamases producing E. coli (ESBL-E. coli) represent a major problem in human and veterinary medicine, particular in nosocomial infections. Additionally an onset of community-acquired ESBL-E. coli infections and an emergence in livestock farming has been observed in recent years, suggesting a successful transmission as well as persistence of ESBL-E. coli strains outside clinical settings. Another parallel worldwide phenomenon is the spread of ESBL-E. coli into the environment beyond human and domesticated animal populations, and this seems to be directly influenced by antibiotic practice. This might be a collateral consequence of the community-onset of ESBL-E. coli infections but can result (a) in a subsequent colonization of wild animal populations which can turn into an infectious source or even a reservoir of ESBL-E. coli, (b) in a contribution of wildlife to the spread and transmission of ESBL-E. coli into fragile environmental niches, (c) in new putative infection cycles between wildlife, domesticated animals and humans, and (d) in problems in the medical treatment of wildlife. This review aims to summarize the current knowledge on ESBL-E. coli in wildlife, in turn underlining the need for more large scale investigations, in particular sentinel studies to monitor the impact of multiresistant bacteria on wildlife. PMID:22203818

  12. Risk Factors for Sporadic Shiga Toxin–producing Escherichia coli Infections in Children, Argentina1

    PubMed Central

    Rivas, Marta; Sosa-Estani, Sergio; Rangel, Josefa; Caletti, Maria G.; Vallés, Patricia; Roldán, Carlos D.; Balbi, Laura; Marsano de Mollar, Maria C.; Amoedo, Diego; Miliwebsky, Elizabeth; Chinen, Isabel; Hoekstra, Robert M.; Mead, Paul

    2008-01-01

    We evaluated risk factors for sporadic Shiga toxin–producing Escherichia coli (STEC) infection among children in Argentina. We conducted a prospective case–control study in 2 sites and enrolled 150 case-patients and 299 controls. The median age of case-patients was 1.8 years; 58% were girls. Serotype O157:H7 was the most commonly isolated STEC. Exposures associated with infection included eating undercooked beef, living in or visiting a place with farm animals, and contact with a child <5 years of age with diarrhea. Protective factors included the respondent reporting that he or she always washed hands after handling raw beef and the child eating more than the median number of fruits and vegetables. Many STEC infections in children could be prevented by avoiding consumption of undercooked beef, limiting exposure to farm animals and their environment, not being exposed to children with diarrhea, and washing hands after handling raw beef. PMID:18439359

  13. Shiga Toxin–producing Escherichia coli Serotype O78:H– in Family, Finland, 2009

    PubMed Central

    Lienemann, Taru; Salo, Eeva; Rimhanen-Finne, Ruska; Rönnholm, Kai; Taimisto, Mari; Hirvonen, Jari J.; Tarkka, Eveliina; Kuusi, Markku

    2012-01-01

    Shiga toxin–producing Escherichia coli (STEC) is a pathogen that causes gastroenteritis and bloody diarrhea but can lead to severe disease, such as hemolytic uremic syndrome (HUS). STEC serotype O78:H– is rare among humans, and infections are often asymptomatic. We detected a sorbitol-fermenting STEC O78:H–stx1c:hlyA in blood and fecal samples of a 2-week-old boy who had bacteremia and HUS and in fecal samples of his asymptomatic family members. The phenotypic and genotypic characteristics and the virulence properties of this invasive STEC were investigated. Our findings demonstrate that contrary to earlier suggestions, STEC under certain conditions can invade the human bloodstream. Moreover, this study highlights the need to implement appropriate diagnostic methods for identifying the whole spectrum of STEC strains associated with HUS. PMID:22469631

  14. Longitudinal monitoring of extended-spectrum-beta-lactamase/AmpC-producing Escherichia coli at German broiler chicken fattening farms.

    PubMed

    Laube, H; Friese, A; von Salviati, C; Guerra, B; Käsbohrer, A; Kreienbrock, L; Roesler, U

    2013-08-01

    Antimicrobial resistance of Escherichia coli to modern beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBL) and/or plasmid-mediated AmpC beta-lactamases (AmpC) represents an emerging and increasing resistance problem that dramatically limits therapeutic options in both human and veterinary medicine. The presence of ESBL/AmpC genes in commensal E. coli from food-producing animals like broilers may pose a human health hazard. However, there are no data available concerning the prevalence of ESBL/AmpC-producing E. coli in German broiler flocks using selective methods. In this longitudinal study, samples were taken from seven conventional broiler fattening farms at three different times within one fattening period. Various samples originating from the animals as well as from their direct environment in the barn were investigated for the occurrence of ESBL/AmpC-producing E. coli. Average detection levels of 51, 75, and 76% in animal samples collected during the three samplings in the course of the fattening period demonstrate a colonization of even 1-day-old chicks, as well as a continuous significant (P < 0.001) increase in prevalence thereafter. The detection frequencies in housing environmental samples were relatively high, with an increase over time, and ranged between 54.2 and 100%. A total of 359 E. coli isolates were characterized by PCR and partly via the disc diffusion method. This study shows that prevalence of ESBL/AmpC-producing E. coli increases during the fattening period of the broiler flocks examined. Both colonized day-old chicks and contaminated farm environments could represent significant sources of ESBL/AmpC-producing E. coli in German broiler fattening farms.

  15. Real-time isothermal detection of Shiga toxin-producing Escherichia coli using recombinase polymerase amplification.

    PubMed

    Murinda, Shelton E; Ibekwe, A Mark; Zulkaffly, Syaizul; Cruz, Andrew; Park, Stanley; Razak, Nur; Paudzai, Farah Md; Ab Samad, Liana; Baquir, Khairul; Muthaiyah, Kokilah; Santiago, Brenna; Rusli, Amirul; Balkcom, Sean

    2014-07-01

    Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

  16. Emergence of KPC-2-producing Escherichia coli isolates in an urban river in Harbin, China.

    PubMed

    Xu, Guofeng; Jiang, Yue; An, Wei; Wang, Hongdong; Zhang, Xiuying

    2015-09-01

    Three KPC-2-producing Escherichia coli (E1, E2, and E3) were recovered from water samples of an urban river in the city of Harbin, China. Antimicrobial susceptibility was determined by broth microdilution. Molecular characterization and genetic relatedness of the isolates were determined by polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and PCR-directed phylotyping. Plasmids were analyzed by conjugation, S1-PFGE, Southern blotting and PCR-based replicon typing (PBRT). The genetic environment of the bla KPC-2 gene was determined using PCR and sequencing. PCR analyses revealed that the E1 isolate carried the bla KPC-2, bla CMY-2, bla TEM-1, bla CTX-M-14, and qnrB2 genes and belonged to sequence type ST410, phylogenetic type A; the E2 isolate was assigned to ST131-B2 and carried the bla KPC-2, bla TEM-1, bla CTX-M-3, bla DHA-1, aac(6')-Ib-cr, and qnrS1 genes; while the E3 isolate was of ST648-D and possessed bla KPC-2, bla TEM-1, bla OXA-1, bla CTX-M-15, armA, and aac(6')-Ib-cr genes. PFGE demonstrated that each of the three KPC-2-producing E. coli isolates exhibited an individual XbaI patterns. The bla KPC-2 gene was located on plasmids of 60-140 kb with IncA/C, IncN, or non-typeable replicon types. The genetic environment of bla KPC-2 of the three strains was consistent with the genetic structure of bla KPC-2 on the plasmid pKP048.

  17. Mexican unpasteurised fresh cheeses are contaminated with Salmonella spp., non-O157 Shiga toxin producing Escherichia coli and potential uropathogenic E. coli strains: A public health risk.

    PubMed

    Guzman-Hernandez, Rosa; Contreras-Rodriguez, Araceli; Hernandez-Velez, Rosa; Perez-Martinez, Iza; Lopez-Merino, Ahide; Zaidi, Mussaret B; Estrada-Garcia, Teresa

    2016-11-21

    Fresh cheeses are a main garnish of Mexican food. Consumption of artisanal fresh cheeses is very common and most of them are made from unpasteurised cow milk. A total of 52 fresh unpasteurised cheeses of five different types were purchased from a variety of suppliers from Tabasco, Mexico. Using the most probable number method, 67% and 63% of samples were positive for faecal coliforms and E. coli, respectively; revealing their low microbiological quality. General hygienic conditions and practices of traditional cheese manufacturers were poor; most establishments had unclean cement floors, all lacked windows and doors screens, and none of the food-handlers wore aprons, surgical masks or bouffant caps. After analysing all E. coli isolates (121 strains) for the presence of 26 virulence genes, results showed that 9 (17%) samples were contaminated with diarrheagenic E. coli strains, 8 harboured non-O157 Shiga toxin producing E. coli (STEC), and one sample contained both STEC and diffusely adherent E. coli strains. All STEC strains carried the stx1 gene. Potential uropathogenic E. coli (UPEC) strains were isolated from 15 (29%) samples; the most frequent gene combination was fimA-agn43. Two samples were contaminated with Salmonella. The results demonstrated that unpasteurised fresh cheeses produced in Tabasco are of poor microbiological quality and may frequently harbour foodborne pathogens. Food safety authorities in Mexico need to conduct more rigorous surveillance of fresh cheeses. Furthermore, simple and inexpensive measures as establishing programs emphasizing good hand milking practices and hygienic manufacturing procedures may have a major effect on improving the microbiological quality of these food items.

  18. Use of ramification amplification assay for detection of Escherichia coli O157:H7 and other E. coli Shiga toxin-producing strains.

    PubMed

    Li, Fan; Zhao, Chunyan; Zhang, Wandi; Cui, Shenghui; Meng, Jianghong; Wu, Josephine; Zhang, David Y

    2005-12-01

    Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) strains are important human pathogens that are mainly transmitted through the food chain. These pathogens have a low infectious dose and may cause life-threatening illnesses. However, detection of this microorganism in contaminated food or a patient's stool specimens presents a diagnostic challenge because of the low copy number in the sample. Often, a more sensitive nucleic acid amplification method, such as PCR, is required for rapid detection of this microorganism. Ramification amplification (RAM) is a recently introduced isothermal DNA amplification technique that utilizes a circular probe for target detection and achieves exponential amplification through the mechanism of primer extension, strand displacement, and ramification. In this study, we synthesized a circular probe specific for the Shiga toxin 2 gene (stx(2)). Our results showed that as few as 10 copies of stx(2) could be detected, indicating that the RAM assay was as sensitive as conventional PCR. We further tested 33 isolates of E coli O157:H7, STEC, Shigella dysenteriae, and nonpathogenic E. coli by RAM assay. Results showed that all 27 STEC isolates containing the stx(2) gene were identified by RAM assay, while S. dysenteriae and nonpathogenic E. coli isolates were undetected. The RAM results were 100% in concordance with those of PCR. Because of its simplicity and isothermal amplification, the RAM assay could be a useful method for detecting STEC in food and human specimens.

  19. Contact precautions for preventing nosocomial transmission of ESBL-producing Escherichia coli - a point/counterpoint review.

    PubMed

    Tschudin-Sutter, Sarah; Lucet, Jean-Christophe; Mutters, Nico T; Tacconelli, Evelina; Zahar, Jean Ralph; Harbarth, Stephan

    2017-04-03

    Contact precautions have been recommended for hospitalized patients colonized or infected with extendend-spectrum beta-lactamase (ESBL)-producing Escherichia coli. Despite such recommendations, a steady, worldwide increase of ESBL-E. coli has been reported. We discuss arguments in favor and against contact precautions for ESBL- E.coli-carriers.Healthcare settings with high ESBL-E.coli colonization pressure, extended hospital stay and close contact between vulnerable patients may serve as amplification platform further accelerating transmission. However, the evidence base for justifying the implementation of contact precautions for all ESBL-E.coli carriers remains weak.Until more high-level evidence is available, we support the attitude that hospitals and countries should carefully evaluate their decision on whether to implement contact precautions for ESBL-E.coli carriers. It is likely that a large majority of patients and wards do not need to rely on contact precautions for preventing nosocomial ESBL-E.coli transmission in non-epidemic settings, without harming patient-safety, providing sufficient compliance with standard precautions and ongoing surveillance.

  20. Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomes of a diverse set of Shiga toxin-producing E. coli strains and the presence of 38 plasmids among all the isolates were determined. Among the novel plasmids found, there were eight that encoded resistance genes to antibiotics, including aminoglycosides, carbapenems, penicillins, cephalosp...

  1. Ciprofloxacin-resistant, CTX-M-15-producing Escherichia coli ST131 clone in extraintestinal infections in Italy.

    PubMed

    Cerquetti, M; Giufrè, M; García-Fernández, A; Accogli, M; Fortini, D; Luzzi, I; Carattoli, A

    2010-10-01

    Quinolone and β-lactam resistance mechanisms and clonal relationships were characterized among Escherichia coli isolates resistant to ciprofloxacin and extended-spectrum cephalosporins associated with human extra-intestinal infections in Rome. The E. coli. ST131 clone was found to be prevalent. This clone invariably carried a specific pattern of substitutions in the topoisomerase genes and all isolates but one produced CTX-M-15. One ST131 isolate produced SHV-12. The new ST131 variant described here is of particular concern because it combines fluoroquinolone resistance and chromosomally encoded CTX-M-15.

  2. Characterization of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Strains Involved in Maternal-Fetal Colonization: Prevalence of E. coli ST131

    PubMed Central

    Birgy, André; Mariani-Kurkdjian, Patricia; Bidet, Philippe; Doit, Catherine; Genel, Nathalie; Courroux, Céline; Bingen, Edouard

    2013-01-01

    Maternal-fetal Escherichia coli infections, such as neonatal bacteremia and meningitis, are important causes of morbidity and mortality. From 2006 to 2010, we studied newborns and their mothers who were colonized with E. coli in a French hospital in order to document (i) the epidemiology and genetic characteristics of extended-spectrum-beta-lactamase (ESBL)-producing E. coli strains, (ii) the prevalence of associated virulence genes, (iii) the prevalence of clone sequence type 131 (ST131), and (iv) the genetic relationship among ESBL-producing strains. Among the 2,755 E. coli cultures recovered from vaginal or neonatal samples, 68 were ESBL producers (2.46%). We found a wide diversity of ESBL genes, with the majority being blaCTX-M-14, blaCTX-M-1, and blaCTX-M-15, distributed among the 4 main phylogenetic groups. Genes encoding virulence factors were found in 90.7% of the isolates, with ≥2 virulence genes present in 76% of cases. The prevalence of ST131 among ESBL-producing E. coli isolates was 9.4% (6/64). Five of these 6 ST131 isolates possessed blaCTX-M-15 enzymes (and also were resistant to quinolones), and one possessed blaCTX-M-2 enzymes. Two possessed virulence genes, suggesting the presence of pathogenicity island IIJ96 (PAI IIJ96)-like domains. Pulsed-field gel electrophoresis (PFGE) revealed a high level of genomic diversity overall, except for 3 closely related isolates belonging to clonal group ST131. Repetitive PCR showed that the six ST131 isolates were closely related to ST131 control strains (>95% similarity). This study shows a high prevalence of ESBL-producing E. coli strains and clonal group ST131 in the French maternal-fetal population. These results suggest a widespread distribution of ESBL enzymes in the community and highlight the early transmission between mothers and neonates. These findings are worrisome, especially for this particularly vulnerable population. PMID:23515552

  3. [An effective scheme to produce recombinant uracil-DNA glycosylase of Escherichia coli for PCR diagnostics].

    PubMed

    Dmitrochenko, A E; Turiianskaia, O M; Gilep, A A; Usanov, S A; Iantsevich, A V

    2014-01-01

    An effective scheme has been developed to produce recombinant uracil-DNA glycosylase of Escherichia coli K12 intended to be used for PCR diagnostics, making it possible to achieve a high yield of the end product using a two-stage purification. The gene encoding this enzyme was cloned into the pCWori vector within the same reading frame with six residues of histidine in the C-erminal sequence. Using this vector and the E. coli DH5alpha, a host-vector expression system has been developed and conditions for protein synthesis have been optimized. To purify the protein, metal affinity chromatography with further dialysis was used to remove imidazole. The enzyme yield was no less than 60 mg of the end protein per 1 L of the culture medium. The concordance between amino acid sequences of the recombinant and native enzymes was proved by peptide mass fingerprinting and mass spectrometry. A rapid test to determine the activity of the enzyme preparation was suggested. It was found that the activity of 1.0 mg of the recombinant protein is no less than 3 x 10(3) units. The recombinant enzyme was most stable at pH 8.0 and an ionic strength of the solution equal to 200 mM; it lost its activity completely for 10 min at 60 degrees C. Storage during 1 h at 20 degrees C resulted in the loss of no more than 30% of activity. In the enzyme preparation, the activity of DNase was absent. The free energy of the unfolding of the protein globule of the recombinant uracil-DNA glycosylase is 23.1 +/- 0.2 kJ/mol. The data obtained indicate that the recombinant enzyme may be recommended for use in PCR diagnostics to prevent the appearance of false positive results caused by pollution of the reaction mixture by products of the preceding reactions.

  4. Molecular characterization of integrons in clinical isolates of betalactamase-producing Escherichia coli and Klebsiella pneumoniae in Iran.

    PubMed

    Zeighami, Habib; Haghi, Fakhri; Hajiahmadi, Fahimeh

    2015-06-01

    Integrons are considered to play a significant role in the evolution and spread of antibiotic resistance genes. A total of 349 clinical isolates of Escherichia coli and Klebsiella pneumoniae were investigated for molecular characterization of integrons and betalactamases. Antimicrobial susceptibility testing was also performed as the Clinical and Laboratory Standards Institute (CLSI) guidelines. The frequency of extended spectrum betalactamases (ESBL) or metallo-betalactamases (MBL)-producing isolates, patient demographics, and the susceptibility to various antimicrobial agents were described. BlaCTX-M was the most frequently detected betalactamase in all isolates. Moreover, MBL producing K. pneumoniae carried blaIMP and blaVIM at 100 and 41·6%, respectively but no MBL-positive E. coli was detected. Class 1 integrons were more frequent among E. coli and K. pneumoniae isolates in comparison with class 2 integrons and the frequency of intI2 in K. pneumoniae was significantly higher than E. coli isolates. Five different resistance gene arrays were identified among class 1 integrons. Dihydrofolate reductase (dfrA) and aminoglycoside adenyltransferase (aad) gene cassettes were found to be predominant in the class 1 integrons. These results indicate that class 1 integrons are widespread among ESBL-producing isolates of K. pneumoniae and E. coli and appropriate surveillance and control measures are essential to prevent further dissemination of these elements among Enterobacteriaceae in our country.

  5. High-virulence CMY-2- and CTX-M-2-producing avian pathogenic Escherichia coli strains isolated from commercial turkeys.

    PubMed

    da Silva, Ketrin Cristina; Cunha, Marcos Paulo Vieira; Cerdeira, Louise; de Oliveira, Maria Gabriela Xavier; de Oliveira, Mirela Caroline Vilela; Gomes, Cleise Ribeiro; Lincopan, Nilton; Knöbl, Terezinha; Moreno, Andrea Micke

    2017-01-01

    This study reports the high-virulence phylogenetic backgrounds of CMY-2- and CTX-M-2-producing avian pathogenic Escherichia coli strains isolated from turkeys sent to slaughter and condemned by airsacculitis in Brazil. Among 300 air sac samples, seven E. coli strains produced plasmid-mediated CMY-2-type AmpC, of which three carried also the blaCTX-M-2 Extended Spectrum Beta-Lactamase encoding gene. Interestingly, the transfer of the blaCMY-2 gene was positive for three E. coli strains, being associated with the presence of IncI1 plasmids. The complete sequence of the representative pJB10 plasmid revealed that the blaCMY-2 gene was within a transposon-like element in the classical genetic environment consisting of tnpA-blaCMY-2-blc-sugE structure. This plasmid with 94-kb belonged to the sequence type (ST) 12 among IncI1 plasmids, which has been associated with the worldwide spread of blaCMY-2 among Salmonella enterica and E. coli. Furthermore, to the best of our knowledge, this is the first complete sequence of a CMY-2-encoding plasmid derived from an Escherichia coli isolated from food-producing animals in Latin America.

  6. Outbreaks of non-O157 Shiga toxin-producing Escherichia coli infection: USA.

    PubMed

    Luna-Gierke, R E; Griffin, P M; Gould, L H; Herman, K; Bopp, C A; Strockbine, N; Mody, R K

    2014-11-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections are increasingly detected, but sources are not well established. We summarize outbreaks to 2010 in the USA. Single-aetiology outbreaks were defined as ⩾2 epidemiologically linked culture-confirmed non-O157 STEC infections; multiple-aetiology outbreaks also had laboratory evidence of ⩾2 infections caused by another enteric pathogen. Twenty-six states reported 46 outbreaks with 1727 illnesses and 144 hospitalizations. Of 38 single-aetiology outbreaks, 66% were caused by STEC O111 (n = 14) or O26 (n = 11), and 84% were transmitted through food (n = 17) or person-to-person spread (n = 15); food vehicles included dairy products, produce, and meats; childcare centres were the most common setting for person-to-person spread. Of single-aetiology outbreaks, a greater percentage of persons infected by Shiga toxin 2-positive strains had haemolytic uraemic syndrome compared with persons infected by Shiga toxin 1-only positive strains (7% vs. 0·8%). Compared with single-aetiology outbreaks, multiple-aetiology outbreaks were more frequently transmitted through water or animal contact.

  7. Detection of shiga-toxin producing E. coli (STEC) in leafy greens sold at local retail markets in Alexandria, Egypt.

    PubMed

    Khalil, Rowaida K S; Gomaa, Mohamed A E; Khalil, Mahmoud I M

    2015-03-16

    Leafy green vegetables, a popular and an indispensable ingredient of the daily menus of Egyptians' diets, currently presents a great concern in terms of microbiological hazards. To the best of our knowledge, this is the first report that provides scientific evidence for prevalence of shiga-toxigenic Escherichia coli (STEC) in leafy greens sold at open air local retail markets and superstores in the Egyptian environment. A total of 486 conventional and organic leafy green samples that are eaten raw were collected from different areas in Alexandria, evaluated for total E. coli counts (ECCs), and screened for E. coli O157:H7 using conventional and molecular methods. Recovery of E. coli (≥10(2)CFU/g) from all studied types of leafy greens was indicative of fecal contamination. Total ECCs in conventional samples ranged from 5.47 to 2.56 log CFU/g. Based on their inability to ferment sorbitol on CT-SMAC media, 26 presumptive E. coli O157 isolates were detected in 71.4% (270/378) of the studied conventional samples. From all studied organic samples, only 2 types (organic cabbage and parsley, 16.7%) were contaminated with presumptive E. coli O157. All 28 isolates were further serotyped as E. coli O157 by latex agglutination test, and biochemically confirmed as E. coli. Multiplex PCR assays confirmed the ability of 21.4% (6/28) of the E. coli O157 strains to produce shiga-toxins (Stxs), and their virulence markers were as follows: stx1, 66.6% (4/6); stx2, 50% (3/6); stx1/stx2, 16.7% (1/6); eaeA, 83.3% (5/6); and hlyA, 16.7% (1/6). Only 2 strains recovered from conventional and organic parsley could possibly be classified as E. coli O157:H7 based on the presence of stx-genes (either stx1 or stx2 or both). Results of the present research highlight that high E. coli loads, together with recovery of STEC O157 isolates could pose serious health risks to the produce consumers. This emphasizes the urgent need for health authorities to value and utilize the existing knowledge to

  8. Acid Resistance and molecular characterization of Escherichia coli O157:H7 and different non-O157 Shiga toxin-producing E. coli serogroups

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the acid resistance (AR) of seven non-O157 Shiga toxin-producing E. coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121 and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and 30°...

  9. Acid resistance and molecular characterization of Escherichia coli O157:H7 and different Non-O157 shiga toxin-producing E. coli serogroups

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the acid resistance (AR) of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121, and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and...

  10. Epidemiology of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli in Japan: Characteristics of community-associated versus healthcare-associated ESBL E. coli.

    PubMed

    Hayakawa, Kayoko; Nagamatsu, Maki; Mezaki, Kazuhisa; Sugiki, Yuko; Kutsuna, Satoshi; Takeshita, Nozomi; Yamamoto, Kei; Mawatari, Momoko; Fujiya, Yoshihiro; Ohmagari, Norio

    2017-02-01

    Data on community-associated extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (CA-ESBLEC) infections in Japan are scarce. We compared the clinical and microbiological epidemiology of CA-ESBLEC infections with that of healthcare-associated-ESBLEC infections among 76 patients with ESBLEC infections. We identified a high prevalence (26%) of CA-ESBLEC infections in Japan; only a small proportion (15%) of patients with CA-ESBLEC infections had recent exposure to antibiotics.

  11. Bactericidal Effect of Selected Antidiarrhoeal Medicinal Plants on Intracellular Heat-Stable Enterotoxin-Producing Escherichia coli

    PubMed Central

    Birdi, Tannaz J.; Brijesh, S.; Daswani, Poonam G.

    2014-01-01

    Diarrhoeal diseases due to enterotoxigenic Escherichia coli continue to be a cause of global concern. Medicinal plants have been gaining popularity as promising antidiarrhoeal agents. In the present study, four antidiarrhoeal plants, viz. Aegle marmelos, Cyperus rotundus, Psidium guajava and Zingiber officinale were screened against a heat-stable toxin-producing enterotoxigenic E. coli strain. Decoctions of these plants were studied for their effect on intracellular killing of the bacterial strain using murine monocytic cell line, J774. [3H] thymidine release assay was used to evaluate the apoptotic/necrotic effect. All plants at concentrations <1% enhanced intracellular killing of the bacteria by J774 cells. However, at higher concentrations, the decoctions induced apoptosis in J774 cells. The study demonstrates that these plants could control diarrhoea caused by heat-stable toxin-producing enterotoxigenic E. coli through their immunomodulatory effect. PMID:25035535

  12. CTX-M-15 Extended-Spectrum β-Lactamase in a Shiga Toxin-Producing Escherichia coli Isolate of Serotype O111:H8

    PubMed Central

    Haenni, Marisa; Saras, Estelle; Auvray, Frédéric; Forest, Karine; Oswald, Eric; Madec, Jean-Yves

    2012-01-01

    We report the discovery of a CTX-M-15-producing Escherichia coli (STEC) of serogroup O111:H8, a major serotype responsible for human enterohemorrhagic Escherichia coli (EHEC) infections. In line with the recent CTX-M-15/O104:H4 E. coli outbreak, these data may reflect an accelerating spread of resistance to expanded-spectrum cephalosporins within the E. coli population, including STEC isolates. PMID:22156432

  13. Comparison of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Drinking Well Water and Pit Latrine Wastewater in a Rural Area of China

    PubMed Central

    Gao, Yanxia

    2016-01-01

    The present study was conducted to gain insights into the occurrence and characteristics of extended-spectrum beta-lactamase- (ESBL-) producing Escherichia coli (E. coli) from drinking well water in the rural area of Laiwu, China, and to explore the role of the nearby pit latrine as a contamination source. ESBL-producing E. coli from wells were compared with isolates from pit latrines in the vicinity. The results showed that ESBL-producing E. coli isolates, with the same antibiotic resistance profiles, ESBL genes, phylogenetic group, plasmid replicon types, and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) fingerprints, were isolated from well water and the nearby pit latrine in the same courtyard. Therefore, ESBL-producing E. coli in the pit latrine may be a likely contributor to the presence of ESBL-producing E. coli in rural well water. PMID:27965975

  14. Virulence markers of Shiga-like toxin-producing Escherichia coli strains originating from healthy domestic animals of different species.

    PubMed Central

    Beutin, L; Geier, D; Zimmermann, S; Karch, H

    1995-01-01

    Shiga-like toxin (verotoxin)-producing strains of Escherichia coli (SLTEC) originating from healthy cattle, sheep, goats, pigs, cats, and dogs were investigated for properties which are related to virulence of E. coli for humans. The slt-II (Shiga-like toxin II) and slt-IIc genes were frequent in SLTEC from healthy cattle and dogs but were rarely found in SLTEC from other animals. The slt-IIe gene was detected only in porcine SLTEC. SLTEC from goats and SLTEC from sheep were found to carry different SLT-II determinants which were not further characterized genetically. Sixty (28.8%) of 208 SLTEC from healthy animals showed diffuse adherence to HEp-2 cells. However, none of the strains was positive for genes specific for the local adherence (eaf), diffuse adherence (daa), or enteroaggregative (EAggEC) E. coli type. Only 3 (1.4%) of the 208 SLTEC were positive for attaching and effacing E. coli (eae) sequences. The enterohemolytic phenotype was present in 128 of the 208 SLTEC. Almost all enterohemolytic animal SLTEC were found to carry DNA sequences specific for the plasmid-encoded enterohemorrhagic E. coli hemolysin of E. coli O157. Bacteriophage-associated enterohemolysin (Ehly1 and Ehly2)-specific sequences were detected only in 14.4% of the 208 SLTEC and were linked with certain serotypes. The SLTEC from healthy animals constitute a very heterogeneous group of E. coli, and many of these strains appeared to be specific for their hosts. The absence of eae sequences in most animal SLTEC could indicate that these strains are less virulent for humans than the classical eae-positive enterohemorrhagic E. coli types. PMID:7538509

  15. Effect of curli expression and hydrophobicity of E. coli O157:H7 on attachment to fresh produce surfaces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aim: To investigate the effect of curli expression on cell hydrophobicity, biofilm formation, and attachment to cut and intact fresh produce surfaces. Methods and Results: Five E. coli O157:H7 strains were evaluated for curli expression, hydrophobicity, biofilm formation, and attachment of E. co...

  16. Virulence Factors and Phenotypical Traits of Verotoxin-Producing Escherichia coli Strains Isolated from Asymptomatic Human Carriers

    PubMed Central

    Stephan, R.; Untermann, F.

    1999-01-01

    Fourteen verotoxin-producing Escherichia coli strains isolated from stool samples of 14 different asymptomatic human carriers were further characterized. A variety of serotypes was found, but none of the strains belonged to serogroup O157. Only one isolate carried most of the virulence genes that are associated with increased pathogenicity. PMID:10203524

  17. Thermal inactivation of Shiga toxin-producing Escherichia coli within cubed beef steaks following cooking on a griddle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to quantify thermal inactivation of Shiga toxin-producing Escherichia coli (STEC) cells within knitted/cubed beef steaks following cooking on a non-stick griddle. Both faces of each beef cutlet (ca. 64 g; ca. 8.5 cm L X 10.5 cm W X 0.75 cm H) were surface inoculated (...

  18. Characterization of Shiga toxin-producing Escherichia coli associated with two multi-state foodborne outbreaks in 2006

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the fall of 2006 two multi-state outbreaks of E. coli serotype O157:H7 infection occurred that involved contaminated spinach and contaminated lettuce. In this study, we compare 7 Shiga toxin-producing isolates associated with those two outbreaks to a collection of food, environmental, and animal ...

  19. Virulence gene profiles of shiga toxin-producing Escherichia coli isolated from fecal samples of finishing swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) are important pathogens responsible for food-borne outbreaks and serious illness including hemorrhagic colitis and hemolytic uremic syndrome. Certain STEC serogroups may cause edema disease in swine; and similar to cattle, swine have been shown to be a ...

  20. Genotypic Characterization of Shiga Toxin-Producing Escherichia coli (STEC) Strains Recovered from Farm Animal Feces in Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Technical Abstract and Interpretive Summary: Provide electronically in Word. Sixty-three strains of Shiga toxin-producing Escherichia coli (STEC) were recovered from farm animal feces in distinct regions in the Culiacan Valley, an important agricultural region in Mexico for horticultural crops that...

  1. Genome Sequences of 64 Non-O157:H7 Shiga Toxin-Producing Escherichia coli Strains

    PubMed Central

    Toro, Magaly; Cao, Guojie; Rump, Lydia; Nagaraja, T. G.; Meng, Jianghong

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) strains are human pathogens. Although >400 non-O157 serotypes have been involved in human disease, whole-genome sequencing information is missing for many serotypes. We sequenced 64 STEC strains comprising 38 serotypes, isolated from clinical sources, animals, and environmental samples, to improve the phylogenetic understanding of these important foodborne pathogens. PMID:26430026

  2. Classification of non-O157 shiga toxin-producing escherichia coli(STEC) serotypes with hyperspectral microscope imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. A conventional microbiological method for cell counting is laborious and needs long time for the results. Since ...

  3. Shiga toxin-producing E. coli (STEC) in swine: prevalence over the finishing period and characterization of the STEC isolates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This descriptive longitudinal study was conducted to investigate the fecal shedding of Shiga toxin-producing E. coli (STEC) in finishing swine and to characterize the swine STEC isolates that were recovered. Three cohorts of finishing swine (n=50/cohort; total 150 pigs) were included in the longitu...

  4. Rapid detection of E. coli produced shiga-like toxins by lateral flow immunoassay in multiple food matrices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxigenic E. coli (STEC) produce shiga-like toxins (Stx) that can cause human disease and death. The STEC serotype O157:H7 is a well-recognized foodborne contaminant and effective detection methods have been established. However, the emergence of non-O157 STEC strains has necessitated the deve...

  5. Molecular characterization of shiga toxin-producing E. coli (STEC) from finishing swine in a longitudinal study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing E. coli (STEC) infections are a critical public health concern because they can cause severe clinical outcomes, such as hemolytic uremic syndrome, in humans. Determining the presence or absence of virulence genes is essential in assessing the potential pathogenicity of STEC str...

  6. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibody array was developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli O serogroups. Sensitivity of the array was 10**5 CFU, and the limit of detection of serogroups in ground beef was 1-10 CFU following 12 h of enrichment. The array utilized a minimal amount...

  7. Shiga toxin-producing Escherichia coli in meat: a preliminary simulation study on detection capabilities for three sampling methods

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this simulation study is to determine which sampling method (Cozzini core sampler, core drill shaving, and N-60 surface excision) will better detect Shiga Toxin-producing Escherichia coli (STEC) at varying levels of contamination when present in the meat. 1000 simulated experiments...

  8. Rapid latex particle agglutination test for Escherichia coli strains of porcine origin producing heat-labile enterotoxin.

    PubMed Central

    Finkelstein, R A; Yang, Z S; Moseley, S L; Moon, H W

    1983-01-01

    A latex particle agglutination test previously shown to be suitable for the rapid identification of Escherichia coli strains of human origin producing heat-labile enterotoxin (R. A. Finkelstein and Z. Yang, J. Clin. Microbiol. 18:23-28) is equally applicable to strains of porcine origin. PMID:6361056

  9. Draft Genome Sequence of Five Shiga Toxin-Producing Escherichia coli Strains Isolated from Wild Deer in Japan

    PubMed Central

    Ikeda, Tetsuya; Yamamoto, Shiori; Kabeya, Hidenori; Sugiyama, Hiromu; Takai, Shinji

    2017-01-01

    ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is one of the major foodborne pathogens. Having observed the wide distribution of this pathogen in wild deer, we report here the draft genome sequence of five STEC strains isolated from wild deer (Cervus nippon yesoensis) in Hokkaido, Japan. PMID:28254967

  10. Distribution and detection of Shiga toxin-producing Escherichia coli (STEC) during an industrial grinding process of beef trim

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During the grinding and packaging processes, it is important to understand how Shiga toxin-producing Escherichia coli (STEC) would be distributed and how well it could be detected in beef trim. This study is important because it shows what would happen if contaminated meat is allowed into a commerc...

  11. Biotinylation of environmentally isolated Shiga toxin-producing Escherichia coli (STEC) – specific bacteriophages for biosensor and biocontrol applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Like common bacteriophages, Shiga toxin-producing Escherichia coli (STEC) bacteriophages are viruses that recognize and bind to specific bacterial host (STEC) for propagation. They co-exist with STEC hosts, which cause epidemic food and waterborne illnesses, but may act as host populations limiting ...

  12. Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by gold nanoparticle optical sensing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga-toxin producing Escherichia coli (STEC) strains (“Big Six” – O26, O45, O103, O111, O121, O145, and O157) represent significant groups of pathogens responsible for foodborne diseases. The objective of this study was to develop a colorimetric optical sensing assay that can simultaneously detect ...

  13. Multidrug-Resistant and Extended Spectrum Beta-Lactamase-Producing Escherichia coli in Dutch Surface Water and Wastewater

    PubMed Central

    Blaak, Hetty; Lynch, Gretta; Italiaander, Ronald; Hamidjaja, Raditijo A.; Schets, Franciska M.; de Roda Husman, Ana Maria

    2015-01-01

    Objective The goal of the current study was to gain insight into the prevalence and concentrations of antimicrobial resistant (AMR) Escherichia coli in Dutch surface water, and to explore the role of wastewater as AMR contamination source. Methods The prevalence of AMR E. coli was determined in 113 surface water samples obtained from 30 different water bodies, and in 33 wastewater samples obtained at five health care institutions (HCIs), seven municipal wastewater treatment plants (mWWTPs), and an airport WWTP. Overall, 846 surface water and 313 wastewater E. coli isolates were analysed with respect to susceptibility to eight antimicrobials (representing seven different classes): ampicillin, cefotaxime, tetracycline, ciprofloxacin, streptomycin, sulfamethoxazole, trimethoprim, and chloramphenicol. Results Among surface water isolates, 26% were resistant to at least one class of antimicrobials, and 11% were multidrug-resistant (MDR). In wastewater, the proportions of AMR/MDR E. coli were 76%/62% at HCIs, 69%/19% at the airport WWTP, and 37%/27% and 31%/20% in mWWTP influents and effluents, respectively. Median concentrations of MDR E. coli were 2.2×102, 4.0×104, 1.8×107, and 4.1×107 cfu/l in surface water, WWTP effluents, WWTP influents and HCI wastewater, respectively. The different resistance types occurred with similar frequencies among E. coli from surface water and E. coli from municipal wastewater. By contrast, among E. coli from HCI wastewater, resistance to cefotaxime and resistance to ciprofloxacin were significantly overrepresented compared to E. coli from municipal wastewater and surface water. Most cefotaxime-resistant E. coliisolates produced ESBL. In two of the mWWTP, ESBL-producing variants were detected that were identical with respect to phylogenetic group, sequence type, AMR-profile, and ESBL-genotype to variants from HCI wastewater discharged onto the same sewer and sampled on the same day (A1/ST23/CTX-M-1, B23/ST131/CTX-M-15, D2/ST405/CTX

  14. Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers.

    PubMed

    Murinda, Shelton E; Nguyen, Lien T; Landers, Tippi L; Draughon, F Ann; Mathew, Alan G; Hogan, Joseph S; Smith, K Larry; Hancock, Dale D; Oliver, Stephen P

    2004-01-01

    The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between

  15. Inactivation of Shiga toxin-producing Escherichia coli O104:H4 using cold atmospheric pressure plasma.

    PubMed

    Baier, Matthias; Janssen, Traute; Wieler, Lothar H; Ehlbeck, Jörg; Knorr, Dietrich; Schlüter, Oliver

    2015-09-01

    From cultivation to the end of the post-harvest chain, heat-sensitive fresh produce is exposed to a variety of sources of pathogenic microorganisms. If contaminated, effective gentle means of sanitation are necessary to reduce bacterial pathogen load below their infective dose. The occurrence of rare or new serotypes raises the question of their tenacity to inactivation processes. In this study the antibacterial efficiency of cold plasma by an atmospheric pressure plasma-jet was examined against the Shiga toxin-producing outbreak strain Escherichia coli O104:H4. Argon was transformed into non-thermal plasma at a power input of 8 W and a gas flow of 5 L min(-1). Basic tests were performed on polysaccharide gel discs, including the more common E. coli O157:H7 and non-pathogenic E. coli DSM 1116. At 5 mm treatment distance and 10(5) cfu cm(-2) initial bacterial count, plasma reduced E. coli O104:H4 after 60 s by 4.6 ± 0.6 log, E. coli O157:H7 after 45 s by 4.5 ± 0.6 log, and E. coli DSM 1116 after 30 s by 4.4 ± 1.1 log. On the surface of corn salad leaves, gentle plasma application at 17 mm reduced 10(4) cfu cm(-2) of E. coli O104:H4 by 3.3 ± 1.1 log after 2 min, whereas E. coli O157:H7 was inactivated by 3.2 ± 1.1 log after 60 s. In conclusion, plasma treatment has the potential to reduce pathogens such as E. coli O104:H4 on the surface of fresh produce. However, a serotype-specific adaptation of the process parameters is required.

  16. Development of a faster method for detection of Shiga toxin producing E. coli using Tetrahymena thermophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While most STEC outbreaks are caused by E. coli O157, non-O157 STECs are increasingly being implicated. Selective agar for E. coli O157 is commercially available but none detect non-O157 STEC. Currently, regulatory agencies screen for non-O157 STECs by enriching foods overnight, spreading aliquots o...

  17. Development of biphasic medium for detection of Shiga toxin producing E. coli using Tetrahymena thermophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E. coli O157 has long been the leading cause of major foodborne STEC outbreaks but recently non-O157 STECs are increasingly implicated. Selective media for E. coli O157 are commercially available but none detect non-O157 STEC. Currently, regulatory agencies screen for non-O157 STECs by enriching foo...

  18. Persistence of Escherichia coli O157:H7 in major leafy green producing soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Persistence of Escherichia coli O157:H7 in 32 (16 organically managed and 16 conventionally managed) soils from California (CA) and Arizona (AZ) was investigated. Results showed that the longest survival (ttd, time needed to reach detection limit, 100 CFU/g dry soil) of E. coli O157:H7 was observed ...

  19. Comparative Genome Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Sequence Type 131 Strains from Nepal and Japan

    PubMed Central

    Miyoshi-Akiyama, Tohru; Sherchan, Jatan Bahadur; Doi, Yohei; Nagamatsu, Maki; Sherchand, Jeevan B.; Tandukar, Sarmila; Ohmagari, Norio; Kirikae, Teruo; Ohara, Hiroshi

    2016-01-01

    ABSTRACT The global spread of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) has largely been driven by the pandemic sequence type 131 (ST131). This study aimed to determine the molecular epidemiology of their spread in two Asian countries with contrasting prevalence. We conducted whole-genome sequencing (WGS) of ESBL-E. coli ST131 strains collected prospectively from Nepal and Japan, two countries in Asia with a high and low prevalence of ESBL-E. coli, respectively. We also systematically compared these genomes with those reported from other regions using publicly available WGS data for E. coli ST131 strains. Further, we conducted phylogenetic analysis of these isolates and all genome sequence data for ST131 strains to determine sequence diversity. One hundred five unique ESBL-E. coli isolates from Nepal (February 2013 to July 2013) and 76 isolates from Japan (October 2013 to September 2014) were included. Of these isolates, 54 (51%) isolates from Nepal and 11 (14%) isolates from Japan were identified as ST131 by WGS. Phylogenetic analysis based on WGS suggested that the majority of ESBL-E. coli ST131 isolates from Nepal clustered together, whereas those from Japan were more diverse. Half of the ESBL-E. coli ST131 isolates from Japan belonged to virotype C, whereas half of the isolates from Nepal belonged to a virotype other than virotype A, B, C, D, or E (A/B/C/D/E). The dominant sublineage of E. coli ST131 was H30Rx, which was most prominent in ESBL-E. coli ST131 isolates from Nepal. Our results revealed distinct phylogenetic characteristics of ESBL-E. coli ST131 spread in the two geographical areas of Asia, indicating the involvement of multiple factors in its local spread in each region. IMPORTANCE The global spread of ESBL-E. coli has been driven in large part by pandemic sequence type 131 (ST131). A recent study suggested that, within E. coli ST131, certain sublineages have disseminated worldwide with little association

  20. Occurrence of generic E. coli, E. coli O157:H7 and Salmonella spp. in water and sediment from leafy green produce farms and streams on the Central California coast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Irrigation with water of poor microbiological quality can elevate levels of bacteria on produce. This study aimed to identify climate and management covariates associated with generic E. coli in irrigation water on leafy green produce farms and to measure the prevalence of E. coli O157:H7 and Salmon...

  1. Antibiotic resistance and integrons in Shiga toxin-producing Escherichia coli (STEC)

    PubMed Central

    Colello, Rocío; Etcheverría, Analía I.; Conza, Jose A. Di; Gutkind, Gabriel O.; Padola, Nora L.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome in humans (HUS). Cattle are the main reservoir of STEC and transmission to humans occurs through contaminated food and water. Antibiotics are used in pig production systems to combat disease and improve productivity and play a key role in the dissemination of antibiotic resistance genes to the bacteria. Integrons have been identified in resistant bacteria allowing for the acquisition and dissemination of antibiotic resistance genes. STEC strains isolated from humans and animals have developed antibiotic resistance. In our laboratory, 21 non-157 STEC strains isolated from pigs were analyzed to detect class 1 and 2 integrons by PCR. Eight carried integrons, 7 of them harbored intl2. In another study 545 STEC strains were also analyzed for the presence of intl1 and intl2 . Strains carrying intl1 belonged to isolates from environment (n = 1), chicken hamburger (n = 2), dairy calves (n = 4) and pigs (n = 8). Two strains isolated from pigs harbored intl2 and only one intl1 / intl2 , highlighting the presence of intl2 in pigs. The selection for multiresistant strains may contribute to the emergence of antibiotic resistant pathogens and facilitate the spreading of the mobile resistance elements to other bacteria. PMID:26221083

  2. Shiga toxin-producing Escherichia coli (STEC) O157 outbreak, The Netherlands, September-October 2005.

    PubMed

    Doorduyn, Y; de Jager, C M; van der Zwaluw, W K; Friesema, I H M; Heuvelink, A E; de Boer, E; Wannet, W J B; van Duynhoven, Y T H P

    2006-07-01

    In September 2005, the first national food-related outbreak of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157 was investigated in the Netherlands. A total of 21 laboratory-confirmed cases (including one secondary case), and another 11 probable cases (two primary and nine secondary cases) were reported in patients who became ill between 11 September and 10 October 2005. Preliminary investigation suggested consumption of a raw beef product, steak tartare (in the Netherlands also known as "filet americain"), and contact with other symptomatic persons as possible risk factors. A subsequent case-control study supported the hypothesis that steak tartare was the source of the outbreak (matched odds ratio (OR) 272, 95% confidence interval (CI) 3-23,211). Consumption of ready-to-eat vegetables was also associated with STEC O157 infection (matched OR 24, 95% CI 1.1-528), but was considered a less likely source, as only 40% of the cases were exposed. Samples of steak tartare collected from one chain of supermarkets where it is likely that most patients (67%) bought steak tartare, all tested negative for STEC O157. However, sampling was done three days after the date of symptom onset of the last reported case. Since 88% of the cases became ill within a two week period, point source contamination may explain these negative results. It is concluded that steak tartare was the most likely cause of the first national food-related outbreak of STEC O157 in the Netherlands.

  3. Systemic effects of Subtilase cytotoxin produced by Escherichia coli O113:H21.

    PubMed

    Seyahian, E Abril; Oltra, Gisela; Ochoa, Federico; Melendi, Santiago; Hermes, Ricardo; Paton, James C; Paton, Adrienne W; Lago, Nestor; Castro Parodi, Mauricio; Damiano, Alicia; Ibarra, Cristina; Zotta, Elsa

    2017-03-01

    Subtilase cytotoxin (SubAB) is a member of the AB5 cytotoxin family and is produced by certain strains of Shiga toxigenic Escherichia coli. The toxin is known to be lethal to mice, but the pathological mechanisms that contribute to Uremic Hemolytic Syndrome (HUS) are poorly understood. In this study we show that intraperitoneal injection of a sublethal dose of SubAB in rats triggers a systemic response, with ascitic fluid accumulation, heart hypertrophy and damage to the liver, colon and kidney. SubAB treated rats presented microalbuminuria 20 days post inoculation. At this time we found disruption of the glomerular filtration barrier and alteration of the protein reabsorption mechanisms of the proximal tubule. In the kidney, SubAB also triggered an epithelial to mesenchymal transition (Wuyts et al., 1996). These findings indicate that apart from direct cytotoxic effects on renal tissues, SubAB causes significant damage to the other organs, with potential consequences for HUS pathogenesis.

  4. Risk Factors for Shiga Toxin-Producing Escherichia coli-Associated Human Diseases.

    PubMed

    Rivas, Marta; Chinen, Isabel; Miliwebsky, Elizabeth; Masana, Marcelo

    2014-10-01

    We have reviewed the risk factors for the occurrence of Shiga toxin-producing Escherichia coli (STEC)-associated human diseases. The analysis of STEC surveillance data and trends shows differences in frequency and severity of the illnesses across countries, whereas the economic and social costs for the affected families, the community, and the health system are better estimated in developed countries. The occurrence of STEC infections is determined by the interaction of the pathogen, the reservoirs, and the biological, cultural, and behavioral aspects of the host. The main risk factors identified in earlier case-control and population-based studies were dietary behaviors and beef consumption. However, in recent years, other risky exposures have also emerged, like the consumption of raw vegetables and sprouts, working or camping in rural areas, visiting farms, and person-to-person transmission. Epidemiological changes have also been determined by the intensification of cattle production, the increase in centralized food production and distribution, and the growth in the volume of international trade of foods. The main lessons learned from recent large outbreaks are knowledge of virulence determinants of new pathogenic strains, recognition of new vehicles of infection, development of new methodologies for detecting STEC in foods and humans, improvement in food regulations and hygiene guidelines, new therapeutic approaches in the treatment of infected patients, establishment of continuous educational programs for food consumers, and enhanced cooperation and teamwork of regional and international networks.

  5. Classification of Shiga toxin-producing escherichia coli (STEC) serotypes with hyperspectral microscope imagery

    NASA Astrophysics Data System (ADS)

    Park, Bosoon; Windham, William R.; Ladely, Scott R.; Gurram, Prudhvi; Kwon, Heesung; Yoon, Seung-Chul; Lawrence, Kurt C.; Narang, Neelam; Cray, William C.

    2012-05-01

    Non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) strains such as O26, O45, O103, O111, O121 and O145 are recognized as serious outbreak to cause human illness due to their toxicity. A conventional microbiological method for cell counting is laborious and needs long time for the results. Since optical detection method is promising for realtime, in-situ foodborne pathogen detection, acousto-optical tunable filters (AOTF)-based hyperspectral microscopic imaging (HMI) method has been developed for identifying pathogenic bacteria because of its capability to differentiate both spatial and spectral characteristics of each bacterial cell from microcolony samples. Using the AOTF-based HMI method, 89 contiguous spectral images could be acquired within approximately 30 seconds with 250 ms exposure time. From this study, we have successfully developed the protocol for live-cell immobilization on glass slides to acquire quality spectral images from STEC bacterial cells using the modified dry method. Among the contiguous spectral imagery between 450 and 800 nm, the intensity of spectral images at 458, 498, 522, 546, 570, 586, 670 and 690 nm were distinctive for STEC bacteria. With two different classification algorithms, Support Vector Machine (SVM) and Sparse Kernel-based Ensemble Learning (SKEL), a STEC serotype O45 could be classified with 92% detection accuracy.

  6. Improved traceability of Shiga-toxin-producing Escherichia coli using CRISPRs for detection and typing.

    PubMed

    Delannoy, Sabine; Beutin, Lothar; Fach, Patrick

    2016-05-01

    Among strains of Shiga-toxin-producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are frequently associated with severe clinical illness in humans. The development of methods for their reliable detection from complex samples such as food has been challenging thus far, and is currently based on the PCR detection of the major virulence genes stx1, stx2, and eae, and O-serogroup-specific genes. However, this approach lacks resolution. Moreover, new STEC serotypes are continuously emerging worldwide. For example, in May 2011, strains belonging to the hitherto rarely detected STEC serotype O104:H4 were identified as causative agents of one of the world's largest outbreak of disease with a high incidence of hemorrhagic colitis and hemolytic uremic syndrome in the infected patients. Discriminant typing of pathogens is crucial for epidemiological surveillance and investigations of outbreaks, and especially for tracking and tracing in case of accidental and deliberate contamination of food and water samples. Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of short, highly conserved DNA repeats separated by unique sequences of similar length. This distinctive sequence signature of CRISPRs can be used for strain typing in several bacterial species including STEC. This review discusses how CRISPRs have recently been used for STEC identification and typing.

  7. Survival of Shiga toxin-producing Escherichia coli and Stx bacteriophages in moisture enhanced beef.

    PubMed

    Langsrud, Solveig; Heir, Even; Rode, Tone Mari

    2014-07-01

    Moisture enhancement of meat through injection is a technology to improve the sensory properties and the weight of meat. However, the technology may increase the risk of food borne infections. Shiga toxin-producing Escherichia coli (STEC) or bacteriophages carrying cytotoxin genes (Shiga toxin genes, stx), which is normally only present on the surface of intact beef, may be transferred to the inner parts of the muscle during the injection process. Pathogens and bacteriophages surviving the storage period may not be eliminated in the cooking process since many consumers prefer undercooked beef. Measures to increase the microbial food safety of moisture enhanced beef may include sterilization or washing of the outer surface of the meat before injection, avoiding recycling of marinade and addition of antimicrobial agents to the marinade. This paper reviews the literature regarding microbial safety of moisture enhanced beef with special emphasis on STEC and Stx bacteriophages. Also, results from a European Union research project, ProSafeBeef (Food-CT-16 2006-36241) are presented.

  8. Peri- and Postharvest Factors in the Control of Shiga Toxin-Producing Escherichia coli in Beef.

    PubMed

    Moxley, Rodney A; Acuff, Gary R

    2014-12-01

    Certain Shiga toxin-producing Escherichia coli (STEC) strains are important causes of food-borne disease, with hemorrhagic colitis and, in some cases, hemolytic-uremic syndrome as the clinical manifestations of illness. Six serogroups and one serotype of STEC (O26, O45, O103, O111, O121, O145, and O157:H7) are responsible for the vast majority of cases in the United States. Based on recent data for all food commodities combined, 55.3% and 50.0% of the outbreaks of STEC O157 and non-O157 in the United States, respectively, are attributable to beef as a food source. Consequently, the U.S. Department of Agriculture, Food Safety and Inspection Service declared these organisms as adulterants in raw, nonintact beef. In North America, cattle are a major reservoir of STEC strains, with organisms shed in the feces and contaminated hides of the animals being the main vehicle for spread to carcasses at slaughter. A number of peri- and postharvest interventions targeting STEC have been developed, and significant progress has been made in improving the microbiological quality of beef in the past 20 years as a result. However, continued improvements are needed, and accurate assessment of these interventions, especially for non-O157 STEC, would greatly benefit from improvements in detection methods for these organisms.

  9. Antibiotic resistance and integrons in Shiga toxin-producing Escherichia coli (STEC).

    PubMed

    Colello, Rocío; Etcheverría, Analía I; Di Conza, Jose A; Gutkind, Gabriel O; Padola, Nora L

    2015-03-01

    Shiga toxin-producing Escherichia coli (STEC) cause hemorrhagic colitis (HC) and hemolytic-uremic syndrome in humans (HUS). Cattle are the main reservoir of STEC and transmission to humans occurs through contaminated food and water. Antibiotics are used in pig production systems to combat disease and improve productivity and play a key role in the dissemination of antibiotic resistance genes to the bacteria. Integrons have been identified in resistant bacteria allowing for the acquisition and dissemination of antibiotic resistance genes. STEC strains isolated from humans and animals have developed antibiotic resistance. In our laboratory, 21 non-157 STEC strains isolated from pigs were analyzed to detect class 1 and 2 integrons by PCR. Eight carried integrons, 7 of them harbored intl2. In another study 545 STEC strains were also analyzed for the presence of intl1 and intl2 . Strains carrying intl1 belonged to isolates from environment (n = 1), chicken hamburger (n = 2), dairy calves (n = 4) and pigs (n = 8). Two strains isolated from pigs harbored intl2 and only one intl1 / intl2 , highlighting the presence of intl2 in pigs. The selection for multiresistant strains may contribute to the emergence of antibiotic resistant pathogens and facilitate the spreading of the mobile resistance elements to other bacteria.

  10. Faecal shedding of CTX-M-producing Escherichia coli in horses receiving broad-spectrum antimicrobial prophylaxis after hospital admission.

    PubMed

    Damborg, Peter; Marskar, Peter; Baptiste, Keith E; Guardabassi, Luca

    2012-01-27

    The objective of this longitudinal study was to investigate the occurrence and genetic background of faecal Escherichia coli resistant to cefotaxime (CTX) in horses receiving broad-spectrum antimicrobial prophylaxis after admission to a veterinary teaching hospital. The ten horses enrolled in the study were treated with cefquinome either alone (n=4) or in combination with metronidazole (n=3) or other antimicrobial agents (n=3). CTX-resistant coliforms in faeces collected before, during and after treatment were quantified on selective MacConkey agar supplemented with CTX, and a colony isolated randomly from each positive sample was characterized by pulsed-field gel electrophoresis, and by PCR detection and sequencing of bla(TEM), bla(SHV), bla(CTX-M) and bla(CMY). All horses were negative for CTX-resistant coliforms at admission but became positive within the first three days of treatment. The average faecal densities of CTX-resistant coliforms increased significantly following antimicrobial prophylaxis (P<0.001). Genetic characterization of 29 faecal isolates revealed that this effect was due to proliferation of E. coli producing either CTX-M-1 (n=28) or CTX-M-14 (n=1). Five CTX-M-1 isolates produced additional β-lactamases (TEM-1, CMY-34 and the novel variant CMY-53). Shedding of CTX-M-producing E. coli appeared intermittent in four horses and persisted two weeks after antimicrobial treatments in five of six patients tested after discharge from hospital. Nosocomial transmission was suggested by finding five identical CTX-M-1-producing E. coli pulsotypes in multiple horses. The originality of the study lies in the unanticipated high frequency and genetic diversity of CTX-M-producing E. coli observed in the faecal flora of hospitalized patients receiving broad-spectrum antimicrobial prophylaxis.

  11. Clinical isolates of uropathogenic Escherichia coli ST131 producing NDM-7 metallo-β-lactamase in China.

    PubMed

    Wang, Lian-Hui; Liu, Pan-Pan; Wei, Dan-Dan; Liu, Yang; Wan, La-Gen; Xiang, Tian-Xin; Zhang, Yu-Juan

    2016-07-01

    Here we report five cases of NDM-7-producing Escherichia coli from patients with bacteriuria in a teaching hospital in mainland China. Two isolates belonged to sequence type 131 (ST131), simultaneously carrying blaCTX-M-15, blaSHV-11, blaTEM-1 and qnrS1. The blaNDM-7 gene was located on a conjugative IncX3-type plasmid bearing blaTEM-1 and qnrS1. These findings indicate the spread of NDM-7 metallo-β-lactamase in a highly resistant and virulent E. coli sequence type in China.

  12. Contribution of urease to colonization by Shiga toxin-producing Escherichia coli.

    PubMed

    Steyert, Susan R; Kaper, James B

    2012-08-01

    Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen with a low infectious dose that colonizes the colon in humans and can cause severe clinical manifestations such as hemolytic-uremic syndrome. The urease enzyme, encoded in the STEC chromosome, has been demonstrated to act as a virulence factor in other bacterial pathogens. The NH(3) produced as urease hydrolyzes urea can aid in buffering bacteria in acidic environments as well as provide an easily assimilated source of nitrogen that bacteria can use to gain a metabolic advantage over intact microflora. Here, we explore the role of urease in STEC pathogenicity. The STEC urease enzyme exhibited maximum activity near neutral pH and during the stationary-growth phase. Experiments altering growth conditions performed with three phylogenetically distinct urease-positive strains demonstrated that the STEC ure gene cluster is inducible by neither urea nor pH but does respond to nitrogen availability. Quantitative reverse transcription-PCR (qRT-PCR) data indicate that nitrogen inhibits the transcriptional response. The deletion of the ure gene locus was constructed in STEC strain 88-0643, and the ure mutant was used with the wild-type strain in competition experiments in mouse models to examine the contribution of urease. The wild-type strain was twice as likely to survive passage through the acidic stomach and demonstrated an enhanced ability to colonize the intestinal tract compared to the ure mutant strain. These in vivo experiments reveal that, although the benefit STEC gains from urease expression is modest and not absolutely required for colonization, urease can contribute to the pathogenicity of STEC.

  13. Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5′ Nuclease PCR Assay

    PubMed Central

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization of non-O157 VTEC isolates are essential for improving our knowledge of these organisms. In the present study, we detected VTEC isolates in bovine fecal samples by a duplex 5′ nuclease PCR assay (real-time PCR) that targets vtx1 and vtx2. VTEC isolates were obtained by colony replication by use of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5′ nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays included assays for the detection of verocytotoxin genes (vtx1, vtx2), pO157-associated genes (ehxA, katP, espP, and etpD), a recently identified adhesin (saa), intimin (eae, all variants), seven subtypes of eae, four subtypes of tir, and three subtypes of espD. A number of reference strains (VTEC and enteropathogenic E. coli strains) and VTEC strains isolated from calves were tested to validate the PCR assays. The expected virulence profiles were detected for all reference strains. In addition, new information on the subtypes of LEE genes was obtained. For reference strains as well as bovine isolates, a consistent relationship between subtypes of the LEE genes was found, so that a total of seven different combinations of these were recognized (corresponding to the seven subtypes of eae). Isolates with 15 different serogroup-virulence profiles were isolated from 16 calves. Among these, 53% harbored LEE and 73% harbored factors carried by the large virulence plasmid. One LEE-negative isolate had the gene for the adhesin Saa. The most common virulence profile among the bovine

  14. Sequential necrotizing fasciitis caused by the monomicrobial pathogens Streptococcus equisimilis and extended-spectrum beta-lactamase-producing Escherichia coli.

    PubMed

    Endo, Akiko; Matsuoka, Ryosuke; Mizuno, Yasushi; Doi, Asako; Nishioka, Hiroaki

    2016-08-01

    Necrotizing fasciitis is a rapidly progressing bacterial infection of the superficial fascia and subcutaneous tissue that is associated with a high mortality rate and is caused by a single species of bacteria or polymicrobial organisms. Escherichia coli is rarely isolated from patients with monomicrobial disease. Further, there are few reports of extended-spectrum beta-lactamase (ESBL)-producing E. coli associated with necrotizing fasciitis. We report here our treatment of an 85-year-old man who was admitted because of necrotizing fasciitis of his right thigh. Streptococcus equisimilis was detected as a monomicrobial pathogen, and the infection was cured by amputation of the patient's right leg and the administration of antibiotics. However, 5 days after discontinuing antibiotic therapy, he developed necrotizing fasciitis on his right upper limb and died. ESBL-producing E. coli was the only bacterial species isolated from blood and skin cultures. This case demonstrates that ESBL-producing E. coli can cause monomicrobial necrotizing fasciitis, particularly during hospitalization and that a different bacterial species can cause disease shortly after a previous episode.

  15. Biodiesel production from different algal oil using immobilized pure lipase and tailor made rPichia pastoris with Cal A and Cal B genes.

    PubMed

    Bharathiraja, B; Ranjith Kumar, R; PraveenKumar, R; Chakravarthy, M; Yogendran, D; Jayamuthunagai, J

    2016-08-01

    In this investigation, oil extraction was performed in marine macroalgae Gracilaria edulis, Enteromorpha compressa and Ulva lactuca. The algal biomass was characterized by Scanning Electron Microscopy and Fourier Transform-Infra Red Spectroscopy. Six different pre-treatment methods were carried out to evaluate the best method for maximum oil extraction. Optimization of extraction parameters were performed and high oil yield was obtained at temperature 55°C, time 150min, particle size 0.10mm, solvent-to-solid ratio 6:1 and agitation rate 500rpm. After optimization, 9.5%, 12.18% and 10.50 (g/g) of oil extraction yield was achieved from the respective algal biomass. The rate constant for extraction was obtained as first order kinetics, by differential method. Stable intracellular Cal A and Cal B lipase producing recombinant Pichia pastoris was constructed and used as biocatalyst for biodiesel production. Comparative analysis of lipase activity and biodiesel yield was made with immobilized Candida antarctica lipase.

  16. Escherichia coli common pilus (ECP) targets arabinosyl residues in plant cell walls to mediate adhesion to fresh produce plants.

    PubMed

    Rossez, Yannick; Holmes, Ashleigh; Lodberg-Pedersen, Henriette; Birse, Louise; Marshall, Jacqueline; Willats, William G T; Toth, Ian K; Holden, Nicola J

    2014-12-05

    Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

  17. Outbreak of verocytotoxin-producing E. coli O145 and O26 infections associated with the consumption of ice cream produced at a farm, Belgium, 2007.

    PubMed

    De Schrijver, K; Buvens, G; Possé, B; Van den Branden, D; Oosterlynck, O; De Zutter, L; Eilers, K; Piérard, D; Dierick, K; Van Damme-Lombaerts, R; Lauwers, C; Jacobs, R

    2008-02-14

    In October 2007, an outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145 and E. coli O26 occurred among consumers of ice cream produced and sold in September 2007 at a farm in the province of Antwerp (Belgium). The ice cream was consumed at two birthday parties and also eaten at the farm. Five children, aged between two and 11 years, developed haemolytic uraemic syndrome (HUS), and seven other co-exposed persons contracted severe diarrhoea. In three of the five HUS cases VTEC O145 infections were laboratory confirmed, one in association with VTEC O26. Identical isolates of E. coli O145 and O26 were detected with PCR and PFGE in faecal samples of patients and in ice cream leftovers from one of the birthday parties, in faecal samples taken from calves, and in samples of soiled straw from the farm at which the ice cream was produced. Ice cream was made from pasteurised milk and most likely contaminated by one of food handlers.

  18. HlyF Produced by Extraintestinal Pathogenic Escherichia coli Is a Virulence Factor That Regulates Outer Membrane Vesicle Biogenesis.

    PubMed

    Murase, Kazunori; Martin, Patricia; Porcheron, Gaëlle; Houle, Sébastien; Helloin, Emmanuelle; Pénary, Marie; Nougayrède, Jean-Philippe; Dozois, Charles M; Hayashi, Tetsuya; Oswald, Eric

    2016-03-01

    Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.

  19. Current status of extended spectrum β-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis in Okinawa prefecture, Japan.

    PubMed

    Nakama, Rika; Shingaki, Aoi; Miyazato, Hiroko; Higa, Rikako; Nagamoto, Chota; Hamamoto, Kouta; Ueda, Shuhei; Hachiman, Teruyuki; Touma, Yuki; Miyagi, Kazufumi; Kawahara, Ryuji; Toyosato, Takehiko; Hirai, Itaru

    2016-05-01

    Enterobacteriaceae producing extended spectrum β-lactamase (ESBL) are distributed worldwide. In this study, 114 ESBL-producing Enterobacteriaceae were isolated by analyzing 1672 clinical isolates of Enterobacteriaceae collected from an Okinawa prefectural hospital in Japan between June 2013 and July 2014. The overall prevalence of ESBL-producing Enterobacteriaceae was 6.8%; the prevalence of different bacterial species among the ESBL-producing isolates was as follows: 11.5% Escherichia coli (90 of 783 isolates), 6.2% Klebsiella pneumoniae (19 of 307 isolates), and 11.1% Proteus mirabilis (5 of 45 isolates). The ESBL types blaCTX-M-1, -3, -15, -2, -14, -27, and mutants of blaSHV-1 were detected. Among them, blaCTX-M-15 (33.3%), blaCTX-M-14 (27.8%) and blaCTX-M-27 (33.3%) were dominant in the E. coli isolates, whereas a blaSHV mutant which possessed four mutations (Tyr7Phe, Leu35Gln, Gly238Ser and Glu240Lys) in the amino acid sequence of SHV-1 dominated in the K. pneumoniae isolates (11 of 19, 57.9%). The pandemic E. coli ST131 clone was found to constitute 3.3% of the overall examined isolates and 62.2% of the ESBL-producing E. coli isolates. Our results suggest that the genetic combination of blaCTX-M, and blaSHV and antibiotics-resistant profile were different from that in other regions such as other areas of Japan, Asia, Europe, and North America, especially in the ESBL-producing K. pneumoniae isolates and in the E. coli B2-O25b-ST131 isolates possessing blaCTX-M-15 (40.7% of the E. coli B2-O25b-ST131 isolates). Taken together, our results indicate that the ESBL-producing Enterobacteriaceae in Okinawa, Japan, might be of a unique nature.

  20. Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli

    PubMed Central

    2013-01-01

    Background Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies. Results We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells. Conclusion A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification. PMID:23638724

  1. Prevalence of ESBL producing Escherichia Coli and Klebsiella species with their co-resistance pattern to antimicrobials.

    PubMed

    Biswas, T; Das, M; Mondal, R; Raj, H J; Mondal, S

    2013-04-01

    Extended spectrum β-lactamase producing bacteria are potential emerging pathogens and continue to be a major challenge in clinical setup worldwide. In the present study an attempt was made to study the prevalence of extended spectrum β-lactamase producing Escherichia coli and Klebsiella species from clinical isolates in a rural tertiary care hospital in West Bengal, India with their antimicrobial susceptibility as well as co-resistance pattern to different antimicrobials. A total of 179 Escherichia coli and 62 Klebsiella isolates recovered from various clinical samples of urine, pus, aural swabs and respiratory secretions (including sputum) for a period of six months were subjected to routine antimicrobial susceptibility testing and also tested for extended spectrum β-lactamase production as per NCCLS recommendations. Extended spectrum β-lactamase was detected in 32.40% of Escherichia coli and 40.32% of Klebsiella species isolates. Urine, pus and respiratory samples were common source of extended spectrum β-lactamase producers and resistance rate of these organisms to third generation cephalosporins were more than 30 to 40%. Co-resistance pattern of these extended spectrum β-lactamase producers to other commonly used antimicrobials were also statistically significant (p≤0.05). From the study it is concluded that indiscriminate use of third generation cephalosporins may be responsible for the selection of extended spectrum β-lactamase producing multidrug resistant strains in hospital setup and amikacin is a reliable drug against them.

  2. O-antigen and virulence profiling of Shiga toxin-producing Escherichia coli by a rapid and cost-effective DNA microarray colorimetric method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype Shiga toxin-producing Escherichia coli strains, the present study evaluated the use of the ampliPHOX colorimetric detection technology, based on ...

  3. First characterization of CTX-M-15-producing Escherichia coli ST131 and ST405 clones causing community-onset infections in South America.

    PubMed

    Ruiz, Sory J; Montealegre, Maria Camila; Ruiz-Garbajosa, Patricia; Correa, Adriana; Briceño, David F; Martinez, Ernesto; Rosso, Fernando; Muñoz, Martin; Quinn, John P; Cantón, Rafael; Villegas, Maria Virginia

    2011-05-01

    CTX-M-15-producing Escherichia coli has emerged worldwide as an important pathogen associated with community-onset infections, but in South America reports are scarce. We document the presence of CTX-M-15-producing E. coli of the international ST131 and ST405 clones in Colombia and present the first molecular characterization of these isolates in South America.

  4. Presence of ESBL/AmpC -Producing Escherichia coli in the Broiler Production Pyramid: A Descriptive Study

    PubMed Central

    Dierikx, Cindy M.; van der Goot, Jeanet A.; Smith, Hilde E.; Kant, Arie; Mevius, Dik J.

    2013-01-01

    Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one−/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0–24% to 96–100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms. PMID:24244401

  5. Presence of ESBL/AmpC-producing Escherichia coli in the broiler production pyramid: a descriptive study.

    PubMed

    Dierikx, Cindy M; van der Goot, Jeanet A; Smith, Hilde E; Kant, Arie; Mevius, Dik J

    2013-01-01

    Broilers and broiler meat products are highly contaminated with extended spectrum beta-lactamase (ESBL) or plasmid-mediated AmpC beta-lactamase producing Escherichia coli and are considered to be a source for human infections. Both horizontal and vertical transmission might play a role in the presence of these strains in broilers. As not much is known about the presence of these strains in the whole production pyramid, the epidemiology of ESBL/AmpC-producing E. coli in the Dutch broiler production pyramid was examined. Cloacal swabs of Grandparent stock (GPS) birds (one-/two-days (breed A and B), 18 and 31 weeks old (breed A)), one-day old Parent stock birds (breed A and B) and broiler chickens of increasing age (breed A) were selectively cultured to detect ESBL/AmpC-producing isolates. ESBL/AmpC-producing isolates were found at all levels in the broiler production pyramid in both broiler breeds examined. Prevalence was already relatively high at the top of the broiler production pyramid. At broiler farms ESBL/AmpC producing E. coli were still present in the environment of the poultry house after cleaning and disinfection. Feed samples taken in the poultry house also became contaminated with ESBL/AmpC producing E. coli after one or more production weeks. The prevalence of ESBL/AmpC-positive birds at broiler farms increased within the first week from 0-24% to 96-100% independent of the use of antibiotics and stayed 100% until slaughter. In GPS breed A, prevalence at 2 days, 18 weeks and 31 weeks stayed below 50% except when beta-lactam antibiotics were administered. In that case prevalence increased to 100%. Interventions minimizing ESBL/AmpC contamination in broilers should focus on preventing horizontal and vertical spread, especially in relation to broiler production farms.

  6. Presence of Multidrug-Resistant Shiga Toxin-Producing Escherichia coli, Enteropathogenic E. coli and Enterotoxigenic E. coli, on Raw Nopalitos (Opuntia ficus-indica L.) and in Nopalitos Salads from Local Retail Markets in Mexico.

    PubMed

    Gómez-Aldapa, Carlos A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Torres-Vitela, Mdel Refugio; Villarruel-López, Angelica; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

    2016-05-01

    The presence of multidrug-resistant pathogenic bacteria in food is a significant public health concern. Diarrheagenic Escherichia coli pathotypes (DEPs) are foodborne bacteria. In Mexico, DEPs have been associated with diarrheal illness. There is no information about the presence of multidrug-resistant DEPs on fresh vegetables and in cooked vegetable salads in Mexico. "Nopalitos" (Opuntia ficus-indica L.) is a Cactacea extensively used as a fresh green vegetable throughout Mexico. The presence of generic E. coli and multidrug-resistant DEPs on raw whole and cut nopalitos and in nopalitos salad samples was determined. One hundred raw whole nopalitos (without prickles) samples, 100 raw nopalitos cut into small square samples, and 100 cooked nopalitos salad samples were collected from markets. Generic E. coli was determined using the most probable number procedures. DEPs were identified using two multiplex polymerase chain reaction procedures. Susceptibility to 16 antibiotics was tested for the isolated DEP strains by standard test. Of the 100 whole nopalitos samples, 100 cut nopalitos samples, and 100 nopalitos salad samples, generic E. coli and DEPs were identified, respectively, in 80% and 10%, 74% and 10%, and 64% and 8%. Eighty-two DEP strains were isolated from positive nopalitos samples. The identified DEPs included Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). All isolated strains exhibited resistance to at least six antibiotics. To the best of our knowledge, this is the first report of the presence of multidrug-resistant and antibiotic resistance profiles of STEC, ETEC, and EPEC on raw nopalitos and in nopalitos salads in Mexico.

  7. Phage biocontrol of enteropathogenic and shiga toxin-producing Escherichia coli in meat products

    PubMed Central

    Tomat, David; Migliore, Leonel; Aquili, Virginia; Quiberoni, Andrea; Balagué, Claudia

    2013-01-01

    Ten bacteriophages were isolated from faeces and their lytic effects assayed on 103 pathogenic and non-pathogenic Enterobacteriaceae. Two phages (DT1 and DT6) were selected based on their host ranges, and their lytic effects on pathogenic E. coli strains inoculated on pieces of beef were determined. We evaluated the reductions of viable cells of Escherichia coli O157:H7 and non-O157 Shiga toxigenic E. coli strains on meat after exposure to DT6 at 5 and 24°C for 3, 6, and 24 h and the effect of both phages against an enteropathogenic E. coli strain. Significant viable cell reductions, compared to controls without phages, at both temperatures were observed, with the greatest decrease taking place within the first hours of the assays. Reductions were also influenced by phage concentration, being the highest concentrations, 1.7 × 1010 plaque forming units per milliliter (PFU/mL) for DT1 and 1.4 × 1010 PFU/mL for DT6, the most effective. When enteropathogenic E. coli and Shiga toxigenic E. coli (O157:H7) strains were tested, we obtained viable cell reductions of 0.67 log (p = 0.01) and 0.77 log (p = 0.01) after 3 h incubation and 0.80 log (p = 0.01) and 1.15 log (p = 0.001) after 6 h. In contrast, all nonpathogenic E. coli strains as well as other enterobacteria tested were resistant. In addition, phage cocktail was evaluated on two strains and further reductions were observed. However, E. coli bacteriophage insensitive mutants (BIMs) emerged in meat assays. BIMs isolated from meat along with those isolated by using the secondary culture method were tested to evaluate resistance phenotype stability and reversion. They presented low emergence frequencies (6.5 × 10−7–1.8 × 10−6) and variable stability and reversion. Results indicate that isolated phages were stable on storage, negative for all the virulence factors assayed, presented lytic activity for different E. coli virotypes and could be useful in reducing Shiga toxigenic E. coli and enteropathogenic E

  8. Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli.

    PubMed

    Lee, Jin-Hee; Jeong, Ji-Seon; Kim, Sook-Kyung; Song, Jimyeong; Lee, Ji Youn; Baek, Soyun; Choi, Jun-Hyuk

    2016-11-01

    Isotopically labeled proteins have been used as internal standards for mass spectrometry (MS)-based absolute protein quantification. Although this approach can provide highly accurate analyses of proteins of interest within a complex mixture, one of the major limitations of this method is the difficulty in preparing uniformly labeled standards. Human growth hormone (hGH) is one of the most important hormones that circulate throughout the body, and its measurement is primarily of interest in the diagnosis and treatment of growth disorders. In order to provide a useful internal standard for MS-based hGH measurement, we describe an efficient strategy to produce a potentially valuable, stable isotope-labeled hGH with high purity and yield. The strategy involves the following steps: solubilization of hGH under labeling conditions, detection of stable isotope incorporation, large-scale purification, analysis of the labeled protein, and assessment of the labeling efficiency. We show that the yield of soluble hGH under selective isotopic labeling conditions can be greatly increased by optimizing protein expression and extraction. Our efficient method for generating isotopically labeled hGH does not influence the structural integrity of hGH. Finally, we assessed the efficiency of stable isotope labeling at the intact protein level, and the result was further verified by amino acid analysis. These results clearly indicate that our labeling approach allows an almost complete incorporation of (13)C6(15)N4-arginine into the hGH expressed in E.coli without detectable isotope scrambling.

  9. Whole-Genome Sequencing for National Surveillance of Shiga Toxin–Producing Escherichia coli O157

    PubMed Central

    Dallman, Timothy J.; Byrne, Lisa; Ashton, Philip M.; Cowley, Lauren A.; Perry, Neil T.; Adak, Goutam; Petrovska, Liljana; Ellis, Richard J.; Elson, Richard; Underwood, Anthony; Green, Jonathan; Hanage, William P.; Jenkins, Claire; Grant, Kathie; Wain, John

    2015-01-01

    Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations. PMID:25888672

  10. Characterization and Molecular Subtyping of Shiga Toxin-Producing Escherichia coli Strains in Butcher Shops.

    PubMed

    Brusa, Victoria; Costa, Magdalena; Londero, Alejandra; Leotta, Gerardo A; Galli, Lucía

    2017-01-19

    Shiga toxin-producing Escherichia coli (STEC) are important emerging foodborne human pathogens. Ruminants are the main animal reservoir of STEC currently known, and meat can become contaminated at different stages of the production chain. The aim of this work was to subtype and establish the epidemiological relatedness of non-O157 STEC strains isolated from ground beef and the environment in butcher shops before (evaluation stage, 2010-2011 period) and after (verification stage, 2013) implementing improvement actions. Sixty-eight non-O157 STEC strains were tested for eae, saa, ehxA, iha, efa1, toxB, subAB, cdt-V, astA, aggR, and aaiC genes, and stx1 and stx2 variants were determined. Pulsed-field gel electrophoresis (PFGE) was carried out with XbaI and XmaJI. From the 68 strains, 92.6%, 75.0%, 58.8%, 53.5%, 10.3%, 7.3%, and 4.4% were positive for iha, ehxA, subAB, saa, cdt-V, astA, and eae, respectively. All strains were aggR/aaiC-negative. PFGE showed that 19 strains grouped in 9 clusters and 41 showed unique XbaI patterns. During the evaluation stage (2010-2011), we identified clonal strains in different samples, circulating clones in different butcher shops, and more than one different strain in the same butcher shop. The bovine origin of meat and its manufacturing process could not ensure the total absence of all non-O157 STEC serotypes in this foodstuff. Most strains isolated during the evaluation (2010-2011) and verification (2013) stages did not exhibit a genotypic profile associated with human disease. It is necessary to conduct periodic reviews of the new epidemiological information and verify that the analyses of non-O157 STEC in food are appropriate to identify strains affecting the population.

  11. Detection and characterization of Shiga toxin-producing Escherichia coli in captive non-domestic mammals.

    PubMed

    Leotta, Gerardo A; Deza, Natalia; Origlia, Javier; Toma, Claudia; Chinen, Isabel; Miliwebsky, Elizabeth; Iyoda, Sunao; Sosa-Estani, Sergio; Rivas, Marta

    2006-11-26

    Shiga toxin producing-Escherichia coli (STEC) is an important emerging pathogen, and ruminants are recognized as their main natural reservoir. The aim of this work was to establish the frequency of STEC in non-domestic mammals of the Zoo and Botanical Garden of La Plata City, Argentina, and to pheno-genotypically characterize STEC isolates. By polymerase chain reaction (PCR), Shiga toxin (stx) gene sequences were detected in 50.8% of 65 fecal samples. Twenty-five STEC strains were isolated from 38.5% of the Zoo's animals. Ten species of order Cetartiodactyla and one species of order Rodentia were recognized as new STEC carriers. STEC strains belonged to 7 different serotypes including new serotypes O12:H25 and O13:H6. Serotype O146:H28, previously associated with human infections, represented 24% of STEC isolates. The most frequent Shiga toxin identified were type 1c and type 2c. Nineteen strains were positive for iha gene, 8 strains were positive for ehxA gene. Moreover, all strains were positive for lpfAO113 and negative for rfbO157, eae, saa, lpfAO157/OI-141, lpfAO157/OI-154, efa1, and toxB genes. Results obtained by XbaI-pulsed-field gel electrophoresis (XbaI-PFGE) confirmed the transmission of STEC strains among different animal species and suborders. In addition, we observed a potential association between STEC-harboring animal and factors such as belonging to order Cetartiodactyla, living in a pit, and belonging to a non-autochthonous species. This is the first work developed with zoological mammals and STEC in Argentina.

  12. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina.

    PubMed

    Brusa, Victoria; Aliverti, Virginia; Aliverti, Florencia; Ortega, Emanuel E; de la Torre, Julian H; Linares, Luciano H; Sanz, Marcelo E; Etcheverría, Analía I; Padola, Nora L; Galli, Lucía; Peral García, Pilar; Copes, Julio; Leotta, Gerardo A

    2012-01-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfb(O157)] (O157 lipopolysaccharide), fliC(H7) (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx(2)/eae/ehxA/fliC(H7) (83.4%), and for STEC non-O157 the most frequent ones were stx(1)/stx(2)/saa/ehxA (29.7%); stx(2) (29.7%); and stx(2)/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected.

  13. Shiga toxin-producing Escherichia coli in beef retail markets from Argentina

    PubMed Central

    Brusa, Victoria; Aliverti, Virginia; Aliverti, Florencia; Ortega, Emanuel E.; de la Torre, Julian H.; Linares, Luciano H.; Sanz, Marcelo E.; Etcheverría, Analía I.; Padola, Nora L.; Galli, Lucía; Peral García, Pilar; Copes, Julio; Leotta, Gerardo A.

    2013-01-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfbO157] (O157 lipopolysaccharide), fliCH7 (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx2/eae/ehxA/fliCH7 (83.4%), and for STEC non-O157 the most frequent ones were stx1/stx2/saa/ehxA (29.7%); stx2 (29.7%); and stx2/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected. PMID:23346554

  14. Wild Ungulates as Disseminators of Shiga Toxin-Producing Escherichia coli in Urban Areas

    PubMed Central

    Franklin, Alan B.; VerCauteren, Kurt C.; Maguire, Hugh; Cichon, Mary K.; Fischer, Justin W.; Lavelle, Michael J.; Powell, Amber; Root, J. Jeffrey; Scallan, Elaine

    2013-01-01

    Background In 2008, children playing on a soccer field in Colorado were sickened with a strain of Shiga toxin-producing Escherichia coli (STEC) O157:H7, which was ultimately linked to feces from wild Rocky Mountain elk. We addressed whether wild cervids were a potential source of STEC infections in humans and whether STEC was ubiquitous throughout wild cervid populations in Colorado. Methodology/Principal Findings We collected 483 fecal samples from Rocky Mountain elk and mule deer in urban and non-urban areas. Samples testing positive for STEC were higher in urban (11.0%) than non-urban (1.6%) areas. Elk fecal samples in urban areas had a much higher probability of containing STEC, which increased in both urban and non-urban areas as maximum daily temperature increased. Of the STEC-positive samples, 25% contained stx1 strains, 34.3% contained stx2, and 13% contained both stx1 and stx2. Additionally, eaeA genes were detected in 54.1% of the positive samples. Serotypes O103, and O146 were found in elk and deer feces, which also have the potential to cause human illness. Conclusions/Significance The high incidence of stx2 strains combined with eaeA and E-hyl genes that we found in wild cervid feces is associated with severe human disease, such as hemolytic uremic syndrome. This is of concern because there is a very close physical interface between elk and humans in urban areas that we sampled. In addition, we found a strong relationship between ambient temperature and incidence of STEC in elk feces, suggesting a higher incidence of STEC in elk feces in public areas on warmer days, which in turn may increase the likelihood that people will come in contact with infected feces. These concerns also have implications to other urban areas where high densities of coexisting wild cervids and humans interact on a regular basis. PMID:24349083

  15. Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Parsons, Brendon D.; Zelyas, Nathan; Berenger, Byron M.; Chui, Linda

    2016-01-01

    Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world. Given the public health importance of STEC, effective detection, characterization and typing is critical to any medical laboratory system. While non-O157 serotypes account for the majority of STEC infections, frontline microbiology laboratories may only screen for STEC using O157-specific agar-based methods. As a result, non-O157 STEC infections are significantly under-reported. This review discusses recent advances on the detection, characterization and typing of STEC with emphasis on work performed at the Alberta Provincial Laboratory for Public Health (ProvLab). Candidates for the detection of all STEC serotypes include chromogenic agars, enzyme immunoassays (EIA) and quantitative real time polymerase chain reaction (qPCR). Culture methods allow further characterization of isolates, whereas qPCR provides the greatest sensitivity and specificity, followed by EIA. The virulence gene profiles using PCR arrays and stx gene subtypes can subsequently be determined. Different non-O157 serotypes exhibit markedly different virulence gene profiles and a greater prevalence of stx1 than stx2 subtypes compared to O157:H7 isolates. Finally, recent innovations in whole genome sequencing (WGS) have allowed it to emerge as a candidate for the characterization and typing of STEC in diagnostic surveillance isolates. Methods of whole genome analysis such as single nucleotide polymorphisms and k-mer analysis are concordant with epidemiological data and standard typing methods, such as pulsed-field gel electrophoresis and multiple-locus variable number tandem repeat analysis while offering additional strain differentiation. Together these findings highlight improved strategies for STEC detection using currently available systems and the development of novel approaches for future surveillance. PMID:27148176

  16. Carriage and Fecal Counts of Cefotaxime M-Producing Escherichia coli in Pigs: a Longitudinal Study

    PubMed Central

    Damborg, Peter; Andreasen, Margit; Nielsen, Søren Saxmose; Guardabassi, Luca

    2013-01-01

    Current knowledge on extended-spectrum beta-lactamases (ESBLs) in animals is based largely on cross-sectional studies and qualitative data. The aim of this longitudinal study was to elucidate carriage proportions and fecal counts of ESBL-producing Escherichia coli in pigs during the production cycle. At each of three ESBL-positive single-sited farrow-to-finisher pig farms (farms A, B, and C) included in the study, individual fecal samples were taken from 17 to 20 sows 1 week before farrowing and from 2 piglets of each sow's litter four times from birth to slaughter (as piglets, weaners, and finishers). Cefotaxime (CTX)-resistant coliforms in feces were counted on MacConkey agar containing 2 μg/ml CTX and characterized for the presence of ESBL-encoding genes by PCR and sequencing. CTX-M-positive pigs were detected in all age groups at farms A (blaCTX-M-9 group, compatible with blaCTX-M-14/17) and B (blaCTX-M-1 group, compatible with blaCTX-M-1/61), whereas only three weaners were positive at farm C (blaCTX-M-1 group, compatible with blaCTX-M-1/61). A significant decrease in carriage was detected during the production cycle, with on average 50% carriage immediately after birth, 58% just before weaning, 29% during weaning, and 12% during finishing. The observed reduction in numbers of CTX-M-positive pigs was accompanied by a significant reduction in mean fecal counts of CTX-resistant coliforms from ∼107 CFU/g in piglets to ∼103 CFU/g in finishers (P < 0.001). These findings provide novel information about the epidemiology of ESBLs at the farm level and have important implications for assessments of risks of meat contamination during slaughter. PMID:23160131

  17. Inactivation of Shiga toxin-producing Escherichia coli in lean ground beef by gamma irradiation.

    PubMed

    Sommers, Christopher; Rajkowski, Kathleen T; Scullen, O Joseph; Cassidy, Jennifer; Fratamico, Pina; Sheen, Shiowshuh

    2015-08-01

    In this study the radiation resistance of 40 Shiga Toxin-Producing Escherichia coli (STEC) isolates which contained various combinations of the shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eae), and hemolysin (ehx) genes were determined. The STEC were suspended in lean ground beef and irradiated at 4 °C. D10 values, the radiation dose needed to reduce 1 log (90%) of a microorganism, ranged from 0.16 to 0.48 kGy, with a mean of 0.31 kGy for the 40 isolates. Isolates associated with illness outbreaks had a mean D10 of 0.27 kGy, while non-outbreak isolates had a mean D10 of 0.36 kGy (p < 0.05). The presence or absence of stx1, stx2, or both stx1 and 2 had no affect on D10 (p > 0.05). The presence (0.30 kGy) or absence (0.35 kGy) of ehx had no affect on D10 (p > 0.05). However, the mean D10 of isolates lacking eae (0.37 kGy) were significantly higher than those containing eae (0.27 kGy) (p < 0.05). There was no difference in D10 for isolates lacking eae regardless of whether or not they were associated with a foodborne illness outbreak (p > 0.05). It may be possible to use some of the STEC isolates which lacked eae, ehx, or both (D10 > 0.30) as avirulent surrogates in food irradiation research. The data presented in this study provides risk assessors data for metagenomic analysis as well as food and radiation processors with valuable information to control of STEC in meat.

  18. Edema disease as a model for systemic disease induced by Shiga toxin-producing E. coli.

    PubMed

    Cornick, N A; Matise, I; Samuel, J E; Bosworth, B T; Moon, H W

    1999-01-01

    Edema disease (ED) is a naturally occurring disease of weaned pigs caused by host adapted strains of E. coli that produce Shiga toxin (STEC). We determined the temporal and quantitative relationships between intestinal colonization by STEC, levels of Shiga toxin (Stx2e) in the gut, in the blood, and clinical manifestations of ED. Bacterial colonization (10(8) CFU/cm ileum) was highest 4 days post inoculation (pi) in animals that did not develop clinical disease and 6 days pi in animals with clinical signs of ED. The mean time for the development of clinical signs of ED was 6 days pi (range 4-10). Average peak titers of Stx2e in the ileum were 1:16,384 in asymptomatic animals and 1:32,768 in clinical animals. Titers of Stx2e in the feces reflected the toxin titers in the ileum but were lower. Intestinal titers of Stx2e and the density of bacterial colonization were predictive of clinical ED for a group of animals but not for individuals. Approximately 50% of the pigs that had Stx2e titers of > or = 1:4096 and a bacterial density of > or = 10(6) CFU/cm in their ileum, had clinical ED. Pigs that had intestinal Stx2e titers < 1:4096 were asymptomatic. Stx2e was detected in the red cell fraction of blood from some of the pigs with clinical ED and in some that were asymptomatic. Stx2e was not detected in the serum of any animals. ED may be a useful model for predicting the temporal and quantitative relationships between bacterial colonization, Stx levels in the gut and blood and systemic disease for STEC in other species.

  19. Comparative Exposure Assessment of ESBL-Producing Escherichia coli through Meat Consumption

    PubMed Central

    Pielaat, Annemarie; Smid, Joost H.; van Duijkeren, Engeline; Vennemann, Francy B. C.; Wijnands, Lucas M.; Chardon, Jurgen E.

    2017-01-01

    The presence of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli (EEC) in food animals, especially broilers, has become a major public health concern. The aim of the present study was to quantify the EEC exposure of humans in The Netherlands through the consumption of meat from different food animals. Calculations were done with a simplified Quantitative Microbiological Risk Assessment (QMRA) model. The model took the effect of pre-retail processing, storage at the consumers home and preparation in the kitchen (cross-contamination and heating) on EEC numbers on/in the raw meat products into account. The contribution of beef products (78%) to the total EEC exposure of the Dutch population through the consumption of meat was much higher than for chicken (18%), pork (4.5%), veal (0.1%) and lamb (0%). After slaughter, chicken meat accounted for 97% of total EEC load on meat, but chicken meat experienced a relatively large effect of heating during food preparation. Exposure via consumption of filet americain (a minced beef product consumed raw) was predicted to be highest (61% of total EEC exposure), followed by chicken fillet (13%). It was estimated that only 18% of EEC exposure occurred via cross-contamination during preparation in the kitchen, which was the only route by which EEC survived for surface-contaminated products. Sensitivity analysis showed that model output is not sensitive for most parameters. However, EEC concentration on meat other than chicken meat was an important data gap. In conclusion, the model assessed that consumption of beef products led to a higher exposure to EEC than chicken products, although the prevalence of EEC on raw chicken meat was much higher than on beef. The (relative) risk of this exposure for public health is yet unknown given the lack of a modelling framework and of exposure studies for other potential transmission routes. PMID:28056081

  20. Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E. coli (STEC) infections in the Netherlands, January 2008 to December 2011.

    PubMed

    Friesema, I; van der Zwaluw, K; Schuurman, T; Kooistra-Smid, M; Franz, E; van Duynhoven, Y; van Pelt, W

    2014-05-01

    The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx2f is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O157 cases and 351 STEC non-O157 cases, including 87 stx2f STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. stx2f STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease.

  1. Conventional curing practices reduce generic Escherichia coli and Salmonella spp. on dry bulb onions produced with contaminated irrigation water.

    PubMed

    Emch, Alexander W; Waite-Cusic, Joy G

    2016-02-01

    Food Safety Modernization Act (FSMA) has emphasized microbial risks associated with irrigation water. Treasure Valley (eastern Oregon/western Idaho) has the highest yield of dry bulb onions in the country; however, their irrigation water is often non-compliant with current industry and proposed federal standards for fresh produce. Conventional curing practices may provide a mechanism to mitigate irrigation water quality to comply with FSMA regulations. Dry bulb onions were grown in Owyhee silt loam and Semiahmoo muck soils in greenhouses and irrigated with water containing a cocktail of rifampicin-resistant generic Escherichia coli and Salmonella spp. (4.80 log CFU/ml). To mimic conventional practices, mature onions remained undisturbed in soil without irrigation for 12 days prior to being lifted and cured for 16 additional days. Surviving generic E. coli and Salmonella spp. were selectively enumerated on using standard plating (Hektoen Enteric Agar with rifampicin; HE + rif) or most probable number (lactose broth with rifampicin; HE + rif) methods. Generic E. coli and Salmonella spp. on onions decreased 0.19-0.26 log CFU/g·d during the initial 12 days of finishing. At lifting, generic E. coli and Salmonella spp. had been reduced to <1 CFU/g and persisted through the end of curing. This study demonstrates conventional curing practices as an effective mitigation strategy for dry bulb onions produced with water of poor microbiological quality.

  2. Escherichia coli can produce recombinant chitinase in the soil to control the pathogenesis by Fusarium oxysporum without colonization.

    PubMed

    Chung, Soohee; Kim, Sang-Dal

    2007-03-01

    Fusarium wilt of cucumbers was effectively controlled by Escherichia coli expressing an endochitinase gene (chiA), and the rate was as effective (60.0%) as the wildtype strain S. proteamaculans 3095 (55.0%) where the gene was cloned. However, live cells of soil inoculated E. coli host harboring the chiA gene did not proliferate but declined 100-fold from 108 CFU during the first week and showed less than 10 cells after day 14, suggesting that E. coli was able to express and produce the chitinase enzyme to the soil even as the population was gradually decreasing. Because the majority of the strains was alive for only a short period of time and the Fusarium-affected seedlings showed symptoms of wilting within 7-10 days, it seems that the pathogen control was decided early after the introduction of the biocontrol agent, eliminating the survival of the antagonist. These results indicated that soil inoculated E. coli could sufficiently express and produce the recombinant protein to control the pathogen, and root or soil colonization of the antagonist might not be a significant factor in determining the efficacy of biological control.

  3. Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli

    PubMed Central

    Andrade, Fernanda B.; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D.; Yamamoto, Bruno B.; Luz, Daniela; Abreu, Patrícia A. E.; Horton, Denise S. P. Q.; Elias, Waldir P.; Ramos, Oscar H. P.; Piazza, Roxane M. F.

    2015-01-01

    Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Methods and Findings Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis. PMID:26154103

  4. O157:H7 and O104:H4 Vero/Shiga toxin-producing Escherichia coli outbreaks: respective role of cattle and humans

    PubMed Central

    2012-01-01

    An enteroaggregative Verotoxin (Vtx)-producing Escherichia coli strain of serotype O104:H4 has recently been associated with an outbreak of haemolytic-uremic syndrome and bloody diarrhoea in humans mainly in Germany, but also in 14 other European countries, USA and Canada. This O104:H4 E. coli strain has often been described as an enterohaemorrhagic E. coli (EHEC), i.e. a Vtx-producing E. coli with attaching and effacing properties. Although both EHEC and the German O104:H4 E. coli strains indeed produce Vtx, they nevertheless differ in several other virulence traits, as well as in epidemiological characteristics. For instance, the primary sources and vehicles of typical EHEC infections in humans are ruminants, whereas no animal reservoir has been identified for enteroaggregative E. coli (EAggEC). The present article is introduced by a brief overview of the main characteristics of Vtx-producing E. coli and EAggEC. Thereafter, the O104:H4 E. coli outbreak is compared to typical EHEC outbreaks and the virulence factors and host specificity of EHEC and EAggEC are discussed. Finally, a renewed nomenclature of Vtx-producing E. coli is proposed to avoid more confusion in communication during future outbreaks and to replace the acronym EHEC that only refers to a clinical condition. PMID:22330148

  5. Occurrence of CTX-M Producing Escherichia coli in Soils, Cattle, and Farm Environment in France (Burgundy Region)

    PubMed Central

    Hartmann, Alain; Locatelli, Aude; Amoureux, Lucie; Depret, Géraldine; Jolivet, Claudy; Gueneau, Eric; Neuwirth, Catherine

    2012-01-01

    CTX-M [a major type of extended-spectrum beta-lactamase (ESBL)] producing Escherichia coli are increasingly involved in human infections worldwide. The aim of this study was to investigate potential reservoirs for such strains: soils, cattle, and farm environment. The prevalence of blaCTX-M genes was determined directly from soil DNA extracts obtained from 120 sites in Burgundy (France) using real-time PCR. blaCTX-M targets were found in 20% of the DNA extracts tested. Samples of cattle feces (n = 271) were collected from 182 farms in Burgundy. Thirteen ESBL-producing isolates were obtained from 12 farms and further characterized for the presence of bla genes. Of the 13 strains, five and eight strains carried blaTEM-71 genes and blaCTX-M-1 genes respectively. Ten strains of CTX-M-1 producing E. coli were isolated from cultivated and pasture soils as well as from composted manure within two of these farms. The genotypic analysis revealed that environmental and animal strains were clonally related. Our study confirms the occurrence of CTX-M producing E. coli in cattle and reports for the first time the occurrence of such strains in cultivated soils. The environmental competence of such strains has to be determined and might explain their long term survival since CTX-M isolates were recovered from a soil that was last amended with manure 1 year before sampling. PMID:22408639

  6. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011–2012, Russia

    PubMed Central

    Kartsev, Nikolay N.; Fursova, Nadezhda K.; Pachkunov, Dmitry M.; Bannov, Vasiliy A.; Eruslanov, Boris V.; Svetoch, Edward A.; Dyatlov, Ivan A.

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011–2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates—to two drugs, one isolate—to three drugs, two isolates—to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  7. Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

    PubMed

    Kartsev, Nikolay N; Fursova, Nadezhda K; Pachkunov, Dmitry M; Bannov, Vasiliy A; Eruslanov, Boris V; Svetoch, Edward A; Dyatlov, Ivan A

    2015-01-01

    Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and

  8. Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms

    PubMed Central

    Solà-Ginés, Marc; González-López, Juan José; Cameron-Veas, Karla; Piedra-Carrasco, Nuria; Cerdà-Cuéllar, Marta

    2015-01-01

    Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained blaCTX-M-1, 18 contained blaCTX-M-14, and 1 contained blaCTX-M-9. ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches. PMID:25795670

  9. Houseflies (Musca domestica) as Vectors for Extended-Spectrum β-Lactamase-Producing Escherichia coli on Spanish Broiler Farms.

    PubMed

    Solà-Ginés, Marc; González-López, Juan José; Cameron-Veas, Karla; Piedra-Carrasco, Nuria; Cerdà-Cuéllar, Marta; Migura-Garcia, Lourdes

    2015-06-01

    Flies may act as potential vectors for the spread of resistant bacteria to different environments. This study was intended to evaluate the presence of Escherichia coli strains resistant to cephalosporins in flies captured in the areas surrounding five broiler farms. Phenotypic and molecular characterization of the resistant population was performed by different methods: MIC determination, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylotyping. The presence of extended-spectrum beta-lactamase (ESBL) genes, their plasmid location, and the mobile genetic elements involved in their mobilization were studied. Additionally, the presence of 35 genes associated with virulence was evaluated. Out of 682 flies captured, 42 yielded ESBL-producing E. coli. Of these isolates, 23 contained bla(CTX-M-1), 18 contained bla(CTX-M-14), and 1 contained bla(CTX-M-9). ESBL genes were associated mainly with the presence of the IncI1 and IncFIB replicons. Additionally, all the strains were multiresistant, and five of them also harbored qnrS. Identical PFGE profiles were found for E. coli isolates obtained from flies at different sampling times, indicating a persistence of the same clones in the farm environment over months. According to their virulence genes, 81% of the isolates were considered avian-pathogenic E. coli (APEC) and 29% were considered extraintestinal pathogenic E. coli (ExPEC). The entrance of flies into broiler houses constitutes a considerable risk for colonization of broilers with multidrug-resistant E. coli. ESBLs in flies reflect the contamination status of the farm environment. Additionally, this study demonstrates the potential contribution of flies to the dissemination of virulence and resistance genes into different ecological niches.

  10. Synthetic biology: Tailor-made genetic codes

    NASA Astrophysics Data System (ADS)

    Jewett, Michael C.; Noireaux, Vincent

    2016-04-01

    Expanding the range of amino acids polymerizable by ribosomes could enable new functionalities to be added to polypeptides. Now, the genetic code has been reprogrammed using a reconstituted in vitro translation system to enable synthesis of unnatural peptides with unmatched flexibility.

  11. Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins

    PubMed Central

    Schulz, Steve; Stephan, Anett; Hahn, Simone; Bortesi, Luisa; Jarczowski, Franziska; Bettmann, Ulrike; Paschke, Anne-Katrin; Tusé, Daniel; Stahl, Chad H.; Giritch, Anatoli; Gleba, Yuri

    2015-01-01

    Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway. PMID:26351689

  12. Ventilator-associated pneumonia caused by OXA-48-producing Escherichia coli complicated by ciprofloxacin-associated rhabdomyolysis.

    PubMed

    Grisold, Andrea J; Hoenigl, Martin; Ovcina, Ismar; Valentin, Thomas; Fruhwald, Sonja

    2013-12-01

    We report the emergence of OXA-48 carbapenemase-producing Escherichia coli in Austria causing ventilator-associated pneumonia in a traveler returning from Egypt. Depending on resistance testing, quinolones may remain a therapeutic option for infections caused by these multiple resistant pathogens, as this class of drugs has a favorable safety and tolerability profile when compared to the alternatives. In this patient, however, the clinical course was dramatically complicated by the development of ciprofloxacin-associated rhabdomyolysis.

  13. Characterization of Escherichia coli-Producing Extended-Spectrum β-Lactamase (ESBL) Isolated from Chicken Slaughterhouses in South Korea.

    PubMed

    Lim, Jong-Soo; Choi, Da-Som; Kim, Young-Jo; Chon, Jung-Whan; Kim, Hong-Seok; Park, Hyun-Jung; Moon, Jin-San; Wee, Sung-Hwan; Seo, Kun-Ho

    2015-09-01

    In South Korea, few reports have indicated the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in food-producing animals, particularly in poultry slaughterhouses. In this study, we investigated the occurrence and antibiotic resistance of ESBL-producing E. coli from whole chicken carcasses (n=156) and fecal samples (n=39) of chickens obtained from 2 slaughterhouses. Each sample enriched in buffered peptone water was cultured on MacConkey agar with 2 mg/L cefotaxime and ESBL agar. ESBL production and antibiotic susceptibility were determined using the Trek Diagnostics system. The ESBL genotypes were determined by polymerase chain reaction (PCR) using the bla(SHV), bla(TEM), and bla(CTX-M) gene sequences. Subtyping using a repetitive sequence-based PCR system (DiversiLab™) and multilocus sequence typing (MLST) were used to assess the interspecific biodiversity of isolates. Sixty-two ESBL-producing E. coli isolates were obtained from 156 samples (39.7%). No bla(SHV) genes were detected in any of the isolates, whereas all contained the bla(TEM) gene. Twenty-five strains (40.3%) harbored the CTX-M group 1 gene. The most prevalent MLST sequence type (ST) was ST 93 (14.5%), followed by ST 117 (9.7%) and ST 2303 (8.1%). This study reveals a high occurrence and β-lactams resistance rate of E. coli in fecal samples and whole chickens collected from slaughterhouses in South Korea.

  14. A QMRA for the Transmission of ESBL-Producing Escherichia coli and Campylobacter from Poultry Farms to Humans Through Flies.

    PubMed

    Evers, Eric G; Blaak, Hetty; Hamidjaja, Raditijo A; de Jonge, Rob; Schets, Franciska M

    2016-02-01

    The public health significance of transmission of ESBL-producing Escherichia coli and Campylobacter from poultry farms to humans through flies was investigated using a worst-case risk model. Human exposure was modeled by the fraction of contaminated flies, the number of specific bacteria per fly, the number of flies leaving the poultry farm, and the number of positive poultry houses in the Netherlands. Simplified risk calculations for transmission through consumption of chicken fillet were used for comparison, in terms of the number of human exposures, the total human exposure, and, for Campylobacter only, the number of human cases of illness. Comparing estimates of the worst-case risk of transmission through flies with estimates of the real risk of chicken fillet consumption, the number of human exposures to ESBL-producing E. coli was higher for chicken fillet as compared with flies, but the total level of exposure was higher for flies. For Campylobacter, risk values were nearly consistently higher for transmission through flies than for chicken fillet consumption. This indicates that the public health risk of transmission of both ESBL-producing E. coli and Campylobacter to humans through flies might be of importance. It justifies further modeling of transmission through flies for which additional data (fly emigration, human exposure) are required. Similar analyses of other environmental transmission routes from poultry farms are suggested to precede further investigations into flies.

  15. Molecular epidemiology of carbapenemase-producing Escherichia coli and the prevalence of ST131 subclone H30 in Shanghai, China.

    PubMed

    Zhang, F; Zhu, D; Xie, L; Guo, X; Ni, Y; Sun, J

    2015-06-01

    The molecular characteristics and epidemiology of carbapenemase-producing Escherichia coli (CPEC) isolates from Shanghai, China, were investigated using 21 imipenem-resistant E. coli isolates obtained from a Shanghai teaching hospital from 2011 to 2014. The presence of bla KPC, bla IMP, bla VIM, bla OXA-48, and bla NDM was assessed by polymerase chain reaction (PCR) amplification and sequencing. CPEC isolates were characterized by the Etest®, multilocus sequence typing (MLST), and pulse-field gel electrophoresis (PFGE). Plasmids carrying resistance genes were analyzed by conjugation experiments, replicon typing, plasmid MLST (pMLST), S1 nuclease PFGE (S1-PFGE), and Southern hybridization. The genetic environment of the resistance genes was determined by PCR and sequencing. Among the 21 E. coli isolates, 16 produced carbapenemases; of these, ten isolates transferred carbapenemase-encoding plasmids to recipient bacteria. Nine of the 16 isolates were clonally related, and their PFGE patterns were designated type A. ST131 was the predominant sequence type (11 isolates, 68.8 %); the H30 subclone comprised 81.8 % of the ST131 strains. In all three isolates, bla IMP-4 was located on 50-kb IncN plasmids. All but two bla KPC-2 genes were carried on IncF plasmids of various sizes. Hence, both clone-spread and horizontal transfer mediated the dissemination of carbapenemase-producing genes in the Shanghai isolates.

  16. Detection of Escherichia coli sequence type 131 clonal group among extended-spectrum β-lactamase-producing E. coli using VITEK MS Plus matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Matsumura, Yasufumi; Yamamoto, Masaki; Nagao, Miki; Tanaka, Michio; Takakura, Shunji; Ichiyama, Satoshi

    2015-12-01

    We investigated the performance of the VITEK MS Plus system for the detection of Escherichia coli sequence type 131 (ST131) among extended-spectrum β-lactamase-producing E. coli isolates. The SARAMIS software could discriminate the 67 ST131 isolates from 82 non-ST131 isolates with a sensitivity of 86.6% and a specificity of 95.1%.

  17. Evaluation of a novel antimicrobial solution and its potential for control E. coli O157:H7, non-O157:H7 shiga toxin-producing E. coli, Salmononella spp., and Listeria monocytogenes on beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to b...

  18. Verocytotoxin-producing Escherichia coli in chamois (Rupicapra rupicapra) and cattle in Austria.

    PubMed

    Freidl, Gudrun; Stalder, Gabrielle; Kostić, Tanja; Sessitsch, Angela; Beiglböck, Christoph; Walzer, Christian

    2011-07-01

    We assessed the prevalence of verotoxigenic Escherichia coli (VTEC) in chamois (Rupicapra rupicapra) and livestock grazing on a mountain pasture in Austria during June-August 2009. We detected VTEC throughout the sampling period in high numbers in cattle as well as in chamois, leading to the assumption that the degree of contamination of the environment is high. This is the first report of pathogenic E. coli identified in chamois, implicating chamois as a new potential reservoir of these zoonotic pathogens. Because the study area also serves recreational purposes, there is a risk of humans acquiring infection via direct or indirect contact.

  19. Emergence of an NDM-5-producing clinical Escherichia coli isolate in Egypt.

    PubMed

    Soliman, Ahmed M; Khalifa, Hazim O; Ahmed, Ashraf M; Shimamoto, Toshi; Shimamoto, Tadashi

    2016-07-01

    The first occurrence of New Delhi metallo-β-lactamase 5 (NDM-5), carried on an IncI1-Iγ-type plasmid of >93kb in a multidrug-resistant Escherichia coli strain in Kafr El-Sheikh, Egypt, is reported. The strain was isolated from a wound pus swab from a patient diagnosed with a fracture of the right femur. This E. coli strain was found to belong to sequence type (ST) 5018 and also to carry other resistance genes, including blaCTX-M-15, blaCMY-42, blaOXA-1, and aac(6')-Ib-cr.

  20. Antimicrobial Resistance and Molecular Epidemiology of ESBL-Producing Escherichia coli Isolated from Outpatients in Town Hospitals of Shandong Province, China.

    PubMed

    Miao, Zengmin; Li, Song; Wang, Lei; Song, Wengang; Zhou, Yufa

    2017-01-01

    This study aimed to investigate antimicrobial resistance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) isolated from outpatients in town hospitals of Shandong province, China. Antimicrobial susceptibility of ESBL-producing E. coli was tested using the disk diffusion and resistance genes encoding for β-lactamases (blaTEM, blaCTXM, and blaSHV) were detected by polymerase chain reaction (PCR). Multilocus sequence typing (ST) of ESBL-producing E. coli was analyzed in this study. Our results showed that of 320 E. coli isolates, 201 carried ESBL genes (201/320, 62.8%), and these isolates all carried blaCTX-M genes, the most common being blaCTX-M-14 (116/201, 57.7%), followed by blaCTX-M-55 (47/201, 23.4%) and blaCTX-M-15 (31/201, 15.4%). ESBL-producing E. coli exhibited highly resistant to penicillin derivatives, fluoroquinolones, folate pathway inhibitors, and third-generation cephalosporins, but no carbapenem-resistant isolates were found in this study. Forty-two STs were found among the 201 ESBL-producing E. coli, and the most common ST was ST131 (27/201, 13.4%), followed by ST405 (19/201, 9.5%) and ST69 (15/201, 7.5%). Taken together, a high isolation rate of ESBL-producing E. coli (62.8%) was found among outpatients in town hospitals. blaCTX-M gene was most dominant and was composed of a variety of subtypes. No dominant ST was detected among ESBL-producing E. coli, indicating that these ESBL-producing E. coli isolates derive from different clones.

  1. Antimicrobial Resistance and Molecular Epidemiology of ESBL-Producing Escherichia coli Isolated from Outpatients in Town Hospitals of Shandong Province, China

    PubMed Central

    Miao, Zengmin; Li, Song; Wang, Lei; Song, Wengang; Zhou, Yufa

    2017-01-01

    This study aimed to investigate antimicrobial resistance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) isolated from outpatients in town hospitals of Shandong province, China. Antimicrobial susceptibility of ESBL-producing E. coli was tested using the disk diffusion and resistance genes encoding for β-lactamases (blaTEM, blaCTXM, and blaSHV) were detected by polymerase chain reaction (PCR). Multilocus sequence typing (ST) of ESBL-producing E. coli was analyzed in this study. Our results showed that of 320 E. coli isolates, 201 carried ESBL genes (201/320, 62.8%), and these isolates all carried blaCTX-M genes, the most common being blaCTX-M-14 (116/201, 57.7%), followed by blaCTX-M-55 (47/201, 23.4%) and blaCTX-M-15 (31/201, 15.4%). ESBL-producing E. coli exhibited highly resistant to penicillin derivatives, fluoroquinolones, folate pathway inhibitors, and third-generation cephalosporins, but no carbapenem-resistant isolates were found in this study. Forty-two STs were found among the 201 ESBL-producing E. coli, and the most common ST was ST131 (27/201, 13.4%), followed by ST405 (19/201, 9.5%) and ST69 (15/201, 7.5%). Taken together, a high isolation rate of ESBL-producing E. coli (62.8%) was found among outpatients in town hospitals. blaCTX-M gene was most dominant and was composed of a variety of subtypes. No dominant ST was detected among ESBL-producing E. coli, indicating that these ESBL-producing E. coli isolates derive from different clones. PMID:28174570

  2. Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization.

    PubMed

    Li, Sha; Xu, Hong; Yu, Jianguang; Wang, Yanyuan; Feng, Xiaohai; Ouyang, Pingkai

    2013-10-01

    Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l⁻¹), yeast extract (25.93 g l⁻¹), and corn steep liquor (10.45 g l⁻¹) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW⁻¹) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet⁻¹ h⁻¹. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.

  3. Shiga Toxin-Producing Escherichia coli Isolated from Bovine Mastitic Milk: Serogroups, Virulence Factors, and Antibiotic Resistance Properties

    PubMed Central

    Momtaz, Hassan; Safarpoor Dehkordi, Farhad; Taktaz, Taghi; Rezvani, Amir; Yarali, Sajad

    2012-01-01

    The aim of this study was to detect the virulence factors, serogroups, and antibiotic resistance properties of Shiga toxin-producing Escherichia coli, by using 268 bovine mastitic milk samples which were diagnosed using California Mastitis Test. After E. coli identification, PCR assays were developed for detection of different virulence genes, serogroups, and antibiotic resistance genes of Escherichia coli. The antibiotic resistance pattern was studied using disk diffusion method. Out of 268 samples, 73 (27.23%) were positive for Escherichia coli, and, out of 73 positive samples, 15 (20.54%) were O26 and 11 (15.06%) were O157 so they were the highest while O111 was not detected in any sample so it was the lowest serogroup. Out of 73 STEC strains, 11 (15.06%) and 36 (49.31%) were EHEC and AEEC, respectively. All of the EHEC strains had stx1, eaeA, and ehly, virulence genes, while in AEEC strains stx1 had the highest prevalence (77.77%), followed by eaeA (55.55%). Totally, aadA1 (65.95%) had the highest while blaSHV (6.38%) had the lowest prevalence of antibiotic resistance genes. The disk diffusion method showed that the STEC strains had the highest resistance to penicillin (100%), followed by tetracycline (57.44%), while resistance to cephalothin (6.38%) was the lowest. PMID:23213293

  4. Factors Associated with Shiga Toxin-Producing Escherichia coli Shedding by Dairy and Beef Cattle

    PubMed Central

    Venegas-Vargas, Cristina; Henderson, Scott; Khare, Akanksha; Mosci, Rebekah E.; Lehnert, Jonathan D.; Singh, Pallavi; Ouellette, Lindsey M.; Norby, Bo; Funk, Julie A.; Rust, Steven; Bartlett, Paul C.; Grooms, Daniel

    2016-01-01

    ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome. Cattle are the primary reservoir for STEC, and food or water contaminated with cattle feces is the most common source of infections in humans. Consequently, we conducted a cross-sectional study of 1,096 cattle in six dairy herds (n = 718 animals) and five beef herds (n = 378 animals) in the summers of 2011 and 2012 to identify epidemiological factors associated with shedding. Fecal samples were obtained from each animal and cultured for STEC. Multivariate analyses were performed to identify risk factors associated with STEC positivity. The prevalence of STEC was higher in beef cattle (21%) than dairy cattle (13%) (odds ratio [OR], 1.76; 95% confidence interval [CI], 1.25, 2.47), with considerable variation occurring across herds (range, 6% to 54%). Dairy cattle were significantly more likely to shed STEC when the average temperature was >28.9°C 1 to 5 days prior to sampling (OR, 2.5; 95% CI, 1.25, 4.91), during their first lactation (OR, 1.8; 95% CI, 1.1, 2.8), and when they were <30 days in milk (OR, 3.9; 95% CI, 2.1, 7.2). These data suggest that the stress or the negative energy balance associated with lactation may result in increased STEC shedding frequencies in Michigan during the warm summer months. Future prevention strategies aimed at reducing stress during lactation or isolating high-risk animals could be implemented to reduce herd-level shedding levels and avoid transmission of STEC to susceptible animals and people. IMPORTANCE STEC shedding frequencies vary considerably across cattle herds in Michigan, and the shedding frequency of strains belonging to non-O157 serotypes far exceeds the shedding frequency of O157 strains, which is congruent with human infections in the state. Dairy cattle sampled at higher temperatures, in their first lactation, and early in the milk production stage were

  5. Prevalence of Class D Carbapenemases among Extended-Spectrum β-Lactamases Producing Escherichia coli Isolates from Educational Hospitals in Shahrekord

    PubMed Central

    Damavandi, Mohammad-Sadegh; Latif Pour, Mohammad

    2016-01-01

    Introduction Extended-spectrum β-lactamases (ESBLs) are a set of plasmid-borne, various and quickly evolving enzymes that are a main therapeutic issue now-a-days for inpatient and outpatient treatment. Aim The aim of this study was to determine multi-drug resistance (MDR) and ESBLs producing E. coli strains, prevalence of class D Carbapenemases among ESBLs producing Escherichia coli isolates from educational hospitals in Shahrekord, Iran. Materials and Methods Uropathogenic Escherichia coli strains were isolated from patients with Urinary Tract Infections (UTIs). The agar disc diffusion test was used to characterize the antimicrobial sensitivity of the E. coli isolates. The ESBL positive strains were identified by phenotypic double-disk synergy test, by third-generation cephalosporin in combination with or without clavulanic acid. Multiplex PCR was carried out for detection of the three families of OXA-type carbapenamases including OXA-23, OXA-24, and OXA-48 in E. coli strains. Results All bacterial isolates were susceptible to meropenem. Ninety isolates produced ESBL, 55 E. coli isolates from inpatients, and 35 isolates from outpatients, with a significant association (p< 0.05). The prevalence of OXA-23, OXA-24, and OXA-48 in the ESBLs producing isolates was respectively 21%, 18%, and 11% for inpatients, and 10%, 8%, and 6% for outpatients. Conclusion ESBL-producing E. coli isolates are also a major threat in the clinical setting. The findings of this study indicated the high occurrence of ESBLs and multiple antibiotic resistance in E. coli isolates. PMID:27462579

  6. Characteristics of the Molecular Epidemiology of CTX-M-Producing Escherichia coli Isolated from a Tertiary Hospital in Daejeon, Korea.

    PubMed

    Kim, Semi; Sung, Ji Youn; Cho, Hye Hyun; Kwon, Kye Chul; Koo, Sun Hoe

    2016-09-28

    The aims of this study were to characterize the molecular epidemiological profiles of CTX-M-producing uropathogenic Escherichia coli isolates from a tertiary hospital in Daejeon, Korea, and to investigate the genetic diversity and compare the prevalence of sequence types (STs) in different areas. Extended spectrum β-lactamase-producing E. coli strains isolated from urine were analyzed for CTX-M, integrons, and insertion sequence common regions (ISCRs) by PCR and sequencing. Multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), phylogenetic analysis, and rep-PCR were also used for molecular typing of the isolates. Of 80 CTX-M producers, 31 and 46 expressed CTX-M-15 and CTX-M-14, respectively. MLST analysis indicated that the most prevalent ST was ST131 (n = 34, 42.5%), followed by ST38 (n = 22, 27.5%), ST405 (n = 8, 10.0%), and ST69 (n = 6, 7.5%). Most CTX-M producers harbored class 1 integrons. ST131 strains belonged to phylogenetic group B2 and showed identical rep-PCR patterns, whereas ST69, ST38, and ST405 strains belonged to phylogenetic group D; the ST38 and ST405 strains displayed the same rep-PCR pattern, respectively. ST131 and ST38 isolates showed 21 and 19 distinct types, respectively, by PFGE. In Daejeon, D-ST38 CTX-M-14 producers were relatively more prevalent than in other countries and Korean cities. Our results indicate that CTX-M-producing E. coli isolates belonged mostly to ST131 or ST38 and were more related to hospital-onset than to community-onset infections and that the blaCTX-M gene may vary according to the ST.

  7. Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4 prime -chlorobiphenyl

    SciTech Connect

    Khan, A.A.; Walia, S.K. )

    1992-04-01

    Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4{prime}-chlorobiphenyl (DHCB) from 4-chlorobiphenyl produced 110 {mu}M DHCB. The K{sub m} for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.

  8. Production and regulation of functional amyloid curli fimbriae by Shiga toxin-producing Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Functional amyloid, in the form of adhesive fimbrial proteins termed curli, was first described in Salmonella and Escherichia coli. Curli fibers adhere to various host cells and structural proteins, interact with components of the host immune system, and participate in biofilm formation. Shiga toxin...

  9. Occurrence of ESBL-Producing Escherichia coli in Livestock and Farm Workers in Mecklenburg-Western Pomerania, Germany

    PubMed Central

    Dahms, Carmen; Hübner, Nils-Olaf; Kossow, Annelene; Mellmann, Alexander; Dittmann, Kathleen; Kramer, Axel

    2015-01-01

    In recent years, extended-spectrum β-lactamases (ESBL) producing bacteria have been found in livestock, mainly as asymptomatic colonizers. The zoonotic risk for people working in close contact to animal husbandry has still not been completely assessed. Therefore, we investigated the prevalence of ESBL-producing Escherichia spp. in livestock animals and workers to determine the potential risk for an animal-human cross-transmission.In Mecklenburg-Western Pomerania, northeast Germany, inguinal swabs of 73 individuals with livestock contact from 23 different farms were tested for ESBL-producing Escherichia spp. Two pooled fecal samples per farm of animal origin from 34 different farms (17 pig farms, 11 cattle farms, 6 poultry farms) as well as cloacal swabs of 10 randomly selected broilers or turkeys were taken at each poultry farm. For identification, selective chromogenic agar was used after an enrichment step. Phenotypically ESBL-producing isolates (n = 99) were tested for CTX-M, OXA, SHV and TEM using PCR, and isolates were further characterized using multilocus sequence typing (MLST). In total, 61 diverse isolates from different sources and/or different MLST/PCR results were acquired. Five farm workers (three from cattle farms and two from pig farms) harbored ESBL-producing E. coli. All human isolates harbored the CTX-M β-lactamase; TEM and OXA β-lactamases were additionally detected in two, resp. one, isolates. ESBL-producing Escherichia spp. were found in fecal samples at pig (15/17), cattle (6/11) and poultry farms (3/6). In total, 70.6% (24/36) of the tested farms were ESBL positive. Furthermore, 9 out of 60 cloacal swabs turned out to be ESBL positive. All isolated ESBL-producing bacteria from animal sources were E. coli, except for one E. hermanii isolate. CTX-M was the most prevalent β-lactamase at cattle and pig farms, while SHV predominated in poultry. One human isolate shared an identical MLST sequence type (ST) 3891 and CTX-M allele to the isolate

  10. Occurrence of ESBL-Producing Escherichia coli in Livestock and Farm Workers in Mecklenburg-Western Pomerania, Germany.

    PubMed

    Dahms, Carmen; Hübner, Nils-Olaf; Kossow, Annelene; Mellmann, Alexander; Dittmann, Kathleen; Kramer, Axel

    2015-01-01

    In recent years, extended-spectrum β-lactamases (ESBL) producing bacteria have been found in livestock, mainly as asymptomatic colonizers. The zoonotic risk for people working in close contact to animal husbandry has still not been completely assessed. Therefore, we investigated the prevalence of ESBL-producing Escherichia spp. in livestock animals and workers to determine the potential risk for an animal-human cross-transmission.In Mecklenburg-Western Pomerania, northeast Germany, inguinal swabs of 73 individuals with livestock contact from 23 different farms were tested for ESBL-producing Escherichia spp. Two pooled fecal samples per farm of animal origin from 34 different farms (17 pig farms, 11 cattle farms, 6 poultry farms) as well as cloacal swabs of 10 randomly selected broilers or turkeys were taken at each poultry farm. For identification, selective chromogenic agar was used after an enrichment step. Phenotypically ESBL-producing isolates (n = 99) were tested for CTX-M, OXA, SHV and TEM using PCR, and isolates were further characterized using multilocus sequence typing (MLST). In total, 61 diverse isolates from different sources and/or different MLST/PCR results were acquired. Five farm workers (three from cattle farms and two from pig farms) harbored ESBL-producing E. coli. All human isolates harbored the CTX-M β-lactamase; TEM and OXA β-lactamases were additionally detected in two, resp. one, isolates. ESBL-producing Escherichia spp. were found in fecal samples at pig (15/17), cattle (6/11) and poultry farms (3/6). In total, 70.6% (24/36) of the tested farms were ESBL positive. Furthermore, 9 out of 60 cloacal swabs turned out to be ESBL positive. All isolated ESBL-producing bacteria from animal sources were E. coli, except for one E. hermanii isolate. CTX-M was the most prevalent β-lactamase at cattle and pig farms, while SHV predominated in poultry. One human isolate shared an identical MLST sequence type (ST) 3891 and CTX-M allele to the isolate

  11. Surveillance of ESBL producing multidrug resistant Escherichia coli in a teaching hospital in India

    PubMed Central

    Rath, Shakti; Dubey, Debasmita; Sahu, Mahesh C.; Padhy, Rabindra N

    2014-01-01

    Objective To record nosocomial and community-acquired accounts of antibiotic resistance in Escherichia coli (E. coli) strains, isolated from clinical samples of a teaching hospital by surveillance, over a period of 39 months (November 2009-January 2013). Methods Clinical samples from nosocomial sources, i.e., wards and cabins, intensive care unit (ICU) and neonatal intensive care unit (NICU), and community (outpatient department, OPD) sources of the hospital, were used for isolating strains of E. coli, which were subjected for testing for production of ‘extended spectrum beta-lactamase’-(ESBL) enzyme as well as determining antibiotic sensitivity pattern with 23 antibiotics. Results Of the total 1642 (100%) isolates, 810 (49.33%) strains were from OPD and 832 (50.66%) were from hospital settings. Occurrence of infectious E. coli strains increased in a mathematical progression in community sources, but in nosocomial infections, such values remained almost constant in each quarter. A total of 395 (24.05%) ESBL strains were isolated from the total 810 isolates of community; of the total of 464 (28.25%) isolates of wards and cabins, 199 (12.11%) were ESBL strains; and among the total of 368 (22.41%) isolates of ICU and NICU, ESBLs were 170 (10.35%); the total nosocomial ESBL isolates, 369 (22.47%) were from the nosocomial total of 832 (50.66%) isolates. Statistically, it was confirmed that ESBL strains were equally distributed in community or hospital units. Antibiogram of 23 antibiotics revealed progressive increases of drug-resistance against each antibiotic with the maximum resistance values were recorded against gentamicin: 92% and 79%, oxacillin: 94% and 69%, ceftriaxone: 85% and 58%, and norfloxacin 97% and 69% resistance, in nosocomial and community isolates, respectively. Conclusions This study revealed the daunting state of occurrence of multidrug resistant E. coli and its infection dynamics in both community and hospital settings.

  12. Characteristics of CTX-M Extended-Spectrum β-Lactamase-Producing Escherichia coli Strains Isolated from Multiple Rivers in Southern Taiwan

    PubMed Central

    Chen, Po-An; Hung, Chih-Hsin; Huang, Ping-Chih; Chen, Jung-Ren; Huang, I-Fei; Chen, Wan-Ling; Chiou, Yee-Hsuan; Hung, Wan-Yu

    2016-01-01

    Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli sequence type ST131 has emerged as the leading cause of community-acquired urinary tract infections and bacteremia worldwide. Whether environmental water is a potential reservoir of these strains remains unclear. River water samples were collected from 40 stations in southern Taiwan from February to August 2014. PCR assay and multilocus sequence typing (MLST) analysis were conducted to determine the CTX-M group and sequence type, respectively. In addition, we identified the seasonal frequency of ESBL-producing E. coli strains and their geographical relationship with runoffs from livestock and poultry farms between February and August 2014. ESBL-producing E. coli accounted for 30% of the 621 E. coli strains isolated from river water in southern Taiwan. ESBL-producing E. coli ST131 was not detected among the isolates. The most commonly detected strain was E. coli CTX-M group 9. Among the 92 isolates selected for MLST analysis, the most common ESBL-producing clonal complexes were ST10 and ST58. The proportion of ESBL-producing E. coli was significantly higher in areas with a lower river pollution index (P = 0.025) and regions with a large number of chickens being raised (P = 0.013). ESBL-producing E. coli strains were commonly isolated from river waters in southern Taiwan. The most commonly isolated ESBL-producing clonal complexes were ST10 and ST58, which were geographically related to chicken farms. ESBL-producing E. coli ST131, the major clone causing community-acquired infections in Taiwan and worldwide, was not detected in river waters. PMID:26773082

  13. Effects of cutting process on pork meat contamination by verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7.

    PubMed

    Bouvet, J; Bavai, C; Rossel, R; Le Roux, A; Montet, M P; Ray-Gueniot, S; Mazuy, C; Atrache, V; Vernozy-Rozand, C

    2002-07-25

    The aims of the present study were: (i) to evaluate verotoxin-producing Escherichia coli (VTEC) prevalence in pork cutting meat; (ii) to determine the effects of cutting process on pork meat contamination by VTEC; (iii) to characterise the VTEC strains isolated from pork and pork cutting plants (virulence genes and serotype); and (iv) to compare the strains isolated the same day in the same cutting plant in order to identify the routes of contamination inside the cutting plant. Pork carcasses from three French cutting plants were sampled before carcass cutting (carcass samples), after carcasses were divided into big portions (untrimmed cuts) and after preparation of primal cuts (rindless boneless cuts), and different environmental sites in each cutting plant were sampled at three different times in the work day. Potable water was also collected. PCR detection of stx genes was performed on a total of 2042 samples. In addition, a second PCR specific for E. coli O157:H7 detection was carried out on the stx-positive samples. VTEC strains were recovered from positive samples by colony hybridisation or immunoconcentration, then serotyped, genetically characterised (eae, ehx, stx1, stx2, stx2e, uidA and genes which are associated with virulence) and pulsotyped. No E. coli O157:H7 was detected. Meat contamination decreased from carcass (12%) and primary cuts (19%) to secondary cuts (5%), whereas environmental contamination increased after 2 h of activity (from 3% before the commencement of the work day to 25% and 20%, 2 and 6 h after commencement of cutting). No VTEC isolates harboured eae, ehx and uidA genes. VTEC contamination routes were not clearly identified.

  14. Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

    PubMed Central

    Klein, Eileen J.; Galanakis, Emmanouil; Thomas, Anita A.; Stapp, Jennifer R.; Rich, Shannon; Buccat, Anne Marie; Tarr, Phillip I.

    2015-01-01

    Timely accurate diagnosis of Shiga toxin-producing Escherichia coli (STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targeting stx1, stx2, and rfbEO157 with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagic E. coli [EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC. E. coli O157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 using rfbEO157, and LD-PCR results prompted successful recovery of E. coli O157 (n = 25) and non-O157 STEC (n = 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and that E. coli O157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections. PMID:25926491

  15. Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

    PubMed Central

    Hikoda, Yuna; Fujii, Yuki; Murata, Misato; Miyoshi, Hirotsugu; Ogura, Yoshitoshi; Gotoh, Yasuhiro; Iwata, Taketoshi; Hayashi, Tetsuya; Akiba, Masato

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli. In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria. PMID:26865687

  16. Feed Fermentation with Reuteran- and Levan-Producing Lactobacillus reuteri Reduces Colonization of Weanling Pigs by Enterotoxigenic Escherichia coli.

    PubMed

    Yang, Yan; Galle, Sandra; Le, Minh Hong Anh; Zijlstra, Ruurd T; Gänzle, Michael G

    2015-09-01

    This study determined the effect of feed fermentation with Lactobacillus reuteri on growth performance and the abundance of enterotoxigenic Escherichia coli (ETEC) in weanling piglets. L. reuteri strains produce reuteran or levan, exopolysaccharides that inhibit ETEC adhesion to the mucosa, and feed fermentation was conducted under conditions supporting exopolysaccharide formation and under conditions not supporting exopolysaccharide formation. Diets were chosen to assess the impact of organic acids and the impact of viable L. reuteri bacteria. Fecal samples were taken throughout 3 weeks of feeding; at the end of the 21-day feeding period, animals were euthanized to sample the gut digesta. The feed intake was reduced in pigs fed diets containing exopolysaccharides; however, feed efficiencies did not differ among the diets. Quantification of L. reuteri by quantitative PCR (qPCR) detected the two strains used for feed fermentation throughout the intestinal tract. Quantification of E. coli and ETEC virulence factors by qPCR demonstrated that fermented diets containing reuteran significantly (P < 0.05) reduced the copy numbers of genes for E. coli and the heat-stable enterotoxin in feces compared to those achieved with the control diet. Any fermented feed significantly (P < 0.05) reduced the abundance of E. coli and the heat-stable enterotoxin in colonic digesta at 21 days; reuteran-containing diets reduced the copy numbers of the genes for E. coli and the heat-stable enterotoxin below the detection limit in samples from the ileum, the cecum, and the colon. In conclusion, feed fermentation with L. reuteri reduced the level of colonization of weaning piglets with ETEC, and feed fermentation supplied concentrations of reuteran that may specifically contribute to the effect on ETEC.

  17. An optical biosensor for detection of pathogen biomarkers from Shiga toxin-producing Escherichia coli in ground beef samples

    NASA Astrophysics Data System (ADS)

    Lamoureux, Loreen; Adams, Peter; Banisadr, Afsheen; Stromberg, Zachary; Graves, Steven; Montano, Gabriel; Moxley, Rodney; Mukundan, Harshini

    2014-03-01

    Shiga toxin-producing Escherichia coli (STEC) poses a serious threat to human health through the consumption of contaminated food products, particularly beef and produce. Early detection in the food chain, and discrimination from other non-pathogenic Escherichia coli (E. coli), is critical to preventing human outbreaks, and meeting current agricultural screening standards. These pathogens often present in low concentrations in contaminated samples, making discriminatory detection difficult without the use of costly, time-consuming methods (e.g. culture). Using multiple signal transduction schemes (including novel optical methods designed for amphiphiles), specific recognition antibodies, and a waveguide-based optical biosensor developed at Los Alamos National Laboratory, we have developed ultrasensitive detection methods for lipopolysaccharides (LPS), and protein biomarkers (Shiga toxin) of STEC in complex samples (e.g. beef lysates). Waveguides functionalized with phospholipid bilayers were used to pull down amphiphilic LPS, using methods (membrane insertion) developed by our team. The assay format exploits the amphiphilic biochemistry of lipoglycans, and allows for rapid, sensitive detection with a single fluorescent reporter. We have used a combination of biophysical methods (atomic force and fluorescence microscopy) to characterize the interaction of amphiphiles with lipid bilayers, to efficiently design these assays. Sandwich immunoassays were used for detection of protein toxins. Biomarkers were spiked into homogenated ground beef samples to determine performance and limit of detection. Future work will focus on the development of discriminatory antibodies for STEC serotypes, and using quantum dots as the fluorescence reporter to enable multiplex screening of biomarkers.

  18. High prevalence of CTX-M-15-producing O25b-ST131 Escherichia coli clone in Bulgarian hospitals.

    PubMed

    Markovska, Rumyana; Schneider, Ines; Ivanova, Dobrinka; Keuleyan, Emma; Stoeva, Temenuga; Sredkova, Mariya; Markova, Boyka; Bojkova, Kalina; Gergova, Raina; Bauernfeind, Adolf; Mitov, Ivan

    2012-08-01

    According to the European Antimicrobial Resistance Surveillance System project results, Bulgaria has become one of the European countries with dramatically increasing rates of extended-spectrum beta-lactamase (ESBL) producers. The aim of this work was to investigate the epidemiology of ESBL-producing Escherichia coli clinical isolates in Bulgaria, collected from seven clinical centers in three towns, during two study periods: 2002-2003 and 2006-2009. For 193 ESBL-producing E. coli isolates random amplified polymorphic DNA (RAPD) analyses, phylogenetic typing, and screening for O25b-ST131 isolates were carried out. Antimicrobial susceptibility, ESBL-type and transferability of resistance determinants were analyzed. Four different ESBL-types, namely TEM-139, SHV-12, CTX-M-3, and CTX-M-15 were found. CTX-M-15 dominated, being found in 88% of the isolates. RAPD-typing revealed 35 types, among which type A dominated, comprising 65% of the isolates. Sixty-eight percent of the 193 isolates belonged to the O25b-ST131 clone, to the phylogenetic group B2, mostly showed RAPD-type A (92%) and were found in all participating hospitals. O25b-ST131 isolates predominantly produced CTX-M-15 (96%), and less SHV-12 (n=3) or TEM-139 (n=2). In conclusion, this study demonstrated for the first time the country-wide dissemination of a highly resistant B2 O25b-ST131 CTX-M-15 producing E. coli clone in Bulgaria.

  19. Emergence of Escherichia coli producing extended-spectrum AmpC β-lactamases (ESAC) in animals

    PubMed Central

    Haenni, Marisa; Châtre, Pierre; Madec, Jean-Yves

    2014-01-01

    In both humans and animals, the spread of Extended-Spectrum β-Lactamases (ESBL)/AmpC producers has become a major issue, particularly due to the plasmidic dissemination of most of these genes. Besides, over-expression of the chromosomal ampC gene was largely reported in human and animal Enterobacteriaceae and, more recently, modifications within the coding region of the ampC gene [encoding Extended-spectrum AmpC β-lactamases (ESACs)] were shown to be responsible for an hydrolysis spectrum expanded to oxyiminocephalosporins in humans. In this study, among 6765 cattle E. coli isolates, 28 (0.37%) isolates harboring a reduced susceptibility to cefepime (MICs ranging from 0.5 to 12 μg/ml) were investigated as presumptive ESACs producers. Highly conserved mutations in the promoter/attenuator region were identified at positions −88, −82, −42, −18, −1, and +58. Using sequencing and cloning experiments, amino acid substitutions of the AmpC beta-lactamase were characterized at positions 287 (mostly S287N, but also S287C), 292 (A292V) and 296 (H296P), similarly to data reported in humans. Interestingly, those cattle ESAC-producing E. coli isolates predominantly belonged to the Clonal Complex (CC) 23, thus mirroring what has been described in humans. The driving forces for the selection of ESACs in animals are unknown, and their prevalence needs to be further investigated in the different animal sectors. Considering the over-representation of ESAC-producing E. coli belonging to CC23 in both humans and animals, exchanges of ESAC producers between the two populations may have occurred as well. To our best knowledge, this study is the first report of ESACs in animals worldwide, which should be considered an emerging mechanism contributing to the resistance to extended-spectrum cephalosporins in the animal population. PMID:24592257

  20. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Shiga toxigenic Escherichia coli O104:H4 bares the characteristics of both enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga...

  1. The efficacy of faropenem for patients with acute cystitis caused by extended spectrum β-lactamase producing Escherichia coli.

    PubMed

    Fujino, Keiko; Hiyama, Yoshiki; Uehara, Teruhisa; Ichihara, Koji; Hashimoto, Jiro; Fujii, Satoshi; Shinagawa, Masaaki; Takahashi, Satoshi; Masumori, Naoya

    2016-12-02

    The number of patients with acute cystitis caused by extended spectrum β lactamase (ESBL)-producing Escherichia coli (E. coli) is increasing gradually. Although it is reported that ESBL-producing E. coli are sensitive to faropenem (FRPM), there are few clinical studies on the efficiency of FRPM against acute cystitis caused by the bacteria. Therefore, we retrospectively reviewed the medical charts of patients with acute cystitis caused by ESBL-producing E. coli who were treated with the oral antimicrobial agent faropenem (FRPM) in our institution from June 2011 to May 2015. Ten patients with acute cystitis caused by ESBL producing E. coli were treated with FRPM. Although clinical cure was achieved in 9 of them, it reoccurred in 3. This study revealed that the treatment regimen with FRPM for patients with acute cystitis caused by ESBL-producing E. coli is promising. However, a non-negligible number of recurrences were caused by ESBL-producing E. coli because of the nature of underlying diseases or pathologies in the urinary tract.

  2. Prevalence and Antibiotic Susceptibility Patterns of Extended-Spectrum ß-Lactamase and Metallo-ß-Lactamase-Producing Uropathogenic Escherichia coli Isolates.

    PubMed

    Ghadiri, Hamed; Vaez, Hamid; Razavi-Azarkhiavi, Kamal; Rezaee, Ramin; Haji-Noormohammadi, Mehdi; Rahimi, Ali Asghar; Vaez, Vahid; Kalantar, Enayatollah

    2014-01-01

    Healthcare professionals worldwide have expressed concern over infections by extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL)-producing bacteria. We evaluated the prevalence of ESBL- and MBL-producing Escherichia coli (E. coli) isolated from community-acquired urinary tract infections (UTIs) and their antibiotic-resistance profiles at 3 private laboratories in Tehran, Iran. E. coli isolates were mostly susceptible to meropenem (90.4%) and imipenem (90.0%), followed by amikacin (89.0%) and gentamicin (84.7%). Moreover, we detected that, of the E. coli isolates, 67 (22.3%) were ESBL producers and 21 (7.0%) of E. coli isolates were MBL positive via the imipenem-ethylenediaminetetraacetic acid (EDTA) combined disc test. This report is the first, to our knowledge, on the prevalence of MBL-producing uropathogenic E. coli (UPEC) strains in Iran. The antibiotic resistance of E. coli isolates revealed that 122 (40.7%) were multidrug resistant. The high number of antibiotic-resistant and ß-lactamase-producing UPEC strains necessitates further attention and consideration, particularly MBL-producing strains.

  3. Longitudinal study of CTX-M ESBL-producing E. coli strains on a UK dairy farm.

    PubMed

    Horton, R A; Duncan, D; Randall, L P; Chappell, S; Brunton, L A; Warner, R; Coldham, N G; Teale, C J

    2016-12-01

    The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm.

  4. Hydrogen-producing Escherichia coli strains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress.

    PubMed

    Grube, Mara; Dimanta, Ilze; Gavare, Marita; Strazdina, Inese; Liepins, Janis; Juhna, Talis; Kalnenieks, Uldis

    2014-01-01

    The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the "lactose killing" phenomenon. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid but with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of Fourier transform-infrared spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress.

  5. Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

    PubMed

    Sudhamsu, Jawahar; Kabir, Mariam; Airola, Michael V; Patel, Bhumit A; Yeh, Syun-Ru; Rousseau, Denis L; Crane, Brian R

    2010-09-01

    Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

  6. Direct ertapenem disk screening method for identification of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance swab specimens.

    PubMed

    Lolans, Karen; Calvert, Karen; Won, Sarah; Clark, James; Hayden, Mary K

    2010-03-01

    Klebsiella pneumoniae carbapenemase (KPC) production in Gram-negative bacilli is an increasing problem worldwide. Rectal swab surveillance is recommended as a component of infection prevention programs, yet few screening methods are published. We compared detection of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance specimens by 2 methods: (i) inoculation of swabs in tryptic soy broth containing 2 microg/ml imipenem followed by plating to MacConkey agar (MAC) (method 1) and (ii) streaking swabs on MAC onto which a 10-microg ertapenem disk was then placed (method 2). Simulated rectal swab specimens of challenge isolates from a collection of well-characterized K. pneumoniae and E. coli strains and salvage rectal swab specimens collected from patients at 4 different health care facilities over a 7-month period were tested. The gold-standard comparator was bla(KPC) PCR testing of isolates. Method 1 detected 4/9 (44%) KPC-positive challenge isolates. By method 2, 9/9 KPC-positive challenge isolates exhibited zones of inhibition of < or = 27 mm; all KPC-negative isolates exhibited zones of inhibition greater than 27 mm. The sensitivity and specificity of method 1 for detection of KPC-positive K. pneumoniae and E. coli in 149 rectal swab specimens were 65.6% (95% confidence interval [CI], 46.8% to 80.8%) and 49.6% (95% CI, 40.3% to 58.9%), respectively. With method 2, a zone diameter of < or = 27 mm had a sensitivity of 97.0% (95% CI, 82.5% to 99.8%) and specificity of 90.5% (95% CI, 83.3% to 94.9%) for detection of KPC in rectal swab specimens. Direct ertapenem disk testing is simpler, more sensitive, and more specific than selective broth enrichment with imipenem for detection of KPC-producing K. pneumoniae and E. coli in surveillance specimens.

  7. Life on the outside: role of biofilms in environmental persistence of Shiga-toxin producing Escherichia coli

    PubMed Central

    Vogeleer, Philippe; Tremblay, Yannick D. N.; Mafu, Akier A.; Jacques, Mario; Harel, Josée

    2014-01-01

    Escherichia coli is a heterogeneous species that can be part of the normal flora of humans but also include strains of medical importance. Among pathogenic members, Shiga-toxin producing E. coli (STEC) are some of the more prominent pathogenic E. coli within the public sphere. STEC disease outbreaks are typically associated with contaminated beef, contaminated drinking water, and contaminated fresh produce. These water- and food-borne pathogens usually colonize cattle asymptomatically; cows will shed STEC in their feces and the subsequent fecal contamination of the environment and processing plants is a major concern for food and public safety. This is especially important because STEC can survive for prolonged periods of time outside its host in environments such as water, produce, and farm soil. Biofilms are hypothesized to be important for survival in the environment especially on produce, in rivers, and in processing plants. Several factors involved in biofilm formation such as curli, cellulose, poly-N-acetyl glucosamine, and colanic acid are involved in plant colonization and adherence to different surfaces often found in meat processing plants. In food processing plants, contamination of beef carcasses occurs at different stages of processing and this is often caused by the formation of STEC biofilms on the surface of several pieces of equipment associated with slaughtering and processing. Biofilms protect bacteria against several challenges, including biocides used in industrial processes. STEC biofilms are less sensitive than planktonic cells to several chemical sanitizers such as quaternary ammonium compounds, peroxyacetic acid, and chlorine compounds. Increased resistance to sanitizers by STEC growing in a biofilm is likely to be a source of contamination in the processing plant. This review focuses on the role of biofilm formation by STEC as a means of persistence outside their animal host and factors associated with biofilm formation. PMID:25071733

  8. Life on the outside: role of biofilms in environmental persistence of Shiga-toxin producing Escherichia coli.

    PubMed

    Vogeleer, Philippe; Tremblay, Yannick D N; Mafu, Akier A; Jacques, Mario; Harel, Josée

    2014-01-01

    Escherichia coli is a heterogeneous species that can be part of the normal flora of humans but also include strains of medical importance. Among pathogenic members, Shiga-toxin producing E. coli (STEC) are some of the more prominent pathogenic E. coli within the public sphere. STEC disease outbreaks are typically associated with contaminated beef, contaminated drinking water, and contaminated fresh produce. These water- and food-borne pathogens usually colonize cattle asymptomatically; cows will shed STEC in their feces and the subsequent fecal contamination of the environment and processing plants is a major concern for food and public safety. This is especially important because STEC can survive for prolonged periods of time outside its host in environments such as water, produce, and farm soil. Biofilms are hypothesized to be important for survival in the environment especially on produce, in rivers, and in processing plants. Several factors involved in biofilm formation such as curli, cellulose, poly-N-acetyl glucosamine, and colanic acid are involved in plant colonization and adherence to different surfaces often found in meat processing plants. In food processing plants, contamination of beef carcasses occurs at different stages of processing and this is often caused by the formation of STEC biofilms on the surface of several pieces of equipment associated with slaughtering and processing. Biofilms protect bacteria against several challenges, including biocides used in industrial processes. STEC biofilms are less sensitive than planktonic cells to several chemical sanitizers such as quaternary ammonium compounds, peroxyacetic acid, and chlorine compounds. Increased resistance to sanitizers by STEC growing in a biofilm is likely to be a source of contamination in the processing plant. This review focuses on the role of biofilm formation by STEC as a means of persistence outside their animal host and factors associated with biofilm formation.

  9. FACTORS ASSOCIATED WITH EXTENDED SPECTRUM β-LACTAMASE PRODUCING ESCHERICHIA COLI IN COMMUNITY-ACQUIRED URINARY TRACT INFECTION AT HOSPITAL EMERGENCY DEPARTMENT, BANGKOK, THAILAND.

    PubMed

    Savatmorigkorngul, Sorravit; Poowarattanawiwit, Pongsuree; Sawanyawisuth, Kittisak; Sittichanbuncha, Yuwares

    2016-03-01

    Urinary tract infection or UTI is most commonly caused by Escherichia coli. This study investigated the prevalence of and risk factors for extended spectrum β-lactamase-producing (ESBL) E. coli in community-acquired UTI presenting at the Emergency Department, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. A retrospective review was conducted over a one-year period (2014) of case histories of patients over 15 years of age diagnosed with (n = 159) and without culture-positive (n = 249) ESBL E. coli. Backward stepwise multivariate logistic regression analysis revealed four independent risk factors for UTI caused by ESBL E. coli, namely, urinary catheter use, previous UTI in which ESBL E. coli was present, and previous use of antibiotics cephalosporin and penicillin. This information should be useful in devising future public health prevention and control programs for ESBL E. coli-associated community-acquired UTI.

  10. Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.

    PubMed Central

    Vogel, T; Weisgraber, K H; Zeevi, M I; Ben-Artzi, H; Levanon, A Z; Rall, S C; Innerarity, T L; Hui, D Y; Taylor, J M; Kanner, D

    1985-01-01

    Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein. Images PMID:3909150

  11. Flagella interact with ionic plant lipids to mediate adherence of pathogenic Escherichia coli to fresh produce plants.

    PubMed

    Rossez, Yannick; Holmes, Ashleigh; Wolfson, Eliza B; Gally, David L; Mahajan, Arvind; Pedersen, Henriette L; Willats, William G T; Toth, Ian K; Holden, Nicola J

    2014-07-01

    Bacterial attachment to plant and animal surfaces is generally thought to constitute the initial step in colonization, requiring adherence factors such as flagella and fimbriae. We describe the molecular mechanism underpinning flagella-mediated adherence to plant tissue for the foodborne pathogen, enterohaemorrhagic Escherichia coli. Escherichia coli H7 flagella interacted with a sulphated carbohydrate (carrageenan) on a glycan array, which occurred in a dose-dependent manner. Adherence of E. coli O157 : H-expressing flagella of serotype H7, H6 or H48 to plants associated with outbreaks from fresh produce and to Arabidopsis thaliana, was dependent on flagella interactions with phospholipids and sulpholipids in plasma membranes. Adherence of purified H7 and H48 flagella to carrageenan was reduced at higher concentrations of KH2 PO4 or KCl, showing an ionic basis to the interactions. Purified H7 flagella were observed to physically interact with plasma membranes in spinach plants and in A.thaliana. The results show a specific interaction between E. coli H7, H6 and H48 flagella and ionic lipids in plant plasma membranes. The work extends our understanding of the molecular mechanisms underpinning E.coli flagella targeting of plant hosts and suggests a generic mechanism of recognition common in eukaryotic hosts belonging to different biological kingdoms.

  12. Identification and Molecular Characterization of Antimicrobial-Resistant Shiga Toxin–Producing Escherichia coli Isolated from Retail Meat Products

    PubMed Central

    Li, Ming-Cheng; Wang, Fang

    2011-01-01

    Abstract Ten (2.7%) Shiga toxin–producing Escherichia coli (STEC) were isolated from 370 samples of raw minced beef, mutton, pork, and chicken from the Jilin region of China; and additional 10 E. coli O157:H7 isolates were previously isolated from different Jilin regions. Seventeen of the isolates were multiresistant, exhibiting resistance to ampicillin, ciprofloxacin, tetracycline, sulfamethoxazole-trimethoprim, gentamycin, and streptomycin. Class 1 integrons were detected in nine (45.0%) of the STEC isolates and consisted of serogroups O157, O62, O113, O149, and O70. Integrons containing amplicons of a 0.5–1.5 or 1.0 kb gene cassette were found in seven (77.8%) of the integron-containing isolates. Sequencing analysis revealed that these gene cassettes encode genes conferring resistance to trimethoprim (dfrA1) and streptomycin (aadA1). The 0.5 kb cassette described here was found to encode a putative transporter peptide in the STEC. Seventeen isolates contained plasmids with different bands, and transfer by conjugation between strains of E. coli demonstrated that class 1 integrons located on mobile plasmids could contribute to the emergence and dissemination of antimicrobial resistance to ampicillin, gentamycin, streptomycin, and sulfamethoxazole-trimethoprim amongst STEC. These data revealed the high prevalence of antimicrobial-resistant STEC isolates in Jilin's surrounding regions, providing important and useful surveillance information reflecting antimicrobial selection pressure. PMID:21453117

  13. Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

    PubMed

    Chin, Young-Wook; Seo, Nari; Kim, Jae-Han; Seo, Jin-Ho

    2016-11-01

    2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of β-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc.

  14. Characterization of tailor-made copolymers of oligo(ethylene glycol) methyl ether methacrylate and N,N-dimethylaminoethyl methacrylate as nonviral gene transfer agents: influence of macromolecular structure on gene vector particle properties and transfection efficiency.

    PubMed

    Uzgün, Senta; Akdemir, Ozgür; Hasenpusch, Günther; Maucksch, Christof; Golas, Monika M; Sander, Bjoern; Stark, Holger; Imker, Rabea; Lutz, Jean-François; Rudolph, Carsten

    2010-01-11

    Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F(OEGMA)) but a comparable degree of polymerization (DP approximately 120). Increase of both F(OEGMA) and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

  15. Novel method for the rapid and specific extraction of multiple β2 -agonist residues in food by tailor-made Monolith-MIPs extraction disks and detection by gas chromatography with mass spectrometry.

    PubMed

    Liu, Haibo; Gan, Ning; Chen, Yinji; Ding, Qingqing; Huang, Jie; Lin, Saichai; Cao, Yuting; Li, Tianhua

    2016-09-01

    A quick and specific pretreatment method based on a series of extraction clean-up disks, consisting of molecularly imprinted polymer monoliths and C18 adsorbent, was developed for the specific enrichment of salbutamol and clenbuterol residues in food. The molecularly imprinted monolithic polymer disk was synthesized using salbutamol as a template through a one-step synthesis process. It can simultaneously and specifically recognize salbutamol and clenbuterol. The monolithic polymer disk and series of C18 disks were assembled with a syringe to form a set of tailor-made devices for the extraction of target molecules. In a single run, salbutamol and clenbuterol can be specifically extracted, cleaned, and eluted by methanol/acetic acid/H2 O. The target molecules, after a silylation derivatization reaction were detected by gas chromatography-mass spectrometry. The parameters including solvent desorption, sample pH, and the cycles of reloading were investigated and discussed. Under the optimized extraction and clean-up conditions, the limits of detection and quantitation were determined as 0.018-0.022 and 0.042-0.049 ng/g for salbutamol and clenbuterol, respectively. The assay described was convenient, rapid, and specific; thereby potentially efficient in the high-throughput analysis of β2 -agonists residues in real food samples.

  16. Validation of the Applied Biosystems RapidFinder Shiga Toxin-Producing E. coli (STEC) Detection Workflow.

    PubMed

    Cloke, Jonathan; Matheny, Sharon; Swimley, Michelle; Tebbs, Robert; Burrell, Angelia; Flannery, Jonathan; Bastin, Benjamin; Bird, Patrick; Benzinger, M Joseph; Crowley, Erin; Agin, James; Goins, David; Salfinger, Yvonne; Brodsky, Michael; Fernandez, Maria Cristina

    2016-11-01

    The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant

  17. Occurrence and characterization of Shiga toxin-producing Escherichia coli O157:H7 and other non-sorbitol-fermenting E. coli in cattle and humans in urban areas of Morogoro, Tanzania.

    PubMed

    Lupindu, Athumani M; Olsen, John E; Ngowi, Helena A; Msoffe, Peter L M; Mtambo, Madundo M; Scheutz, Flemming; Dalsgaard, Anders

    2014-07-01

    Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment

  18. Molecular and clinical characterization of plasmid-mediated AmpC β-lactamase-producing Escherichia coli bacteraemia: a comparison with extended-spectrum β-lactamase-producing and non-resistant E. coli bacteraemia.

    PubMed

    Matsumura, Y; Nagao, M; Iguchi, M; Yagi, T; Komori, T; Fujita, N; Yamamoto, M; Matsushima, A; Takakura, S; Ichiyama, S

    2013-02-01

    Plasmid-mediated AmpC β-lactamase-producing Escherichia coli (AmpC-E) bacteraemia was characterized by comparison with bacteraemia caused by extended-spectrum β-lactamase (ESBL)-producing E. coli (ESBL-E) and non-resistant E. coli (NR-E) in the era of the worldwide spread of the CTX-M-15-producing O25b-ST131-B2 clone. Of 706 bloodstream E. coli isolates collected between 2005 and 2010 in three Japanese university hospitals, 111 ESBL screening-positive isolates were analysed for AmpC and ESBL genes by PCR. A case-control study was performed in which the cases consisted of all of the patients with AmpC-E bacteraemia. Phylogenetic groups, sequence types and O25b serotype were determined. Twenty-seven AmpC-E isolates (26 of which were of the CMY-2 type) were identified, and 54 ESBL-E and 54 NR-E isolates were selected for the controls. Nineteen AmpC-E isolates were also positive for ESBL. CTX-M-14 was the most prevalent ESBL type among both the AmpC-E and ESBL-E isolates. The O25b-ST131-B2 clone was the most prevalent among the ESBL-E isolates (26%) and the second most prevalent among the NR-E isolates (13%), but only one O25b-ST131-B2 clone was found among the AmpC-E isolates. Twenty-three different sequence types were identified among the AmpC-E isolates. When compared with bacteraemia with ESBL-E, previous isolation of multidrug-resistant bacteria and intravascular catheterization were independently associated with a lower risk for AmpC-E. When compared with NR-E bacteraemia, prior use of antibiotics was the only significant risk factor for AmpC-E. Unlike the spread of the O25b-ST131-B2 clone between ESBL-E and NR-E, the AmpC-E isolates were not dominated by any specific clone.

  19. Clinical Epidemiology and Molecular Analysis of Extended-Spectrum-β-Lactamase-Producing Escherichia coli in Nepal: Characteristics of Sequence Types 131 and 648

    PubMed Central

    Sherchan, Jatan Bahadur; Miyoshi-Akiyama, Tohru; Ohmagari, Norio; Kirikae, Teruo; Nagamatsu, Maki; Tojo, Masayoshi; Ohara, Hiroshi; Sherchand, Jeevan B.; Tandukar, Sarmila

    2015-01-01

    Recently, CTX-M-type extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains have emerged worldwide. In particular, E. coli with O antigen type 25 (O25) and sequence type 131 (ST131), which is often associated with the CTX-M-15 ESBL, has been increasingly reported globally; however, epidemiology reports on ESBL-producing E. coli in Asia are limited. Patients with clinical isolates of ESBL-producing E. coli in the Tribhuvan University teaching hospital in Kathmandu, Nepal, were included in this study. Whole-genome sequencing of the isolates was conducted to analyze multilocus sequence types, phylotypes, virulence genotypes, O25b-ST131 clones, and distribution of acquired drug resistance genes. During the study period, 105 patients with ESBL-producing E. coli isolation were identified, and the majority (90%) of these isolates were CTX-M-15 positive. The most dominant ST was ST131 (n = 54; 51.4%), followed by ST648 (n = 15; 14.3%). All ST131 isolates were identified as O25b-ST131 clones, subclone H30-Rx. Three ST groups (ST131, ST648, and non-ST131/648) were compared in further analyses. ST648 isolates had a proportionally higher resistance to non-β-lactam antibiotics and featured drug-resistant genes more frequently than ST131 or non-ST131/648 isolates. ST131 possessed the most virulence genes, followed by ST648. The clinical characteristics were similar among groups. More than 38% of ESBL-producing E. coli isolates were from the outpatient clinic, and pregnant patients comprised 24% of ESBL-producing E. coli cases. We revealed that the high resistance of ESBL-producing E. coli to multiple classes of antibiotics in Nepal is driven mainly by CTX-M-producing ST131 and ST648. Their immense prevalence in the communities is a matter of great concern. PMID:25824221

  20. [Detection and characterization of verocytoxin-producing Escherichia coli types (VTEC) from different sources].

    PubMed

    Bülte, M

    2001-01-01

    Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic E. coli (EHEC) belonging to a few serovars embracing strains of O26, O103, O111, O118, O145 and O157 serogroup, respectively. In own investigations 3.791 food specimen of animal origin were investigated by use of an enzyme-immuno-assay (EIA) and the polymerase chain reaction (PCR). All E. coli isolates (n = 459) of food as well as isolates from cattle feces (n = 440), from HUS patients (n = 50) and asymptomatic human carriers (n = 16) were investigated by means of the PCR using primer pairs for verocytotoxin genes (vtx1, vtx2, vtx2c, vtx2d, vtx2e), the E. coli attaching und effacing gene (eae), the enterohemolysin-gene (ehlyA) and the vt2 transporter protein-gene (ile X tRNA). Differences were found in respect to the eae- and the ile X tRNA genes, which could be detected in significantly higher ratios in the isolates from patients and human carriers. Furthermore vtx2d strains were exclusively analyzed to each 25% in food and cattle strains. In six food samples pathogenic strains of serovar O157 were detected whereas some of the cattle strains were estimated to belong to EHEC serovars O26:H11, O118:H- and O157:H7. The own data support the thesis that the risk for human beings is affiliated to a great extent by direct contact with ruminants, followed by person-to-person transmission. Regarding the epidemiological data the thesis that each VTEC strain ist potentially an EHEC strain can not longer be substantiated.

  1. Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains

    PubMed Central

    Brando, R J F; Miliwebsky, E; Bentancor, L; Deza, N; Baschkier, A; Ramos, M V; Fernández, G C; Meiss, R; Rivas, M; Palermo, M S

    2008-01-01

    Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infections are considered a public health problem in both developed and developing countries because of their increasing incidence and the severity of clinical presentation. Approximately 10% of infected patients develop complications such as haemolytic uraemic syndrome (HUS) characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. The precise sequence of events leading to HUS is still understood incompletely. Because of the lack of a reproducible small animal model for EHEC infections, in vivo studies examining EHEC–host early interactions are limited and insufficient. The aim of this study was to characterize the weaned BALB/c mouse as a model of E. coli O157:H7 infection. In this paper we report that human Shiga toxin 2 (Stx2)-producing EHEC strains can adhere to the intestinal epithelium of weaned BALB/c mice, and produce local damage which leads to systemic disease and death in a percentage of infected mice. The lethality of the EHEC strain is closely age-dependent, and is related to the bacterial ability to colonize intestine and to produce Stx2. It can be concluded that the weaned BALB/c mouse can be used as a small animal model to study host early responses, and the role of bacterial pathogenic factors in the induction of systemic disease, thus providing a useful tool for the evaluation of therapeutic or vaccine approaches. PMID:18549440

  2. Shiga Toxin-Producing Escherichia coli in Meat and Vegetable Products in Emilia Romagna Region, Years 2012-2013

    PubMed Central

    Taddei, Roberta; Nocera, Lucia; Ricchi, Matteo; Merialdi, Giuseppe

    2015-01-01

    In 2012-2013 Emilia-Romagna Region introduced a monitoring plan for Shiga toxin-producing Escherichia coli (STEC) in foodstuff. Six hundred eighty-nine meat samples and 273 fruit and vegetable products were analyzed according to ISOTS13136. Pre-enriched samples were tested by multiplex real time PCR targeting the virulence genes eae, stx1 and stx2. Stx2 positive samples were investigated for the presence of serogroup O104 associated gene. O103, O111, O145, O157, O26 associated genes were tested on samples positive for stx in association with eae gene. Isolation of E. coli strains was attempted from samples positive for serogroup-associated genes. Thirty-four meat products (4.9%) resulted positive for stx1 and/or stx2 genes and 46 (6.7%) for stx1 and/or stx2 genes in association with eae gene. Forty-five (6.5%) samples resulted positive at least at one serogroup. Serogroup O103, O104, O111, O145, O157 and O26 genes were detected respectively in 1.3, 0.3, 0.1, 3.9, 2.9 and 2.5% samples; 0.6% samples resulted positive for STEC isolation (2 E. coli O103 and 2 E. coli O157). It is worth noting that STEC virulence genes were detected at high frequency (19%) in fresh pork meat sausages. Four (1.5%) vegetable samples were positive for stx1 and/or stx2 genes and 1 (0.4%) for stx1 and/or stx2 genes in association with eae gene; none resulted positive for the tested serogroups. Only a low number of samples positive by molecular methods were confirmed by cultural isolation. It is therefore of the uttermost importance for appropriate risk management, to be fully aware of the meaning of the analytical result. PMID:27800377

  3. NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China.

    PubMed

    Huang, Ying-Min; Zhong, Lan-Lan; Zhang, Xue-Fei; Hu, Hang-Tong; Li, Yu-Qi; Yang, Xiao-Rong; Feng, Lian-Qiang; Huang, Xi; Tian, Guo-Bao

    2015-08-01

    We identified New Delhi metallo-β-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii.

  4. Applicability of Phylogenetic Methods for Characterizing the Public Health Significance of Verocytotoxin-Producing Escherichia coli Strains▿

    PubMed Central

    Ziebell, Kim; Konczy, Paulina; Yong, Irene; Frost, Shelley; Mascarenhas, Mariola; Kropinski, Andrew M.; Whittam, Thomas S.; Read, Susan C.; Karmali, Mohamed A.

    2008-01-01

    Two phylogenetic methods (multilocus sequence typing [MLST] and a multiplex PCR) were investigated to determine whether phylogenetic classification of verocytotoxin-producing Escherichia coli serotypes correlates with their classification into groups (seropathotypes A to E) based on their relative incidence in human disease and on their association with outbreaks and serious complications. MLST was able to separate 96% of seropathotype D and E serotypes from those that cause serious disease (seropathotypes A to C), whereas the multiplex PCR lacked this level of seropathotype discrimination. PMID:18165362

  5. NDM-1-Producing Citrobacter freundii, Escherichia coli, and Acinetobacter baumannii Identified from a Single Patient in China

    PubMed Central

    Huang, Ying-Min; Zhong, Lan-lan; Zhang, Xue-Fei; Hu, Hang-tong; Li, Yu-qi; Yang, Xiao-rong; Feng, Lian-Qiang; Huang, Xi

    2015-01-01

    We identified New Delhi metallo-β-lactamase (NDM-1)-producing Citrobacter freundii GB032, Escherichia coli GB102, and Acinetobacter baumannii GB661 in urine and stool samples from a single patient in China. Plasmid profiling and Southern blotting indicated that blaNDM-1 from GB032 and that from GB102 were likely located on the same plasmid, while blaNDM-1 from GB661 was located on a very large (>400-kb) plasmid. This case underscores the broad host range of blaNDM-1 and its potential to spread between members of the family Enterobacteriaceae and A. baumannii. PMID:26055374

  6. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-02-24

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding.

  7. Characterization of Multidrug Resistant Extended-Spectrum Beta-Lactamase-Producing Escherichia coli among Uropathogens of Pediatrics in North of Iran

    PubMed Central

    Rezai, Mohammad Sadegh; Salehifar, Ebrahim; Rafiei, Alireza; Rafati, Mohammadreza; Shafahi, Kheironesa

    2015-01-01

    Escherichia coli remains as one of the most important bacteria causing infections in pediatrics and producing extended-spectrum beta-lactamases (ESBLs) making them resistant to beta-lactam antibiotics. In this study we aimed to genotype ESBL-producing E. coli isolates from pediatric patients for ESBL genes and determine their association with antimicrobial resistance. One hundred of the E. coli isolates were initially considered ESBL producing based on their MIC results. These isolates were then tested by polymerase chain reaction (PCR) for the presence or absence of CTX, TEM, SHV, GES, and VEB beta-lactamase genes. About 30.5% of isolated E. coli was ESBL-producing strain. The TEM gene was the most prevalent (49%) followed by SHV (44%), CTX (28%), VEB (8%), and GES (0%) genes. The ESBL-producing E. coli isolates were susceptible to carbapenems (66%) and amikacin (58%) and showed high resistance to cefixime (99%), colistin (82%), and ciprofloxacin (76%). In conclusion, carbapenems were the most effective antibiotics against ESBl-producing E. coli in urinary tract infection in North of Iran. The most prevalent gene is the TEM-type, but the other resistant genes and their antimicrobial resistance are on the rise. PMID:26064896

  8. Evaluation of a loop-mediated isothermal amplification suite for the rapid, reliable, and robust detection of Shiga toxin-producing Escherichia coli in produce.

    PubMed

    Wang, Fei; Yang, Qianru; Qu, Yinzhi; Meng, Jianghong; Ge, Beilei

    2014-04-01

    Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 10(5) to 10(6) CFU per 25 g (i.e., 10(3) to 10(4) CFU per g) in produce, except for strains harboring the stx2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx2 and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.

  9. The long polar fimbriae genes identified in Shiga toxin-producing Escherichia coli are present in other diarrheagenic E. coli and in the standard E. coli collection of reference (ECOR) strains.

    PubMed

    Toma, Claudia; Higa, Naomi; Iyoda, Sunao; Rivas, Marta; Iwanaga, Masaaki

    2006-03-01

    Long polar fimbriae (LPF) are related to type I fimbriae in genetic organization and were first identified in Salmonella enterica serovar Typhimurium. Four lpfA genetic variants designated lpfA(O157/OI-141), lpfA(O157/OI-154), lpfA(O26) and lpfA(O113) have been identified in Shiga toxin-producing Escherichia coli (STEC). In this study, PCR was employed to determine the distribution of STEC-lpfAs in enteropathogenic, enteroaggregative, enterotoxigenic and enteroinvasive E. coli (EPEC, EAEC, ETEC and EIEC) and in the standard E. coli collection of reference (ECOR). Among the 97 diarrheagenic strains from our collection, only 2 EPEC strains of serotypes O55:H7 and O119:NM were positive for both lpfA(O157/OI-141) and lpfA(O157/OI-154). lpfA(O157/OI-141) was also positive in 1 of 25 ETEC strains. lpfA(O113) was present in 51 of 97 strains and lpfA(O26) in 13 of 97 strains of diverse diarrheagenic categories. STEC-lpfAs were also present in non-pathogenic ECOR strains of all phylogenetic groups. This study showed that the lpfA genes identified in the genome of STEC strains are not specific to this category. Our results suggest that there is a relationship between the lpfA variant and the phylogenetic group.

  10. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

    PubMed Central

    Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; da Costa, Elaine Sobral; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells. PMID:27253887

  11. Metabolic engineering of Escherichia coli W3110 to produce L-malate.

    PubMed

    Dong, Xiaoxiang; Chen, Xiulai; Qian, Yuanyuan; Wang, Yuancai; Wang, Li; Qiao, Weihua; Liu, Liming

    2017-03-01

    A four-carbon dicarboxylic acid L-malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L-malate production via one-step L-malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), pyruvate formate lyase (pflB), phosphotransacetylase (pta), and acetate kinase A (ackA) in pta-ackA pathway led to accumulate 20.9 g/L pyruvate. Then, overexpression of NADP(+) -dependent malic enzyme C490S mutant in this multi-deletion mutant resulted in the direct conversion of pyruvate into L-malate (3.62 g/L). Next, deletion of the genes responsible for succinate biosynthesis further enhanced L-malate production up to 7.78 g/L. Finally, L-malate production was elevated to 21.65 g/L with the L-malate yield to 0.36 g/g in a 5 L bioreactor by overexpressing the pos5 gene encoding NADH kinase in the engineered E. coli F0931 strain. This study demonstrates the potential utility of one-step pathway for efficient L-malate production. Biotechnol. Bioeng. 2017;114: 656-664. © 2016 Wiley Periodicals, Inc.

  12. Nickel Promotes Biofilm Formation by Escherichia coli K-12 Strains That Produce Curli▿

    PubMed Central

    Perrin, Claire; Briandet, Romain; Jubelin, Gregory; Lejeune, Philippe; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès; Dorel, Corinne

    2009-01-01

    The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated. PMID:19168650

  13. Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli.

    PubMed

    Einsfeldt, Karen; Baptista, Isis Cavalcante; Pereira, Juliana Christina Castanheira Vicente; Costa-Amaral, Isabele Campos; Costa, Elaine Sobral da; Ribeiro, Maria Cecília Menks; Land, Marcelo Gerardin Poirot; Alves, Tito Lívio Moitinho; Larentis, Ariane Leites; Almeida, Rodrigo Volcan

    2016-01-01

    L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in silico analysis of the protein from Z. mobilis indicated the presence of a signal peptide in the sequence, as well as high identity to other sequences of L-asparaginases with antileukemic activity. The protein was expressed in a bioreactor with a complex culture medium, yielding 0.13 IU/mL extracellular L-asparaginase and 3.6 IU/mL intracellular L-asparaginase after 4 h of induction with IPTG. The cytotoxicity results suggest that recombinant L-asparaginase from Z. mobilis expressed extracellularly in E.coli has a cytotoxic and cytostatic effect on leukemic cells.

  14. Portable microfluidic chip for detection of Escherichia coli in produce and blood

    PubMed Central

    Wang, ShuQi; Inci, Fatih; Chaunzwa, Tafadzwa L; Ramanujam, Ajay; Vasudevan, Aishwarya; Subramanian, Sathya; Chi Fai Ip, Alexander; Sridharan, Banupriya; Gurkan, Umut Atakan; Demirci, Utkan

    2012-01-01

    Pathogenic agents can lead to severe clinical outcomes such as food poisoning, infection of open wounds, particularly in burn injuries and sepsis. Rapid detection of these pathogens can monitor these infections in a timely manner improving clinical outcomes. Conventional bacterial detection methods, such as agar plate culture or polymerase chain reaction, are time-consuming and dependent on complex and expensive instruments, which are not suitable for point-of-care (POC) settings. Therefore, there is an unmet need to develop a simple, rapid method for detection of pathogens such as Escherichia coli. Here, we present an immunobased microchip technology that can rapidly detect and quantify bacterial presence in various sources including physiologically relevant buffer solution (phosphate buffered saline [PBS]), blood, milk, and spinach. The microchip showed reliable capture of E. coli in PBS with an efficiency of 71.8% ± 5% at concentrations ranging from 50 to 4,000 CFUs/mL via lipopolysaccharide binding protein. The limits of detection of the microchip for PBS, blood, milk, and spinach samples were 50, 50, 50, and 500 CFUs/mL, respectively. The presented technology can be broadly applied to other pathogens at the POC, enabling various applications including surveillance of food supply and monitoring of bacteriology in patients with burn wounds. PMID:22679370

  15. Nickel promotes biofilm formation by Escherichia coli K-12 strains that produce curli.

    PubMed

    Perrin, Claire; Briandet, Romain; Jubelin, Gregory; Lejeune, Philippe; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès; Dorel, Corinne

    2009-03-01

    The survival of bacteria exposed to toxic compounds is a multifactorial phenomenon, involving well-known molecular mechanisms of resistance but also less-well-understood mechanisms of tolerance that need to be clarified. In particular, the contribution of biofilm formation to survival in the presence of toxic compounds, such as nickel, was investigated in this study. We found that a subinhibitory concentration of nickel leads Escherichia coli bacteria to change their lifestyle, developing biofilm structures rather than growing as free-floating cells. Interestingly, whereas nickel and magnesium both alter the global cell surface charge, only nickel promotes biofilm formation in our system. Genetic evidence indicates that biofilm formation induced by nickel is mediated by the transcriptional induction of the adhesive curli-encoding genes. Biofilm formation induced by nickel does not rely on efflux mechanisms using the RcnA pump, as these require a higher concentration of nickel to be activated. Our results demonstrate that the nickel-induced biofilm formation in E. coli is an adaptational process, occurring through a transcriptional effect on genes coding for adherence structures. The biofilm lifestyle is obviously a selective advantage in the presence of nickel, but the means by which it improves bacterial survival needs to be investigated.

  16. Prosequence switching: an effective strategy to produce biologically active E. coli heat-stable enterotoxin STh.

    PubMed

    Weiglmeier, Philipp R; Berkner, Hanna; Seebahn, Angela; Vogel, Nico; Schreiber, Rainer; Wöhrl, Birgitta M; Schwarzinger, Stephan; Rösch, Paul

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) infections account for the majority of cases of acute secretory diarrhea. The causative agents are enterotoxins secreted by ETEC, among them is the heat-stable enterotoxin, STh. STh is a 19-amino acid peptide containing three disulfide bonds that stimulates fluid secretion in the bowel by binding to the receptor domain of intestinal guanylyl cyclase C (GC-C). Since GC-C agonists have pharmacologic potential for diagnosis and treatment of disorders such as constipation-predominant irritable bowel syndrome (IBS-C), chronic constipation, and colorectal carcinoma, it is crucial to develop methods for the large-scale production of STh and related peptides. Here, we present a strategy for recombinant expression of STh that relies on the use of the prosequence of human uroguanylin to support proper folding and disulfide bond formation. The chimeric protein CysCys-STh consisting of the propeptide of uroguanylin as N-terminus and the STh peptide as C-terminus was expressed in E. coli, and an efficient purification protocol was developed. Trypsin digestion of this protein released the enterotoxin which could be obtained in high purity. NMR and mass spectrometry confirmed the identity and homogeneity of the toxin, and its biological activity was confirmed by a cell-based in vivo assay. The expression scheme introduced here represents a cost-efficient and scalable way of STh production.

  17. Prevalence and characteristics of the epidemic multiresistant Escherichia coli ST131 clonal group among extended-spectrum beta-lactamase-producing E. coli isolates in Copenhagen, Denmark.

    PubMed

    Olesen, Bente; Hansen, Dennis S; Nilsson, Frida; Frimodt-Møller, Jakob; Leihof, Rikke Fleron; Struve, Carsten; Scheutz, Flemming; Johnston, Brian; Krogfelt, Karen A; Johnson, James R

    2013-06-01

    We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.

  18. Genetic characterization of Shiga toxin-producing Escherichia coli (STEC) and atypical enteropathogenic Escherichia coli (EPEC) isolates from goat's milk and goat farm environment.

    PubMed

    Álvarez-Suárez, María-Elena; Otero, Andrés; García-López, María-Luisa; Dahbi, Ghizlane; Blanco, Miguel; Mora, Azucena; Blanco, Jorge; Santos, Jesús A

    2016-11-07

    The aim of this study was to characterize a collection of 44 Shiga toxin-producing (STEC) and enteropathogenic Escherichia coli (EPEC) isolated from goat milk and goat farm environment. Of the 19 STEC isolates, five (26.3%) carried the stx1 gene, four (21.1%) the stx2 gene and 10 (52.6%) presented both stx genes. Six (31.6%) STEC strains were eae-positive and belonged to serotypes related to severe human disease (O157:H7 and O5:HNM). Another seven STEC strains were of serotype O146:H21 and three of serotype O166:H28, also linked to human disease. The STEC strains isolated from goat milk were of serotypes potentially pathogenic for humans. All the 25 EPEC isolates were considered atypical (aEPEC) and one aEPEC strain was of serotype O26:H11, a serotype frequently isolated in children with diarrhea. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 23 sequence types (ST) were detected, 14 of them newly described. Twelve STs grouped STEC isolates and 11 STs grouped EPEC isolates. Genetic typing by pulsed field gel electrophoresis (PFGE) resulted in 38 patterns which grouped in 10 clusters. Well-defined groups were also observed for strains of pathogenic serotypes. In conclusion, strains of STEC and aEPEC belonging to serotypes related to severe human disease have been detected in goat milk and the goat farm environment. Ruminants are an important reservoir of STEC strains and the role of these animals as carriers of other pathogenic types of E. coli seems to be an emerging concern.

  19. Relative Fecal Abundance of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains and Their Occurrence in Urinary Tract Infections in Women

    PubMed Central

    Lixandru, Brandusa; Cojocaru, Radu; Büke, Çağrı; Paramythiotou, Elisabeth; Angebault, Cécile; Visseaux, Claire; Djuikoue, Ingrid; Erdem, Esra; Burduniuc, Olga; El Mniai, Assiya; Marcel, Candice; Perrier, Marion; Kesteman, Thomas; Clermont, Olivier; Denamur, Erick; Armand-Lefèvre, Laurence; Andremont, Antoine

    2013-01-01

    Extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli) strains are of major concern because few antibiotics remain active against these bacteria. We investigated the association between the fecal relative abundance (RA) of ESBL-producing E. coli (ESBL-RA) and the occurrence of ESBL E. coli urinary tract infections (UTIs). The first stool samples passed after suspicion of UTI from 310 women with subsequently confirmed E. coli UTIs were sampled and tested for ESBL-RA by culture on selective agar. Predictive values of ESBL-RA for ESBL E. coli UTI were analyzed for women who were not exposed to antibiotics when the stool was passed. ESBL E. coli isolates were characterized for ESBL type, phylogroup, relatedness, and virulence factors. The prevalence of ESBL E. coli fecal carriage was 20.3%, with ESBL E. coli UTIs being present in 12.3% of the women. The mean ESBL-RA (95% confidence interval [CI]) was 13-fold higher in women exposed to antibiotics at the time of sampling than in those not exposed (14.3% [range, 5.6% to 36.9%] versus 1.1% [range, 0.32% to 3.6%], respectively; P < 0.001) and 18-fold higher in women with ESBL E. coli UTI than in those with another E. coli UTI (10.0% [range, 0.54% to 100%] versus 0.56% [range, 0.15% to 2.1%[, respectively; P < 0.05). An ESBL-RA of <0.1% was 100% predictive of a non-ESBL E. coli UTI. ESBL type, phylogroup, relatedness, and virulence factors were not found to be associated with ESBL-RA. In conclusion, ESBL-RA was linked to the occurrence of ESBL E. coli UTI in women who were not exposed to antibiotics and who had the same clone of E. coli in urine samples and fecal samples. Especially, a low ESBL-RA appeared to be associated with a low risk of ESBL E. coli infection. PMID:23836184

  20. Relative fecal abundance of extended-spectrum-β-lactamase-producing Escherichia coli strains and their occurrence in urinary tract infections in women.

    PubMed

    Ruppé, Etienne; Lixandru, Brandusa; Cojocaru, Radu; Büke, Cagri; Paramythiotou, Elisabeth; Angebault, Cécile; Visseaux, Claire; Djuikoue, Ingrid; Erdem, Esra; Burduniuc, Olga; El Mniai, Assiya; Marcel, Candice; Perrier, Marion; Kesteman, Thomas; Clermont, Olivier; Denamur, Erick; Armand-Lefèvre, Laurence; Andremont, Antoine

    2013-09-01

    Extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli (ESBL E. coli) strains are of major concern because few antibiotics remain active against these bacteria. We investigated the association between the fecal relative abundance (RA) of ESBL-producing E. coli (ESBL-RA) and the occurrence of ESBL E. coli urinary tract infections (UTIs). The first stool samples passed after suspicion of UTI from 310 women with subsequently confirmed E. coli UTIs were sampled and tested for ESBL-RA by culture on selective agar. Predictive values of ESBL-RA for ESBL E. coli UTI were analyzed for women who were not exposed to antibiotics when the stool was passed. ESBL E. coli isolates were characterized for ESBL type, phylogroup, relatedness, and virulence factors. The prevalence of ESBL E. coli fecal carriage was 20.3%, with ESBL E. coli UTIs being present in 12.3% of the women. The mean ESBL-RA (95% confidence interval [CI]) was 13-fold higher in women exposed to antibiotics at the time of sampling than in those not exposed (14.3% [range, 5.6% to 36.9%] versus 1.1% [range, 0.32% to 3.6%], respectively; P < 0.001) and 18-fold higher in women with ESBL E. coli UTI than in those with another E. coli UTI (10.0% [range, 0.54% to 100%] versus 0.56% [range, 0.15% to 2.1%[, respectively; P < 0.05). An ESBL-RA of <0.1% was 100% predictive of a non-ESBL E. coli UTI. ESBL type, phylogroup, relatedness, and virulence factors were not found to be associated with ESBL-RA. In conclusion, ESBL-RA was linked to the occurrence of ESBL E. coli UTI in women who were not exposed to antibiotics and who had the same clone of E. coli in urine samples and fecal samples. Especially, a low ESBL-RA appeared to be associated with a low risk of ESBL E. coli infection.

  1. Emergence of Multidrug Resistant Extended-Spectrum β-Lactamase Producing Eshcherichia coli Associated With Urinary Tract Infections in Bangladesh

    PubMed Central

    Mowla, Rumana; Imam, K.M. Al-Hasan; Asaduzzaman, Muhammad; Nasrin, Nishat; Raihan, Sheikh Zahir; Chowdhury, A.K. Azad

    2011-01-01

    The incidence of infections due to extended-spectrum β-lactamase (ESBL)–producing Escherichia coli has been increased dramatically in recent years. Treatment is difficult because of frequent multidrug resistance. To identify the sensitivity of commonly used antibiotics, 36 ESBL producing E. coli strains were isolated from young adult female patients in a govt. medical college hospital in Bangladesh. The samples were studied for antimicrobial sensitivity against nine (9) commonly used antibiotics namely ampicillin (amp), trimethoprim-sulfomethoxazole (tms), tetracycline (tet), ciprofloxacin (cip), mecillinum (mel), ceftriaxone (cef), nalidixic acid (nal), Azithromycin (azm) and Chloramphenicol (chl) and the MIC values were determined by agar dilution method. Overall, 72% of the strains were multidrug resistant (MDR) i.e. resistant to two or more drugs. Among 36 strains, 14 isolates were initially found to be resistant against third generation cephalosporin, ceftriaxone. Those were subjected to the test for production of ESBL (Extended Spectrum β-Lactamase) and 7 showed positive results. PMID:24826028

  2. F41 pili as protective antigens of enterotoxigenic Escherichia coli that produce F41, K99, or both pilus antigens.

    PubMed Central

    Runnels, P L; Moseley, S L; Moon, H W

    1987-01-01

    Pigs suckling dams that have been vaccinated with pilus antigen are protected against challenge with enterotoxigenic Escherichia coli (ETEC) strains that express the same pilus antigen. However, some ETEC strains express more than one pilus antigen. Pregnant swine were vaccinated either with E. coli HB101 that harbored a recombinant plasmid coding for F41 expression (F41+) or with the HB101 parent strain that carries the pHC79 vector (F41-). Suckling pigs born to vaccinated dams were challenged with ETEC that expressed either K99, F41, or both pilus antigens. Production of F41 in vivo was demonstrated by immunofluorescence assay of sections of ileum and by seroconversion against F41 antigen by pigs challenged with F41+ and K99+ F41+ ETEC strains. The F41+ vaccine protected against challenge with an F41+ ETEC strain. In contrast, F41+ vaccination did not protect against challenge with K99+ or K99+ F41+ ETEC strains. The F41- vaccine did not protect against challenge with any strain used. The results indicate that K99+ F41+ ETEC strains produce F41 antigen in the small intestine during disease and that F41+ vaccination can be a protective antigen if the challenge strain expresses only F41 antigen, but that F41+ vaccination may not protect against strains that produce both K99 and F41 antigens. PMID:2880807

  3. Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources.

    PubMed

    Miko, Angelika; Delannoy, Sabine; Fach, Patrick; Strockbine, Nancy A; Lindstedt, Björn Arne; Mariani-Kurkdjian, Patricia; Reetz, Jochen; Beutin, Lothar

    2013-12-01

    Sixty-two Escherichia coli strains carrying the wzxO104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n=1), O104:H4 (n=37), O104:H7 (n=5) and O104:H21 (n=11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype. The 38 O104:H4 strains formed a single PFGE-cluster (>83.7% similarity). Thirty-one of these strains were from the European O104:H4 outbreak in 2011. The outbreak strains and older O104:H4 strains from Germany (2001), Georgia and France (2009) clustered together at>86.2% similarity. O104:H4 strains isolated between 2001 and 2009 differed for some plasmid-encoded virulence genes compared to the outbreak strains from 2011. STEC O104:H21 and STEC O104:H7 strains isolated in the U.S. and in Europe showed characteristic differences in their Stx-types, virulence gene and PFGE profiles indicating that these have evolved separately. E. coli K9 strains were not associated with virulence and were heterogeneous for their serotypes and PFGE profiles.

  4. Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals.

    PubMed

    Karczmarczyk, Maria; Martins, Marta; Quinn, Teresa; Leonard, Nola; Fanning, Séamus

    2011-10-01

    Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

  5. [Characterization of first sorbitol-fermenting shiga toxin-producing Escherichia coli O157:H- strain isolated in Poland].

    PubMed

    Jakubczak, Aleksandra; Szych, Jolanta; Januszkiewicz, Kamil

    2008-01-01

    Sorbitol-fermenting shiga toxin-producing E. coli O157:H- strains have emerged as a cause of human disease in many European and non-European countries. The role of SF VTEC O157:H- in the etiology of pediatric HUS and diarrhea is significant. We characterized the first SF VTEC O157:H- strain isolated from 9 year old patient in Poland. Strain possessed many traits characteristics for SF VTEC O157:H-. It fermented sorbitol after overnight incubation and produced beta-glucuronidase. It possessed the stx2, eae-gamma, EhlyA and sfpA genes and did not harbour plasmid-encoded katP and espP genes. Motility was not expressed but the strain possessed the chromosomal fliC locus for H7 antigen. The spread of SF VTEC O157:H- strains demonstrates the need for appropriate procedures for their microbiological diagnosis in Poland.

  6. Diversity of Escherichia coli strains producing extended-spectrum beta-lactamases in Spain: second nationwide study.

    PubMed

    Díaz, Miguel A; Hernández-Bello, José R; Rodríguez-Baño, Jesús; Martínez-Martínez, Luis; Calvo, Jorge; Blanco, Jorge; Pascual, Alvaro

    2010-08-01

    The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in Spain increased 8-fold from 2000 to 2006. ESBL type, clonal relationship, antimicrobial susceptibility, and clinical data about infections caused by ESBLEC are evaluated in a second nationwide study developed in 2006. From 1008 clinical isolates obtained over 2 months from 44 hospitals, 254 were used for further analysis. ESBL production was evaluated by synergy testing, PCR, and sequencing. Antimicrobial activity was evaluated by microdilution. The clonal relationship was evaluated by repetitive extragenic palindromic-PCR (REP-PCR). The O25b subtype and the new afa operon FM955459 were determined by triplex PCR in isolates producing CTX-M-15. Multilocus sequence typing was performed on these isolates. A total of 72% of all ESBLs were of the CTX-M type, 26.8% were of the SHV type, and 1.2% were of the TEM type. The most prevalent ESBLs were CTX-M-14 (119 isolates), SHV-12 (68 isolates), CTX-M-15 (37 isolates), and CTX-M-9 (21 isolates). By REP-PCR, 214 clones were detected. All but five CTX-M-15 ESBLEC isolates corresponded to the international O25b/ST131 clone. This clone had not been detected in the first study (published in 2000). Epidemiological and clinical features were studied in 304 representative patients. A total of 60% of the patients were older than 60 and had nonfatal underlying diseases, and 55% had recently received antibiotics. Urinary tract infections accounted for 71% of cases, and 9% were bacteremic. There has been a significant increase in the prevalence of ESBLEC in Spain, with most of these strains being CTX-M-producing isolates, including the pandemic O25b-ST131. SHV-12-producing E. coli remains an important cause of community-acquired infection.

  7. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli

    SciTech Connect

    Baranzoni, Gian Marco; Fratamico, Pina M.; Gangiredla, Jayanthi; Patel, Isha; Bagi, Lori K.; Delannoy, Sabine; Fach, Patrick; Boccia, Federica; Anastasio, Aniello; Pepe, Tiziana

    2016-04-21

    Similar to ruminants, swine have been shown to be a reservoir for Shiga toxin-producing Escherichia coli (STEC), and pork products have been linked with outbreaks associated with STEC O157 and O111:H-. STEC strains, isolated in a previous study from fecal samples of late-finisher pigs, belonged to a total of 56 serotypes, including O15:H27, O91:H14, and other serogroups previously associated with human illness. The isolates were tested by polymerase chain reaction (PCR) and a high-throughput real-time PCR system to determine the Shiga toxin (Stx) subtype and virulence-associated and putative virulence-associated genes they carried. Select STEC strains were further analyzed using a Minimal Signature E. coli Array Strip. As expected, stx2e (81%) was the most common Stx variant, followed by stx1a (14%), stx2d (3%), and stx1c (1%). The STEC serogroups that carried stx2d were O15:H27, O159:H16 and O159:H-. Similar to stx2a and stx2c, the stx2d variant is associated with development of hemorrhagic colitis and hemolytic uremic syndrome, and reports on the presence of this variant in STEC strains isolated from swine are lacking. Moreover, the genes encoding heat stable toxin (estIa) and enteroaggregative E. coli heat stable enterotoxin-1 (astA) were commonly found in 50 and 44% of isolates, respectively. The hemolysin genes, hlyA and ehxA, were both detected in 7% of the swine STEC strains. Although the eae gene was not found, other genes involved in host cell adhesion, including lpfAO113 and paa were detected in more than 50% of swine STEC strains, and a number of strains also carried iha, lpfAO26, lpfAO157, fedA, orfA, and orfB. Furthermore, the present work provides new insights on the distribution of virulence factors among swine STEC strains and shows that swine may carry Stx1a-, Stx2e-, or Stx2d-producing E. coli with virulence gene profiles

  8. The Accessory Genome of Shiga Toxin-Producing Escherichia coli Defines a Persistent Colonization Type in Cattle

    PubMed Central

    Barth, Stefanie A.; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Wieler, Lothar H.; Pickard, Derek; Belka, Ariane; Berens, Christian

    2016-01-01

    ABSTRACT Shiga toxin-producing Escherichia coli (STEC) strains can colonize cattle for several months and may, thus, serve as gene reservoirs for the genesis of highly virulent zoonotic enterohemorrhagic E. coli (EHEC). Attempts to reduce the human risk for acquiring EHEC infections should include strategies to control such STEC strains persisting in cattle. We therefore aimed to identify genetic patterns associated with the STEC colonization type in the bovine host. We included 88 persistent colonizing STEC (STECper) (shedding for ≥4 months) and 74 sporadically colonizing STEC (STECspo) (shedding for ≤2 months) isolates from cattle and 16 bovine STEC isolates with unknown colonization types. Genoserotypes and multilocus sequence types (MLSTs) were determined, and the isolates were probed with a DNA microarray for virulence-associated genes (VAGs). All STECper isolates belonged to only four genoserotypes (O26:H11, O156:H25, O165:H25, O182:H25), which formed three genetic clusters (ST21/396/1705, ST300/688, ST119). In contrast, STECspo isolates were scattered among 28 genoserotypes and 30 MLSTs, with O157:H7 (ST11) and O6:H49 (ST1079) being the most prevalent. The microarray analysis identified 139 unique gene patterns that clustered with the genoserotypes and MLSTs of the strains. While the STECper isolates possessed heterogeneous phylogenetic backgrounds, the accessory genome clustered these isolates together, separating them from the STECspo isolates. Given the vast genetic heterogeneity of bovine STEC strains, defining the genetic patterns distinguishing STECper from STECspo isolates will facilitate the targeted design of new intervention strategies to counteract these zoonotic pathogens at the farm level. IMPORTANCE Ruminants, especially cattle, are sources of food-borne infections by Shiga toxin-producing Escherichia coli (STEC) in humans. Some STEC strains persist in cattle for longer periods of time, while others are detected only sporadically

  9. A prospective study of exposure to verotoxin-producing Escherichia coli among Canadian children with haemolytic uraemic syndrome. The CPKDRC co-investigators.

    PubMed Central

    Rowe, P. C.; Orrbine, E.; Lior, H.; Wells, G. A.; McLaine, P. N.

    1993-01-01

    Haemolytic uraemic syndrome (HUS) is a leading cause of acute renal failure in childhood. Although infection with Escherichia coli O 157. H7 has been associated with HUS in North America and Europe, only a limited number of studies have examined the role of other verotoxin-producing E. coli (VTEC) serotypes in this condition. To address this issue, we conducted a comprehensive, prospective microbiological study of patients treated for HUS at eight Canadian hospitals in the summer of 1990. Of the 34 consecutive patients with HUS enrolled over 4 months, E. coli O 157. H7 was isolated from the stools of 26, and other E. coli serotypes were isolated from four patients. In four subjects no pathogenic E. coli serotypes were identified on stool culture. Using oligonucleotide probes specific for VT-1 and VT-2, verotoxin genes were detected in the stool isolates of all patients with E. coli O 157. H7, and from two with other E. coli serotypes. Two other patients had at least a fourfold rise in anti-verotoxin antibodies. Strong evidence of exposure to a verotoxin was present in 30/34 (88%). Patients with E. coli O 157. H7 infection were more likely to develop an antibody response to VT-2 than to VT-1 (22/22 vs 12/22; P = 0.002). These results further strengthen the association of HUS with verotoxin-producing E. coli in North America, and confirm that E. coli serotypes other than O 157.H7 are isolated in a small proportion of summertime HUS episodes. PMID:8432313

  10. Prevalence of plasmid-mediated AmpC β-lactamase-producing Escherichia coli and spread of the ST131 clone among extended-spectrum β-lactamase-producing E. coli in Japan.

    PubMed

    Matsumura, Yasufumi; Yamamoto, Masaki; Higuchi, Takeshi; Komori, Toshiaki; Tsuboi, Fusayuki; Hayashi, Akihiko; Sugimoto, Yoshihisa; Hotta, Gou; Matsushima, Aki; Nagao, Miki; Takakura, Shunji; Ichiyama, Satoshi

    2012-08-01

    In 2010, a total of 1327 clinical Escherichia coli isolates from five hospitals in the Kyoto and Shiga regions of Japan were analysed by PCR. The prevalences of plasmid-mediated AmpC β-lactamase (pAmpC)-producers, extended-spectrum β-lactamase (ESBL)-producers and co-producers of pAmpC and ESBL were 1.7%, 9.7% and 0.3%, respectively. Less than one-half of the pAmpC-producers were reported to be resistant to third-generation cephalosporins, cephamycins and β-lactam/β-lactam inhibitors using the old 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints. CMY-2 was the most prevalent pAmpC type (95%), and CTX-M-14 (38%), CTX-M-15 (26%) and CTX-M-27 (19%) were the most prevalent ESBL types. The worldwide O25b-ST131-B2 clone accounted for 11% of pAmpC-producers and 41% of ESBL-producers. The O25b-ST131-B2 clone was characterised by a CTX-M-27- or CTX-M-15-type ESBL and ciprofloxacin-non-susceptibility with quadruple mutations in the quinolone resistance-determining regions (S83L and D87N in GyrA and S80I and E84V in ParC). A significant proportion of pAmpC-producers and the O25b-ST131-B2 clone were found in Japan by a recent regional surveillance programme.

  11. Usability and Performance of CHROMagar STEC Medium in Detection of Shiga Toxin-Producing Escherichia coli Strains

    PubMed Central

    Siitonen, Anja; Kaukoranta, Suvi-Sirkku

    2012-01-01

    The performance and usability of CHROMagar STEC medium (CHROMagar Microbiology, Paris, France) for routine detection of Shiga toxin-producing Escherichia coli (STEC) strains were examined. The ability of the medium to selectively propagate STEC strains differing by their serotypes and virulence genes was studied with a collection of diarrheagenic E. coli isolates (n = 365) consisting of 49 different serotypes and with non-STEC and other bacterial isolates (n = 264). A total of 272 diarrheagenic E. coli (75.0%) isolates covering 24 different serotypes grew on CHROMagar STEC. The highest detection sensitivities were observed within the STEC serogroups O26 (90.0%), O111 (100.0%), O121 (100.0%), O145 (100.0%), and O157 (84.9%), and growth on CHROMagar STEC was highly associated with the presence of the tellurite resistance gene (terD). The specificity of the medium was 98.9%. In addition, CHROMagar STEC was used in parallel with a Shiga toxin-detecting immunoassay (Ridaquick Verotoxin/O157 Combi; R-biopharm, Darmstadt, Germany) to screen fecal specimens (n = 47) collected from patients suffering from hemorrhagic diarrhea. Positive growth on CHROMagar STEC was confirmed by the Premier EHEC enzyme immunoassay (Meridian Bioscience, Inc., Cincinnati, OH), and discrepant results between the two screening methods were confirmed by stx gene-detecting PCR. All 16 of the 47 stool samples that showed positive growth on CHROMagar STEC were also positive in the confirmatory tests. CHROMagar STEC proved to be an interesting option for STEC screening, allowing good detection sensitivity and specificity and permitting strain isolation for further outbreak investigations when required. PMID:22933601

  12. Intestinal Shiga Toxin-Producing Escherichia coli Bacteria Mitigate Bovine Leukemia Virus Infection in Experimentally Infected Sheep

    PubMed Central

    Ferens, Witold A.; Cobbold, Rowland; Hovde, Carolyn J.

    2006-01-01

    Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx-negative E. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >104 CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P < 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease. PMID:16622229

  13. Shiga toxin-producing Escherichia coli (STEC): Zoonotic risks associated with psittacine pet birds in home environments.

    PubMed

    Gioia-Di Chiacchio, R M; Cunha, M P V; Sturn, R M; Moreno, L Z; Moreno, A M; Pereira, C B P; Martins, F H; Franzolin, M R; Piazza, R M F; Knöbl, T

    2016-02-29

    Psittacidae are frequently bred as pets worldwide, but little is known about the zoonotic risks of these animals. The objective of this study was to investigate the presence of Shiga toxin-producing Escherichia coli (STEC) in the feces of psittacine birds housed as pets. A total of 171 fecal samples (67 cockatiels, 59 budgerigars, and 45 agapornis) were cultured. Forty-two (E. coli) strains were identified, and the presence of the eae, stx1, and stx2 genes was determined using PCR. The antimicrobial resistance profiles of the STEC strains were determined using the disk diffusion method and phylogenetic analysis according to the new Clermont phylotyping method. Using these methods, 19.4% (8/42) of the STEC strains were determined to be positive for the eae and stx2 genes. The results revealed a STEC frequency of 4.6% in the birds (8/171), with a percentage of 8.47% in budgerigars (5/59), 4.47% in cockatiels (3/67), and 0% in agapornis (0/45). None of the STEC isolates belonged to the O157 serogroup. Most of the strains were classified as sensitive to the 18 antibiotics tested. None of the strains exhibited a multiresistance profile. In the phylogenetic analysis, two strains were classified as non-typeable, three were classified as B2, two were classified as F, and one was classified as Clade I. Seven of the eight STEC strains showed a clonal profile using AFLP. E. coli strains that are stx2(+) plus eae(+) are usually associated with severe human diseases such as hemorrhagic colitis and hemolytic-uremic syndrome. The STEC-positive results indicate the zoonotic risk of breeding psittacidae in home environments.

  14. Intestinal Shiga toxin-producing Escherichia coli bacteria mitigate bovine leukemia virus infection in experimentally infected sheep.

    PubMed

    Ferens, Witold A; Cobbold, Rowland; Hovde, Carolyn J

    2006-05-01

    Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx-negative E. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >10(4) CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P < 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.

  15. Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem.

    PubMed

    Singh, Pallavi; Sha, Qiong; Lacher, David W; Del Valle, Jacquelyn; Mosci, Rebekah E; Moore, Jennifer A; Scribner, Kim T; Manning, Shannon D

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.

  16. Incidence of Shiga toxin-producing Escherichia coli strains in beef, pork, chicken, deer, boar, bison, and rabbit retail meat.

    PubMed

    Magwedere, Kudakwashe; Dang, Huu Anh; Mills, Edward W; Cutter, Catherine N; Roberts, Elisabeth L; DeBroy, Chitrita

    2013-03-01

    The objective of the current study was to determine the incidence of contamination by the top 7 Shiga toxin-producing Escherichia coli (STEC) O-groups, responsible for the majority of E. coli infections in human beings, in retail meat from different animal species. Samples from ground beef (n = 51), ground pork (n = 16), ground chicken (n = 16), and game meat (deer, wild boar, bison, and rabbit; n = 55) were collected from retail vendors for the detection of 7 STEC O-groups (O26, O45, O103, O111, O121, O145, and O157). Meat samples were tested by using a multiplex polymerase chain reaction assay targeting the wzx gene of O antigen gene clusters of the 7 STEC O-groups. The positive samples were further tested for Shiga toxin genes (stx1 and stx2). Out of a total of 83 ground beef, pork, and chicken samples, 17 (20%) carried O121, 9 (10%) carried O45, 8 (9%) carried O157, 3 (3%) carried O103, and 1 (1%) carried O145. None of the samples were positive for O26, O111, or the stx gene. All 3 white-tailed deer samples (100%) were positive for O45, O103, or both, 2 (10%) out of 20 red deer samples exhibited the presence of O103, and all 3 bison samples were contaminated with either O121, O145, or O157. One sample from ground deer, contaminated with E. coli O45, carried the stx1 gene. This preliminary investigation illustrates the importance of microbiological testing of pathogens in meat products, as well as the recognized need for increased surveillance and research on foodborne pathogens.

  17. Emergence and Outbreaks of CTX-M β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Strains in a Tunisian Hospital▿

    PubMed Central

    Mamlouk, Kelthoum; Boutiba-Ben Boubaker, Ilhem; Gautier, Valérie; Vimont, Sophie; Picard, Bertrand; Ben Redjeb, Saida; Arlet, Guillaume

    2006-01-01

    Sixty-two isolates of Enterobacteriaceae (35 Escherichia coli and 27 Klebsiella pneumoniae isolates) producing CTX-M-type β-lactamases were collected between March 2000 and June 2003 in different wards of Charles Nicolle Hospital in Tunis (Tunisia). Sequencing identified the blaCTX-M-15 determinant in 55 isolates and blaCTX-M-16 in 7 isolates. The CTX-M-15-producing strains were isolated in several wards and consisted mainly of two successive clonal groups of E. coli and a major clonal group of K. pneumoniae. The second clonal group of E. coli belonged to phylogenetic group B2 and harbored more virulence factors than the first clonal group. Among the 22 transconjugants or electroporants obtained with selected E. coli and K. pneumoniae CTX-M-15-producing strains, a predominant plasmid restriction pattern was obtained with 17 isolates. The four CTX-M-16-producing strains of E. coli yielded the same pulsed-field gel electrophoresis (PFGE) pattern, while the three CTX-M-16-producing strains of K. pneumoniae yielded two different PFGE patterns. All of the CTX-M-16-producing isolates were recovered in the pediatric ward and had the same plasmid restriction pattern. PMID:16957046

  18. Emergence and outbreaks of CTX-M beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital.

    PubMed

    Mamlouk, Kelthoum; Boutiba-Ben Boubaker, Ilhem; Gautier, Valérie; Vimont, Sophie; Picard, Bertrand; Ben Redjeb, Saida; Arlet, Guillaume

    2006-11-01

    Sixty-two isolates of Enterobacteriaceae (35 Escherichia coli and 27 Klebsiella pneumoniae isolates) producing CTX-M-type beta-lactamases were collected between March 2000 and June 2003 in different wards of Charles Nicolle Hospital in Tunis (Tunisia). Sequencing identified the bla(CTX-M-15) determinant in 55 isolates and bla(CTX-M-16) in 7 isolates. The CTX-M-15-producing strains were isolated in several wards and consisted mainly of two successive clonal groups of E. coli and a major clonal group of K. pneumoniae. The second clonal group of E. coli belonged to phylogenetic group B2 and harbored more virulence factors than the first clonal group. Among the 22 transconjugants or electroporants obtained with selected E. coli and K. pneumoniae CTX-M-15-producing strains, a predominant plasmid restriction pattern was obtained with 17 isolates. The four CTX-M-16-producing strains of E. coli yielded the same pulsed-field gel electrophoresis (PFGE) pattern, while the three CTX-M-16-producing strains of K. pneumoniae yielded two different PFGE patterns. All of the CTX-M-16-producing isolates were recovered in the pediatric ward and had the same plasmid restriction pattern.

  19. Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

    PubMed

    Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

    2017-07-15

    Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h.

  20. Exploiting the power of OMICS approaches to produce E. coli O157 vaccines

    PubMed Central

    Kalita, Anjana; Kalita, Mridul; Torres, Alfredo G

    2014-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains are well-documented human pathogens and causative agents of diarrheal episodes and hemorrhagic colitis. The serotype O157:H7 is highly virulent and responsible for both outbreaks and sporadic cases of diarrhea. Because antibiotic treatment is contraindicated against this pathogen, development of a human vaccine could be an effective intervention in public health. In our recent Infection and Immunity paper, we applied integrated approaches of in silico genome wide search combined with bioinformatics tools to identify and test O157 vaccine candidates for their protective effect on a murine model of gastrointestinal infection. Using genomic/immunoinformatic approaches that are further described here, we categorized vaccine candidates as high, medium, and low priorities, and demonstrate that some high priority candidates were able to significantly induce Th2 cytokines and reduce EHEC colonization. Using the STRING database, we have recently evaluated the vaccine candidates and predict functional protein interactions, determining whether correlations exist for the development of a multi-subunit vaccine, targeting different pathways against EHEC O157:H7. The overall approach is designed to screen potential vaccine candidates against EHEC; however, the methodology can be quickly applied to many other intestinal pathogens. PMID:25621619

  1. Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

    PubMed

    You, Weon-Kyoo; So, Seung-Ho; Sohn, Young-Doug; Lee, Hyosil; Park, Doo-Hong; Chung, Soo-Il; Chung, Kwang-Hoe

    2004-07-01

    Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

  2. Characteristics of shiga toxin-producing Escherichia coli isolated from Swiss raw milk cheese within a 3-year monitoring program.

    PubMed

    Zweifel, C; Giezendanner, N; Corti, S; Krause, G; Beutin, L; Danuser, J; Stephan, R

    2010-01-01

    Food is an important vehicle for transmission of Shiga toxin-producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cow's, goat's, and ewe's milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx(1) was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx(2) group, mainly stx(2) and stx(2vh-a/b). Production of Stx(2) and Stx(2vh-a/b) subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.

  3. Monte Carlo simulation for evaluation of the efficacy of carbapenems and new quinolones against ESBL-producing Escherichia coli.

    PubMed

    Nakamura, Tatsuya; Shimizu, Chihiro; Kasahara, Mayumi; Okuda, Kazuyuki; Nakata, Chiyo; Fujimoto, Hiroko; Okura, Hiroe; Komatsu, Masaru; Shimakawa, Kouichi; Sueyoshi, Noriyuki; Ura, Toshiro; Satoh, Kaori; Toyokawa, Masahiro; Wada, Yasunao; Orita, Tamaki; Kofuku, Tomomi; Yamasaki, Katsutoshi; Sakamoto, Masako; Nishio, Hisaaki; Kinoshita, Shohiro; Takahashi, Hakuo

    2009-02-01

    Extended-spectrum beta-lactamase (ESBL)-producing bacteria are known to be resistant to penicillins, cephalosporins, and monobactams because of their substrate specificity, and these bacteria are sensitive only to a narrow range of antimicrobial agents. The present study was undertaken to evaluate the efficacy of carbapenems and the new quinolones against ESBL-producing Escherichia coli, using a Monte Carlo simulation based on the pharmacokinetic/pharmacodynamic (PK/PD) theory. The time above MIC (TAM, %) served as the PK/PD parameter for carbapenems, with the target level set at 40%. The AUC/MIC served as the PK/PD parameter for the new quinolones, with the target level set at more than 125. In the analysis of drug sensitivity, the MIC50 of all carbapenems other than imipenem was low (0.03 microg/ml), while the MIC50 of the new quinolones was higher (1-2 microg/ml). The probability of achieving the PK/PD target with carba penems after two doses at the usual dose level, as determined by the Monte Carlo simulation, was high for each of the carbapenems tested (99.0% for biapenem, 99.60% for meropenem, and 95.03% for doripenem), except for imipenem. Among the new quinolones, the highest probability of achieving the PK/PD target was obtained with pazufloxacin (42.90%). Thus, the results of the present study have revealed that carbapenems are effective at the regular dose and can be used as the first-choice antibiotics for ESBL-producing E. coli because the resistance ratios for carbapenems are low compared to those of the new quinolones.

  4. Persistence and prevalence of pathogenic and extended-spectrum beta-lactamase-producing Escherichia coli in municipal wastewater treatment plant receiving slaughterhouse wastewater.

    PubMed

    Diallo, Alpha Amadou; Brugère, Hubert; Kérourédan, Monique; Dupouy, Véronique; Toutain, Pierre-Louis; Bousquet-Mélou, Alain; Oswald, Eric; Bibbal, Delphine

    2013-09-01

    We compared the prevalence of pathogenic and extended-spectrum beta-lactamase (ESBL) - producing Escherichia coli in effluents of a municipal wastewater treatment plant (WWTP) receiving wastewater from a slaughterhouse. A total of 1248 isolates were screened for the presence of virulence genes associated with enterohemorrhagic E. coli (EHEC) (stx1, stx2, and eae) and extraintestinal pathogenic E. coli (ExPEC) (sfa/focDE, kpsMT K1, hlyA, papEF, afa/draBC, clbN, f17A and cnf). The prevalence of atypical enteropathogenic E. coli (EPEC) was 0.7%, 0.2% and 0.5% in city wastewater, slaughterhouse wastewater and in the treated effluent, respectively. One stx1a and stx2b-positive E. coli isolate was detected in city wastewater. The prevalence of ExPEC was significantly higher in city wastewater (8.4%), compared to slaughterhouse wastewater (1.2%). Treatment in the WWTP did not significantly impact the prevalence of ExPEC in the outlet effluent (5.0%) compared to city wastewater. Moreover, the most potentially pathogenic ExPEC were isolated from city wastewater and from the treated effluent. ESBL-producing E. coli was also mainly detected in city wastewater (1.7%), compared to slaughterhouse wastewater (0.2%), and treated effluent (0.2%). One ESBL-producing E. coli, isolated from city wastewater, was eae-β1 positive. These results showed that pathogenic and/or ESBL-producing E. coli were mainly detected in human wastewater, and at a lesser extend in animal wastewater. Treatment failed to eliminate these strains which were discharged into the river, and then these strains could be transmitted to animals and humans via the environment.

  5. Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype

    PubMed Central

    Rahal, Elias A.; Fadlallah, Sukayna M.; Nassar, Farah J.; Kazzi, Natalie; Matar, Ghassan M.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise. PMID:25853096

  6. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  7. Secondary transmissions during the outbreak of Shiga toxin-producing Escherichia coli O104 in Hesse, Germany, 2011.

    PubMed

    Hauri, Am; Gotsch, U; Strotmann, I; Krahn, J; Bettge-Weller, G; Westbrock, Hj; Bellinger, O; Uphoff, H

    2011-08-04

    During the recent outbreak of Shiga toxin-producing Escherichia coli (STEC) O104:H4 in Germany most cases notified in the State of Hesse (6 million inhabitants) were linked to satellite clusters or had travelled to the outbreak area in northern Germany. Intensified surveillance was introduced to rapidly identify cases not linked to known clusters or cases and thus to obtain timely information on possible further contaminated vehicles distributed in Hesse, as well to describe the risk of secondary transmission among known cases. As of 2 August 2011* [corrected], 56 cases of haemolytic uraemic syndrome (HUS) including two fatal cases, and 124 cases of STEC gastroenteritis meeting the national case definitions have been reported in Hesse. Among the 55 HUS and 81 STEC gastroenteritis cases thatmet the outbreak case definition, one HUS case and eight STEC gastroenteritis cases may have acquired their infection through secondary transmission. They include six possible transmissions within the family, two possible nosocomial and one possible laboratory transmission. Our results do not suggest an increased transmissibility of the outbreak strain compared to what is already known about E. coli O157 and other STEC serotypes.

  8. High Prevalence of Escherichia coli-Producing CTX-M-15 Extended-Spectrum Beta-Lactamases in Poultry and Human Clinical Isolates in Romania.

    PubMed

    Maciuca, Iuliana E; Williams, Nicola J; Tuchilus, Cristina; Dorneanu, Olivia; Guguianu, Eleonora; Carp-Carare, Catalin; Rimbu, Cristina; Timofte, Dorina

    2015-12-01

    Use of antibiotics in food animals may contribute to development and spread of resistant organisms, particularly so in some countries. The aim of this study was two-fold; first, to establish the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chicken production in a region within Romania. Second, to study the relatedness of ESBL-producing E. coli isolates recovered from broilers, abattoir workers where the chickens were slaughtered and from the human clinical specimens from two regional hospitals. The results indicated a very high (69%) rate of carriage of ESBL and AmpC-producing E. coli in chickens with 36% CTX-M producers. Sequencing showed that chickens in Romania have the highest worldwide prevalence (53%) of blaCTX-M-15 reported in poultry E. coli isolates. The majority (53%) of the extended-spectrum cephalosporin-resistant E. coli carried plasmid-mediated blaampC genes, mostly blaCMY-2 type, one of the highest prevalences reported in Europe. The predominant CTX-M type found in the human clinical E. coli isolates was blaCTX-M-15 and most isolates coharbored blaOXA-1, blaTEM, and aac(6')-ib-cr. The majority (60%) of the human clinical isolates belonged to the pandemic virulent clone B2-ST131. The clonal relationship between broiler and the human CTX-M-producing E. coli isolates was assessed by macrorestriction pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which indicated strain diversity with no common STs found between human and poultry isolates. Moreover, IncI1 was the most prevalent replicon found in broiler ESBL-producing E. coli isolates and also in transconjugants, indicating that plasmids and not clonal spread may play a role in the transfer of blaCTX-M genes. This study identifies a high prevalence of ESBL-producing E. coli from broiler chickens in Romania with a high occurrence incidence of blaCTX-M-15, which reflects the main ESBL type found in human E. coli infections in this

  9. Evaluation of lactic acid as an initial and secondary subprimal intervention for Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli, and a nonpathogenic E. coli surrogate for E. coli O157:H7.

    PubMed

    Pittman, C I; Geornaras, I; Woerner, D R; Nightingale, K K; Sofos, J N; Goodridge, L; Belk, K E

    2012-09-01

    Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.

  10. Bloodstream infections caused by Escherichia coli producing AmpC β-lactamases: epidemiology and clinical features.

    PubMed

    Pascual, V; Alonso, N; Simó, M; Ortiz, G; Garcia, M C; Xercavins, M; Rivera, A; Morera, M A; Miró, E; Espejo, E; Navarro, F; Gurguí, M; Pérez, J; Rodríguez-Carballeira, M; Garau, J; Calbo, E

    2016-12-01

    The aim of the study was to investigate the epidemiology and clinical features of bloodstream infections due to Escherichia coli producing AmpC β-lactamases (AmpC-Ec-BSI). In a multi-centre case-control study, all third-generation-cephalosporin-resistant Escherichia coli BSI (3GC-Ec-BSI) isolates were analysed. Acquired bla AmpC (bla ac-AmpC) detection was done by polymerase chain reaction (PCR) and sequencing. Chromosomal bla AmpC (bla c-AmpC) expression was quantified by real-time PCR. Cases were patients with AmpC-Ec-BSI. Controls were patients with cephalosporin-susceptible E. coli BSI, matched 1:1 by sex and age. Demographics, comorbidities, intrinsic and extrinsic risk factors for antimicrobial resistance, clinical presentation and outcomes were investigated. Among 841 E. coli BSI, 17 were caused by AmpC-Ec (2 %). Eleven isolates (58.8 %) had bla ac-AmpC and six were bla c-AmpC overproducers. The mean age of cases was 66.2 years and 71 % were men. Cases were more frequently healthcare-related (82 vs. 52 % controls, p < 0.05) and presented more intrinsic and extrinsic risk factors. At least one risk factor was present in 94.1 % of cases vs. 41.7 % of controls (p = 0.002). Severity and length of stay (LOS) were higher among cases (mean Pitt Score 2.6 vs. 0.38 in controls, p = 0.03; LOS 17.5 days vs. 6 in controls, p = 0.02). Inappropriate empirical therapy (IET) was administered to 70.6 % of cases and 23.5 % of controls (p < 0.003). No differences were found in terms of cure rate at the 14th day and mortality. Bloodstream infections due to AmpC-Ec (mostly plasmid-mediated) are infrequent in our area. AmpC-Ec-BSI affects mainly patients with intrinsic risk factors and those with previous antibiotic exposure. A high proportion received IET.

  11. Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4.

    PubMed

    Nagy, Attila; Xu, Yunfeng; Bauchan, Gary R; Shelton, Daniel R; Nou, Xiangwu

    2016-07-16

    The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.

  12. Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli.

    PubMed

    Frech, M; Schlichting, I; Wittinghofer, A; Chardin, P

    1990-04-15

    The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.

  13. An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

    PubMed Central

    Aguilar-Yáñez, José M.; Portillo-Lara, Roberto; Mendoza-Ochoa, Gonzalo I.; García-Echauri, Sergio A.; López-Pacheco, Felipe; Bulnes-Abundis, David; Salgado-Gallegos, Johari; Lara-Mayorga, Itzel M.; Webb-Vargas, Yenny; León-Angel, Felipe O.; Rivero-Aranda, Ramón E.; Oropeza-Almazán, Yuriana; Ruiz-Palacios, Guillermo M.; Zertuche-Guerra, Manuel I.; DuBois, Rebecca M.; White, Stephen W.; Schultz-Cherry, Stacey; Russell, Charles J.; Alvarez, Mario M.

    2010-01-01

    Background The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy. Methodology/Principal Findings We expressed the globular HA receptor binding domain, referred to here as HA63–286-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA63–286-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model. Conclusions/Significance Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy. PMID:20661476

  14. Characterization and virulence potential of serogroup O113 Shiga toxin-producing Escherichia coli strains isolated from beef and cattle in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) of serotype O113:H21 have caused severe diseases but are unusual in that they do not produce the intimin protein required for adherence to intestinal epithelial cells. Strains of serogroup O113 are one of the most common STEC found in ground beef and be...

  15. Risk of Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing Escherichia coli, and Campylobacter spp. in Food Animals and Their Products in Qatar.

    PubMed

    Mohammed, Hussni O; Stipetic, Korana; Salem, Ahmed; McDonough, Patrick; Chang, Yung Fu; Sultan, Ali

    2015-10-01

    Escherichia coli O157:H7, non-O157 E. coli, and Campylobacter spp. are among the top-ranked pathogens that threaten the safety of food supply systems around the world. The associated risks and predisposing factors were investigated in a dynamic animal population using a repeat-cross-sectional study design. Animal and environmental samples were collected from dairy and camel farms, chicken processing plants, and abattoirs and analyzed for the presence of these pathogens using a combination of bacterial enrichment and real-time PCR tests without culture confirmation. Data on putative risk factors were also collected and analyzed. E. coli O157:H7 was detected by PCR at higher levels in sheep and camel feces than in cattle feces (odds ratios [OR], 6.8 and 21.1, respectively). Although the genes indicating E. coli O157:H7 were detected at a relatively higher rate (4.3%) in fecal samples from dairy cattle, they were less common in milk and udder swabs from the same animals (1 and 2%, respectively). Among the food adulterants, E. coli O103 was more common in cattle fecal samples, whereas O26 was more common in sheep feces and O45 in camel feces compared with cattle (OR, 2.6 and 3.1, respectively). The occurrence of E. coli in the targeted populations differed by the type of sample and season of the year. Campylobacter jejuni and Campylobacter coli were more common in sheep and camel feces than in cattle feces. Most of the survey and surveillance of E. coli focused on serogroup O157 as a potential foodborne hazard; however, based on the PCR results, non-O157 Shiga toxin-producing E. coli serotypes appeared to be more common, and efforts should be made to include them in food safety programs.

  16. The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh.

    PubMed

    Schroder, W; Burger, M; Edwards, C; Douglas, M; Innes, D; Beacham, I R; Burns, D M

    2001-09-11

    Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.

  17. Immunization of swine with heat-stable Escherichia coli enterotoxin coupled to a carrier protein does not protect suckling pigs against an Escherichia coli strain that produces heat-stable enterotoxin.

    PubMed Central

    Moon, H W; Baetz, A L; Giannella, R A

    1983-01-01

    Pregnant swine were immunized parenterally with purified heat-stable Escherichia coli enterotoxin that was made antigenic by coupling it to bovine immunoglobulin G. Immunized swine had high titers of antitoxin in serum and colostrum as measured by radioimmunoassay. However, the heat-stable enterotoxin neutralizing titers of the serum and colostrum from immunized swine were comparatively low. Newborn pigs suckling their immunized dams were not protected against challenge with porcine enterotoxigenic E. coli that produce heat-stable toxin but do not produce heat-labile toxin. PMID:6339398

  18. Molecular characterization of ESBL-producing Escherichia coli isolates from hospital- and community-acquired infections in NW Mexico.

    PubMed

    Miranda-Romero, Ana Laura; Silva-Sanchez, Jesus; Garza-Ramos, Ulises; Barrios, Humberto; Sánchez-Pérez, Alejandro; Reyna-Flores, Fernando

    2017-01-01

    We investigated the molecular characteristics of ESBL-producing E. coli (ESBL-PEc) isolates from two hospitals and community settings in Ciudad Obregon, Sonora, Mexico. Between 2011 and 2014, thirty-seven ESBL-PEc isolates were collected. The major encoded ESBL was the blaCTX-M-15 gene (97%); followed by 13.5% of the blaSHV-12 gene, and 5.5% encoded the blaTLA-1 gene. The PMQR gene aac(6´)-Ib-cr was detected in 97% of the isolates and the qnrB gene, in one isolate. The ESBL-PEc isolates corresponded to phylogenetic group B2, ST131. Our results highlight the dissemination of ESBL-PEc isolates in northwest Mexico (Ciudad Obregon, Sonora).

  19. Engineering of a tyrosol-producing pathway, utilizing simple sugar and the central metabolic tyrosine, in Escherichia coli.

    PubMed

    Satoh, Yasuharu; Tajima, Kenji; Munekata, Masanobu; Keasling, Jay D; Lee, Taek Soon

    2012-02-01

    Metabolic engineering was applied to the development of Escherichia coli capable of synthesizing tyrosol (2-(4-hydroxyphenyl)ethanol), an attractive phenolic compound with great industrial value, from glucose, a renewable carbon source. In this strain, tyrosine, which was supplied not only from the culture medium but also from the central metabolism, was converted into tyrosol via three steps: decarboxylation, amine oxidation, and reduction. The engineered strain synthesized both tyrosol and 4-hydroxyphenylacetate (4HPA), but disruption of the endogenous phenylacetaldehyde dehydrogenase gene shut off 4HPA production and improved the production of tyrosol as a sole product. The engineered mutant strain was capable of producing 0.5 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation.

  20. Shiga toxin-producing Escherichia coli infection: temporal and quantitative relationships among colonization, toxin production, and systemic disease.

    PubMed

    Cornick, N A; Matise, I; Samuel, J E; Bosworth, B T; Moon, H W

    2000-01-01

    Edema disease, a naturally occurring disease of swine caused by Shiga toxin-producing Escherichia coli (STEC), was used as a model for the sequence of events that occur in the pathogenesis of STEC infection. The mean time from production of levels of Shiga toxin 2e (Stx2e) detectable in the feces (day 1) to the onset of clinical disease (neurologic disturbances or death) was 5 days (range, 3-9). Bacterial colonization and titers of Stx2e in the ileum peaked at 4 days after inoculation in pigs without signs of clinical disease and at 6 days after inoculation in clinically affected pigs. Animals with the greatest risk of progressing to clinical disease tended to have the highest fecal toxin titers (>/=1:4096). Stx2e was detected in the red cell fraction from blood of some pigs showing clinical signs of edema disease but was not detected in the serum or cerebrospinal fluid.

  1. Binding of Soluble Natural Ligands to a Soluble Human T-Cell Receptor Fragment Produced in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Hilyard, Katherine L.; Reyburn, Hugh; Chung, Shan; Bell, John I.; Strominger, Jack L.

    1994-09-01

    An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.

  2. [Occurrence of Salmonella spp. and shigatoxin-producing escherichia coli (STEC) in horse faeces and horse meat products].

    PubMed

    Pichner, Rohtraud; Sander, Andrea; Steinrück, Hartmut; Gareis, Manfred

    2005-01-01

    In order to assess the relevance of horses as a possible reservoir of Salmonella and Shigatoxin-producing Escherichia coli (STEC), 400 samples of horse faeces and 100 samples of horse meat products were examined by PCR-screening methods. Salmonella enterica was not found in any of the samples. One faeces-sample and one horse meat product were proved to be STEC positive. The STEC-strain from faecal origin belonged to the serotype 0113:H21 and had the stx 2c gene and the enterohemolysin gene. The STEC-strain isolated from a horse meat product had the serotype O87:H16 and the stx 2d gene. The results indicate a very low risk for human to get a Salmonella- or EHEC- infection from horses in Germany.

  3. Adherence of curli producing Shiga-toxigenic Escherichia coli to baby spinach leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellular appendages, such as curli fibers have been suggested to be involved in STEC persistence in fresh produce as these curli are critical in biofilm formation and adherence to animal cells. We determined the role of curli in attachment of STEC on spinach leaves. The curli expression by wild-ty...

  4. Characterization of the Shiga toxin-producing Escherichia coli O26 isolated from human in Poland between 1996 and 2014.

    PubMed

    Januszkiewicz, A; Wołkowicz, T; Chróst, A; Szych, J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) O26 infections can be comparable with STEC O157 infections in severity of the acute haemolytic-uremic syndrome HUS and long-term sequelae. Among O26 STEC isolates, highly virulent clone O26:H11/H- Sequence Type 29 (ST 29) emerged in Germany in mid-1990s and spread to European countries. However, up to date, no STEC O26:H11/H- belonging to ST29 has been documented in Poland. In this study, we determined the relationship and clonal structure, stx genotypes, plasmid gene profiles and antimicrobial resistance of nine human STEC O26:H11/H- strains from human patients in Poland between 1996 and 2014. Of the 9 human STEC O26:H11/H- strains, two belonged to ST29 and were isolated from two children with HUS and renal failure with sepsis respectively. These strains showed the molecular characteristics of the emerging human-pathogenic ST29 clone (stx1-, stx2a+, eae+, ehxA+, etpD+, katP-, espP-). The remaining STEC O26:H11/H- strains examined in this study, belonged to ST21, with plasmid genes profiles frequently reported in ST21 strains in Europe. STEC O26 infections with serious human health consequences highlight the need of continuous surveillance of non-O157 STEC and implementation of the diagnostic approaches focused on their detection. Significance and impact of the study: These study provides the first data on the occurrence of emerging Shiga toxin-producing Escherichia coli O26:H11 ST 29 clone in human patients in Poland. Those strains show the molecular characteristics of highly virulent new ST29 pathotype (stx1-, stx2a+, eae+ ehxA+, etpD+, katP-, espP-). These results demonstrated prompt efforts to implement diagnostic approaches detection of those pathogen in the European countries.

  5. The Polymorphic Aggregative Phenotype of Shiga Toxin-Producing Escherichia coli O111 Depends on RpoS and Curli

    PubMed Central

    Diodati, M. E.; Bates, A. H.; Miller, W. G.; Carter, M. Q.; Zhou, Y.

    2015-01-01

    Escherichia coli O111 is an emerging non-O157:H7 serotype of Shiga toxin-producing E. coli (STEC). We previously reported that outbreak and environmental, but not sporadic-case, strains of STEC O111 share a distinct aggregation phenotype (M. E. Diodati, A. H. Bates, M. B. Cooley, S. Walker, R. E. Mandrell, and M. T. Brandl, Foodborne Pathog Dis 12:235−243, 2015, http://dx.doi.org/10.1089/fpd.2014.1887). We show here the natural occurrence of nonaggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae and lack RpoS function but synthesize cellulose. The deletion of csgBAC or rpoS in an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a nonaggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blotting with an anti-CsgA antibody. Immunomicroscopy revealed that curli were undetectable on the cells of the nonaggregative variant and the RpoS mutant but were present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis of rpoS in the aggregative strain and its variant showed a single substitution of threonine for asparagine at amino acid 124. Our results indicate that the multicellular behavior of STEC O111 is RpoS dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of nonaggregative variants may allow the cell population to adapt to conditions benefiting a planktonic lifestyle. PMID:26712542

  6. Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of β-Lactamase-Producing Escherichia coli.

    PubMed

    Baker, Kristin R; Sigurðardóttir, Helga Høeg; Jana, Bimal; Guardabassi, Luca

    2017-03-01

    Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

  7. Dead-end ultrafiltration concentration and IMS/ATP-bioluminescence detection of Escherichia coli O157:H7 in recreational water and produce wash.

    PubMed

    Hunter, Dawn M; Leskinen, Stephaney D; Magaña, Sonia; Schlemmer, Sarah M; Lim, Daniel V

    2011-12-01

    The purpose of this study was to develop a detection method for viable E. coli O157:H7 in fresh produce and recreational water. The method was evaluated using eight samples of produce wash and recreational water with or without spiked E. coli O157:H7 at ≤10(2) CFU·ml(-1) and concentrated using dead-end ultrafiltration (DEUF) to produce primary and secondary retentates. Fifty-four matrix replicates of undiluted secondary retentates or dilutions (1:2 or 1:10 in buffer) were evaluated using an IMS/ATP bioluminescence assay (IMS/ATP). Combining primary and secondary DEUF yielded a 2-4 log(10) increase in E. coli O157:H7 concentrations in spiked samples and resulted in signal-to-noise ratios 2-219 fold higher than controls, depending on the sample type. DEUF increased the concentration of E. coli O157:H7 to within the detectable limits of IMS/ATP. The combined assay provided detection of viable E. coli O157:H7 in produce and recreational water. Accurate detection of microbial pathogens using DEUF and IMS/ATP could reduce disease outbreaks from contaminated water sources and food products.

  8. Emergency surgery for tubo-ovarian abscess identified extended-spectrum beta-lactamase-producing Escherichia coli: the first case presentation revealing causative bacteria.

    PubMed

    Tokumaru, Teppei; Shima, Yasuo; Okabayashi, Takehiro; Hayashi, Kazutoshi; Yamamoto, Yorito; Ozaki, Kazuhide; Iwata, Jun

    We report herein a 41-year-old female with a tubo-ovarian abscess (TOA), which microbial cultures showed to contain extended-spectrum beta-lactamase (ESBL)-producing E. coli, a causative agent of community-acquired infection. The patient initially presented with acute abdominal pain and back pain. Pelvic computed tomography and transvaginal ultrasonography revealed multiple cystic lesions in the bilateral ovaries that suggested TOA. An emergency laparotomy was therefore performed due to the potential for life-threatening septic shock from the TOA-associated pelvic inflammatory disease. Microbial cultures of postoperative fluid discharge from the placed intra-abdominal catheter, vaginal secretions, urine, blood, and feces detected ESBL-producing E.coli. In summary, we successfully performed emergency surgery for life-threatening septic TOA caused by ESBL-producing E. coli infection.

  9. Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR.

    PubMed

    Li, Yong; Mustapha, Azlin

    2004-01-01

    With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.

  10. Bacteraemia due to non-ESBL-producing Escherichia coli O25b:H4 sequence type 131: insights into risk factors, clinical features and outcomes.

    PubMed

    Morales-Barroso, Isabel; López-Cerero, Lorena; Molina, José; Bellido, Mar; Navarro, María Dolores; Serrano, Lara; González-Galán, Verónica; Praena, Julia; Pascual, Alvaro; Rodríguez-Baño, Jesús

    2017-04-01

    The epidemiology and outcomes of bloodstream infections (BSIs) caused by Escherichia coli ST131 isolates not producing extended-spectrum β-lactamases (ESBLs) are not well defined despite being more prevalent than ESBL-producers. In this study, risk factors and the impact on outcome of BSIs caused by non-ESBL-producing ST131 E. coli versus non-ST131 E. coli were investigated. A case-control study was performed in two tertiary centres to identify risk factors for ST131. Molecular methods were used to investigate all E. coli isolates from blood cultures for those belonging to O25b:H4-ST131 clonal group. fimH alleles were characterised in ST131 isolates. Multivariate analysis was performed by logistic regression or Cox regression as appropriate. A total of 33 ST131 E. coli cases and 56 controls were studied. ST131 isolates showed higher rates of resistance to ampicillin and ciprofloxacin; fimH alleles were H30 in 14 isolates (42.4%) and H22 in 12 isolates (36.4%). Only recent surgery (OR = 7.03, 95% CI 1.71-28.84; P = 0.007) and unknown source of bacteraemia (OR = 5.37, 95% CI 0.93-30.81; P = 0.05) were associated with ST131. ST131 isolates showed no association with 30-day mortality, therapeutic failure, presentation with severe sepsis/shock or length of stay. Bacteraemia due to non-ESBL-producing O25b:H4-ST131 E. coli showed few differences in terms of risk factors as well as similar outcome to non-ST131 E. coli. These data support the notion that ST131 strains are not less clinically virulent despite showing increased antimicrobial resistance, but also that they are not more virulent than other clonal groups causing BSI.

  11. Comparative assessment of inoculum effects on the antimicrobial activity of amoxycillin-clavulanate and piperacillin-tazobactam with extended-spectrum beta-lactamase-producing and extended-spectrum beta-lactamase-non-producing Escherichia coli isolates.

    PubMed

    López-Cerero, L; Picón, E; Morillo, C; Hernández, J R; Docobo, F; Pachón, J; Rodríguez-Baño, J; Pascual, A

    2010-02-01

    A significant inoculum-size effect has been observed with piperacillin-tazobactam, and has been associated with beta-lactamase production in extended-spectrum beta-lactamase (ESBL) producers. This association has not been previously studied in the case of amoxycillin-clavulanate. Piperacillin-tazobactam and amoxycillin-clavulanate were compared, using high inocula of susceptible strains either harbouring ESBLs or not. Two non-ESBL-producing and 15 amoxycillin-clavulanate-susceptible and piperacillin-tazobactam-susceptible ESBL-producing Escherichia coli isolates, and their respective transconjugants, were tested in dilution susceptibility tests using standard and 100-fold higher inocula. Three ESBL-producing strains and E. coli ATCC 25922 were selected for time-kill studies using standard and high initial inocula. At high inocula, MICs of piperacillin increased >eight-fold for non-ESBL-producing strains, and MICs of piperacillin-tazobactam (8:1 ratio or with tazobactam fixed at 4 mg/L) increased>eight-fold for all ESBL-producing strains. However, amoxycillin MICs were not affected by a high inoculum with non-ESBL-producing strains, whereas the MICs of amoxycillin-clavulanate (2:1 and 4:1) increased producers, using the broth and agar dilution methods. In kinetic studies at a high inoculum, amoxycillin and amoxycillin-clavulanate were bactericidal against E. coli ATCC 25922, whereas piperacillin and piperacillin-tazobactam yielded decreases of <1 log(10) CFU/mL. Similarly, at a high inoculum, only amoxycillin-clavulanate was able to maintain bactericidal rates of killing over 24 h against the ESBL-positive E. coli isolates. The stability of amoxycillin-clavulanate and the contrasting results obtained with piperacillin-tazobactam against high inocula of ESBL-non-producing and ESBL-producing E. coli strains appear to be related to aspects other than the amount of beta-lactamase production.

  12. Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

    PubMed

    Gibello, Alicia; Díaz de Alba, Paula; Blanco, M Mar; Machuca, Jesus; Cutuli, M Teresa; Rodríguez-Martínez, José Manuel

    2014-09-01

    This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

  13. First description of OXA-48-producing Escherichia coli and the pandemic clone ST131 from patients hospitalised at a military hospital in Algeria.

    PubMed

    Agabou, A; Pantel, A; Ouchenane, Z; Lezzar, N; Khemissi, S; Satta, D; Sotto, A; Lavigne, J-P

    2014-09-01

    The aim of the study was to assess the frequency and diversity of carbapenemases and extended-spectrum β-lactamases (ESBL) produced by Escherichia coli isolates from patients hospitalised in the Regional Military Hospital of Constantine (Algeria). E. coli isolates were collected over a 2-year period from patients presenting E. coli infections. Strains with reduced susceptibility to ertapenem and/or positive for ESBL were characterised with regard to antibiotic resistance, bla genes, phylogenetic groups, O25 serotyping, quinolone resistance, repetitive sequence-based polymerase chain reaction (rep-PCR) profiles and multi-locus sequence typing (MLST). Of the 448 isolated E. coli, 94 (20.9 %) were multidrug-resistant. One of them (1.1 %) produced a bla OXA-48 and was identified as a B1 ST5 strain. The transposon bearing this gene was Tn1999.2. This strain was isolated from a patient coming from a border province with Tunisia, where this carbapenemase is endemic. In addition, 84 (18.8 %) isolates among them produced an ESBL with predominance (97.6 %) of bla CTX-M-15, which was coupled with qnr genes in 10.9 %. ESBL-producing strains were mainly detected in phylogroups D and A. They displayed 20 rep-PCR profiles and all the clonally related isolates were of the same sequence type (ST). Ten strains (9.4 %) belonged to the pandemic clone ST131. This study describes for the first time the presence of OXA-48-producing E. coli and the emergence of the intercontinental ST131 bla CTX-15-producing E. coli strains in Algeria.

  14. Prevalence and genetic diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in nursing homes in Bavaria, Germany.

    PubMed

    Valenza, Giuseppe; Nickel, Silke; Pfeifer, Yvonne; Pietsch, Michael; Voigtländer, Elzbieta; Lehner-Reindl, Verena; Höller, Christiane

    2017-02-01

    Main goal of this study was to determine the prevalence and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae among 156 nursing home residents in Bavaria and to compare the results with healthy individuals from the Bavarian community. Intestinal colonisation by ESBL-producing Escherichia coli was detected in 23 nursing home residents (14.7%) using MacConkey agar supplemented with cefotaxime (1mg/L) for screening and the combined disc method for ESBL confirmation. Antimicrobial susceptibility testing revealed co-resistance to ciprofloxacin in 86.9% of the ESBL-producers. All isolates harboured CTX-M-ESBL with CTX-M-15 (65.2%) and CTX-M-27 (21.7%) as the most common types. Moreover, 16 isolates (69.6%) could be assigned by PCR-typing to the epidemic clonal lineage E. coli O25b-ST131. Further typing by rep-PCR and XbaI-macrorestriction with subsequent pulsed-field gel electrophoresis, respectively, revealed that two or more residents shared the same ESBL-producing E. coli clone in four nursing homes. In conclusion, we could show a high prevalence of ESBL-producing E. coli in Bavarian nursing homes (14.7%) compared to the healthy population (6.3%). Although the prevalence of ESBL-type CTX-M-15 in E. coli was similar in nursing home residents (65.2%) and healthy individuals (46%) the presence of E. coli O25b-ST131 clones differed substantially (69.6% and 14.2%, respectively). Furthermore, this study demonstrates that a person-to-person transmission or a common source of infection for ESBL-producing microorganisms may occur in these facilities. Therefore, basic hygiene measures should be assiduously implemented to prevent the further spread of these multidrug-resistant bacteria.

  15. A Poly-N-acetylglucosamine-Shiga toxin broad-spectrum conjugate vaccine for Shiga toxin-producing Escherichia coli.

    PubMed

    Lu, Xi; Skurnik, David; Pozzi, Clarissa; Roux, Damien; Cywes-Bentley, Colette; Ritchie, Jennifer M; Munera, Diana; Gening, Marina L; Tsvetkov, Yury E; Nifantiev, Nikolay E; Waldor, Matthew K; Pier, Gerald B

    2014-03-25

    Many pathogens produce the β-(1-6)-linked poly-N-acetylglucosamine (PNAG) surface polysaccharide that is being developed as a broadly protective antimicrobial vaccine. However, it is unknown whether systemically injected PNAG vaccines or antibodies would provide protective immunity against pathogens confined to the gastrointestinal tract such as Shiga toxin (Stx)-producing Escherichia coli (STEC), an important group of gastrointestinal (GI) pathogens for which effective immunotherapeutics are lacking. To ascertain whether systemic IgG antibody to PNAG impacts this infectious situation, a vaccine consisting of a synthetic nonamer of nonacetylated PNAG, 9GlcNH2, conjugated to the Shiga toxin 1b subunit (9GlcNH2-Stx1b) was produced. Rabbit antibodies raised to the conjugate vaccine were tested for bacterial killing and toxin neutralization in vitro and protection against infection in infant mice. Cell surface PNAG was detected on all 9 STEC isolates tested, representing 6 STEC serogroups, including E. coli O157:H7. Antibody to the 9GlcNH2-Stx1b conjugate neutralized Stx1 potently and Stx2 modestly. For O157:H7 and O104:H4 STEC strains, antibodies elicited by the 9GlcNH2-Stx1b conjugate possessed opsonic killing and bactericidal activity. Following intraperitoneal injection, antibodies to both PNAG and Stx were needed for infant mouse protection against O157 STEC. These antibodies also mediated protection against the Stx2-producing O104:H4 strain that was the cause of a recent outbreak in Germany, although sufficient doses of antibody to PNAG alone were protective against this strain in infant mice. Our observations suggest that vaccination against both PNAG and Stx, using a construct such as the 9GlcNH2-Stx1b conjugate vaccine, would be protective against a broad range of STEC serogroups. IMPORTANCE The presence of poly-N-acetylglucosamine (PNAG) on many pathogens presents an opportunity to target this one structure with a multispecies vaccine. Whether antibodies to

  16. Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources.

    PubMed

    Martin, Annett; Beutin, Lothar

    2011-03-15

    Shiga toxin-producing strains of Escherichia coli (STEC) cause diarrhoea and haemorrhagic colitis in humans. Most human infections are attributed to consumption of STEC contaminated foodstuff. Food producing animals constitute important reservoirs of STEC and serve as source of food contamination. In this study, we have analyzed 593 foodborne STEC strains for their serotypes and for nine virulence genes (stx1, stx1c, stx1d, stx2, stx2b, stx2e, stx2g, E-hly and eae). The 593 STEC strains grouped into 215 serotypes, and 123 serotypes (57.2%) were represented each by only one STEC isolate. Fifteen serotypes (7.0%) were attributed to 198 (33.3%) of the 593 STEC strains. The foodborne STEC were grouped into different categories in relation to the species of the food producing animal (cattle, pigs, sheep, goats, red deer, wild-boar and hare). Univariate and multivariate statistical analyses revealed significant similarities between the animal origin of the food and the virulence markers of foodborne STEC. Significant associations (p<0.001) were found for stx1 and for stx2 with bovine meat and milk products. The stx2e gene was significantly (p<0.001) associated with STEC from pork and wild boar meat. Stx1c and stx2b genes were significantly (p<0.001) more frequent in STEC from deer meat, as well as from meat and milk products derived from sheep and goats. Using logistic regression models we detected significant (p<0.01) combinations between stx1, stx2 and E-hly genes and STEC from bovine meat. The combination of stx1c and stx2b genes was significant (p<0.001) for STEC derived from red deer, sheep and goat products. The properties of foodborne STEC were compared with published data on faecal STEC from food producing animals. Virulence profiles and serotypes of STEC from food showed remarkable similarities to those of faecal STEC that were from the same animal species. The findings from our study clearly indicate that the food producing animals represent the most important

  17. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  18. Isolation of Shiga Toxin-Producing Escherichia coli from Ground Beef Using Multiple Combinations of Enrichment Broths and Selective Agars.

    PubMed

    Brusa, Victoria; Piñeyro, Pablo E; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Padola, Nora L; Leotta, Gerardo A

    2016-03-01

    Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens, and beef cattle are recognized as the principal reservoir. The aims of this study were (1) to identify the most sensitive combination of selective enrichment broths and agars for STEC isolation in artificially inoculated ground beef samples, and (2) to evaluate the most efficient combination(s) of methods for naturally contaminated ground beef samples. A total of 192 ground beef samples were artificially inoculated with STEC and non-stx bacterial strains. A combination of four enrichment broths and three agars were evaluated for sensitivity, specificity, and positive predictive value for STEC isolation from experimentally inoculated samples. Enrichments with either modified tryptic soy broth (mTSB) containing 8 mg/L novobiocin (mTSB-8) or modified Escherichia coli (mEC) broth followed by isolation in MacConkey agar were the most sensitive combinations for STEC isolation of artificially inoculated samples. Independently, both enrichments media followed by isolation in MacConkey were used to evaluate ground beef samples from 43 retail stores, yielding 65.1% and 58.1% stx-positive samples by RT-PCR, respectively. No difference was observed in the isolate proportions between these two methods (8/25 [32%] and 8/28 [28.6%]). Identical serotypes and stx genotypes were observed in STEC strains isolated from the same samples by either method. In this study, no single enrichment protocol was sufficient to detect all STEC in artificially inoculated samples and had considerable variation in detection ability with naturally contaminated samples. Moreover, none of the single or combinations of multiple isolation agars used were capable of identifying all STEC serogroups in either artificially inoculated or naturally occurring STEC-contaminated ground beef. Therefore, it may be prudent to conclude that there is no single method or combination of isolation methods capable of identifying all STEC serogroups.

  19. Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe.

    PubMed Central

    Heuvelink, A. E.; van de Kar, N. C.; Meis, J. F.; Monnens, L. A.; Melchers, W. J.

    1995-01-01

    Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed. Images Fig. 1 Fig. 2 Fig. 3 PMID:7641823

  20. In vitro Effectiveness of Commercial Bacteriophage Cocktails on Diverse Extended-Spectrum Beta-Lactamase Producing Escherichia coli Strains

    PubMed Central

    Gundogdu, Aycan; Bolkvadze, Darajen; Kilic, Huseyin

    2016-01-01

    The objective of this study is to determine the in vitro susceptibility of Georgian bacteriophage cocktails on multidrug resistant (MDR) extended-spectrum beta-lactamase producing Escherichia coli (ESBL-EC) isolated from patients’ blood and urine cultures. A total of 615 E. coli isolates were included in this study. Phene Plate (PhP)-typing and phylogenetic grouping were used for the typing. Antimicrobial resistance profiles and ESBL production of all isolates were confirmed according to Clinical and Laboratory Standards Institute (CLSI) criteria. The activities of four bacteriophage cocktails (Enko-phage, SES-bacteriophage, Pyo-bacteriophage, and Intesti-bacteriophage) were determined against 142 ESBL-EC using in vitro spot tests. According to this, Enko-phage were active against 87.3% of the tested strains while that ratio was 81.7% for Intesti-bacteriophage, 81.7% for Pyo-bacteriophage, and 59.2% for SES-bacteriophage cocktails. Based on the contingency tests, the phage cocktails were observed to be statistically significantly (p < 0.001) more effective on ESBL-EC strains belonging to phylogenetic groups D and B2. The employed phage cocktails were found to be affective against all tested resistant types. These results are promising especially for the infections that are caused by MDR pathogens that are difficult to treat. As this is a preliminary step to the potential clinical trials to be designed for the country, in vitro confirmation of their success on a MDR ESBL-EC collection should be accepted as an initial action, which is encouraging to consider clinical trials of phage therapy especially in countries which are not introduce phage therapy. PMID:27857711

  1. [Serotype identification and antibiotic susceptibility of Shiga toxin-producing Escherichia coli in the Weishan area in Shandong Province, China].

    PubMed

    Shao, C C; Hu, B; Bi, Z W; Kou, Z Q; Fang, M; Chen, B L; Bi, Z Q

    2017-01-06

    Objective: To determine the serotypes and drug resistance profiles of Shiga toxin-producing Escherichia coli (STEC) in animal stools from the Weishan area in Shandong Province, China. To provide the basis for further study. Methods: Five hundred animal stool samples (from pigs, cattle, sheep, dogs and birds) were collected from the Weishan area and STEC strains were isolated from these samples. Strains were serotyped by a serum agglutination test, and their drug resistance profiles were determined through antimicrobial sensitivity experiments. In this study, PCR was used to detect tetracycline resistance genes (tetA, tetB, tetC, tetD) and beta-lactam resistance genes (blaSHV-1, blaCTX-M, blaTEM). Results: Sixteen strains of STEC were isolated from animal stool samples. Thirteen strains were isolated from pig stool samples, two from bovine stool samples and one from a sheep stool sample. Two of the strains were identified as E. coli O157:H7, and other 14 strains were non-O157 STEC of different serotypes. Antimicrobial sensitivity experiments showed that 15 of the strains were multidrug resistant. The rates of resistance were as follows: nalidixic acid (12/16 strains), sulfisoxazole (11/16), trimethoprim and sulphame-thoxazole (11/16), doxycycline (9/16), azithromycin (9/16), tetracycline (9/16), chloramphenicol (8/16) and streptomycin (8/16). Therefore, nalidixic acid showed the highest rate of resistance among the strains, followed by trimethoprim and sulphame-thoxazole, and sulfisoxazole. Resistance to cefepime or imipenem was not detected. In total, three types of drug resistance genes (tetA, tetB and tetC) were detected among the 16 strains. Conclusion: The results showed that STEC strains isolated from animals in the Weishan area were of a range of serotypes. The 16 strains of STEC isolated from animal stools in this area were resistant to a number of antibiotics, with many strains displaying multidrug resistance.

  2. Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli.

    PubMed

    Stromberg, Loreen R; Stromberg, Zachary R; Banisadr, Afsheen; Graves, Steven W; Moxley, Rodney A; Mukundan, Harshini

    2015-09-01

    Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.

  3. Purification and characterization of Streptococcus sobrinus dextranase produced in recombinant Escherichia coli and sequence analysis of the dextranase gene.

    PubMed Central

    Wanda, S Y; Curtiss, R

    1994-01-01

    The plasmid (pYA902) with the dextranase (dex) gene of Streptococcus sobrinus UAB66 (serotype g) produces a C-terminal truncated dextranase enzyme (Dex) with a multicomplex mass form which ranges from 80 to 130 kDa. The Escherichia coli-produced enzyme was purified and characterized, and antibodies were raised in rabbits. Purified dextranase has a native-form molecular mass of 160 to 260 kDa and specific activity of 4,000 U/mg of protein. Potential immunological cross-reactivity between dextranase and the SpaA protein specified by various recombinant clones was studied by using various antisera and Western blot (immunoblot) analysis. No cross-reactivity was observed. Optimal pH (5.3) and temperature (39 degrees C) and the isoelectric points (3.56, 3.6, and 3.7) were determined and found to be similar to those for dextranase purified from S. sobrinus. The dex DNA restriction map was determined, and several subclones were obtained. The nucleotide sequence of the dex gene was determined by using subclones pYA993 and pYA3009 and UAB66 chromosomal DNA. The open reading frame for dex was 4,011 bp, ending with a stop codon TAA. A ribosome-binding site and putative promoter preceding the start codon were identified. The deduced amino acid sequence of Dex revealed the presence of a signal peptide of 30 amino acids. The cleavage site for the signal sequence was determined by N-terminal amino acid sequence analysis for Dex produced in E. coli chi 2831(pYA902). The C terminus consists of a serine- and threonine-rich region followed by the peptide LPKTGD, 3 charged amino acids, 19 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the cell wall-spanning region, the LPXTGX consensus sequence, and the membrane-anchoring domains of surface-associated proteins of gram-positive cocci. Images PMID:8021165

  4. Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum β-lactamases from clinical samples in the Kinki Region of Japan.

    PubMed

    Nakamura, Tatsuya; Komatsu, Masaru; Yamasaki, Katsutoshi; Fukuda, Saori; Miyamoto, Yugo; Higuchi, Takeshi; Ono, Tamotsu; Nishio, Hisaaki; Sueyoshi, Noriyuki; Kida, Kenji; Satoh, Kaori; Toda, Hirofumi; Toyokawa, Masahiro; Nishi, Isao; Sakamoto, Masako; Akagi, Masahiro; Nakai, Isako; Kofuku, Tomomi; Orita, Tamaki; Wada, Yasunao; Zikimoto, Takuya; Koike, Chihiro; Kinoshita, Shohiro; Hirai, Itaru; Takahashi, Hakuo; Matsuura, Nariaki; Yamamoto, Yoshimasa

    2012-04-01

    In the present study, nonduplicate, clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella spp, and Proteus mirabilis were collected during a 10-year period from 2000 to 2009 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from 0.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from 0% to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 2007. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread.

  5. Amikacin therapy for urinary tract infections caused by extended-spectrum β-lactamase-producing Escherichia coli

    PubMed Central

    Cho, Sung-Yeon; Choi, Su-Mi; Park, Sun Hee; Lee, Dong-Gun; Choi, Jung-Hyun; Yoo, Jin-Hong

    2016-01-01

    Background/Aims: The number of urinary tract infections (UTIs) caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) is increasing. In an outpatient setting, there are limited therapeutic options to treat ESBL-producing pathogens. We evaluated the outcomes of amikacin outpatient parenteral antibiotic therapy (OPAT) for UTIs caused by ESBL-EC in patients not pre-treated with carbapenem. Methods: We retrospectively evaluated the outcomes of amikacin OPAT for UTIs caused by ESBL-EC. Results: From November 2011 to October 2012, eight females, who could not be hospitalized for carbapenem treatment, were treated with amikacin OPAT for nine episodes of non-bacteremic ESBL-EC UTIs. Seven of the eight patients had one or more comorbidities. Of the nine UTI cases, three had symptomatic lower UTIs and six had non-bacteremic upper UTIs. In all of the cases, symptomatic and laboratory improvements were observed following amikacin OPAT. One patient showed a delayed relapse with bilateral microabscesses 3 weeks after treatment cessation; however, a clinical and microbiological cure was eventually reached. All of the patients were able to tolerate amikacin OPAT without any significant nephrotoxicity or ototoxicity. Conclusions: Amikacin OPAT represents a feasible therapeutic option for non-bacteremic UTIs caused by ESBL-EC in settings with limited resources. PMID:26767869

  6. High-level expression and purification of recombinant human growth hormone produced in soluble form in Escherichia coli.

    PubMed

    Levarski, Zdenko; Šoltýsová, Andrea; Krahulec, Ján; Stuchlík, Stanislav; Turňa, Ján

    2014-08-01

    Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. In this work, we have utilized an efficient T7 based expression system to produce high levels of soluble thioredoxin-hGH (Trx-hGH) fusion protein. We outline a relatively simple three step purification process employing two immobilized metal-affinity chromatography and one anion-exchange steps and removal of fusion partner by enterokinase cleavage yielding native hGH. The ability of cell populations to produce quantities of up to 1 g/L of the soluble Trx-hGH fusion protein has been tested in flask cultivations as well as in batch and fed-batch bioreactor runs. The sequence and structure of derived hGH were confirmed by mass spectrometry and circular dichroism and its native function, to induce cell proliferation, was confirmed by employing a Nb2 cell line proliferation assay.

  7. Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease.

    PubMed

    Makino, S; Watarai, M; Tabuchi, H; Shirahata, T; Furuoka, H; Kobayashi, Y; Takeda, Y

    2001-07-01

    Porcine edema disease (ED) is an enterotoxaemia in pigs after weaning, caused by Shiga toxin 2e (Stx2e) producing Escherichia coli. Recently in Japan, outbreaks of ED are re-emerging in pig production. In this study we constructed a mutant that retained immunogenicity but lost Vero cell cytotoxicity, which produced genetically modified toxin (Stx2e*) by replacing glutamate with glutamine at position 167 and arginine with leucine at position 170 of the A subunit. The stx(2e)* gene was replaced with the stx(2e)gene of the wild type virulent strain by homologous recombination. As the parent wild strain was pathogenic to pigs but the mutant was not, the mutant named as YT106 was given to the pigs to examine its protective immunity against ED. All 20 pigs vaccinated with YT106 survived, but only eight of the 20 non-vaccinated pigs survived after the challenge with a wild strain. Also, the eight pigs that survived had decreased rates of gain relative to those of the controls. Blood IgG and intestinal IgA titres increased 3.3 and 1.6 times more than the control, respectively, showing that YT106 might be a good candidate of a live attenuated vaccine strain to protect against ED.

  8. Genotyping of ESBL Producing Uropathogenic Escherichia coli in West of Iran

    PubMed Central

    Darfarin, Gita

    2014-01-01

    Background and Objective. Urinary tract infection (UTI) is one of the most common bacterial infections in the world. Molecular fingerprinting of UTI isolates such as pulsed-Field Gel Electrophoresis using for Clonal distribution and determine of predominant type. The aim of the study was to determine genotyping of ESBL producing UPECs. Material and Methods. 200 UPEC isolates from outpatients with UTI were obtained. Antimicrobial susceptibility and interpretation were performed by disk diffusion. Virulence factors for UPECs were screened by using PCR. UPECs were analyzed by Pulsed-Field Gel Electrophoresis and images analyzed by Phoretix1DPro software. Results. A total of 200 isolates of UPECs, 24.5% (n = 49) of isolates, were positive for ESBL production. Resistance ranged from 0% for amikacin and imipenem to over 93.9% for carbenicillin and ampicillin. Frequencies of haemagglutination, haemolysin, and hydrophobicity were 51%, 18.3%, and 14.28%, respectively. A total of 10 different genotypes were obtained, which include nine common clones and one single clone. Conclusion. We confirmed the prevalence of virulence phenotyping especially Haemagglutination among UPEC strains and that it can also contribute to virulence in these strains. Large diversity in genotypes was observed in the isolates that could be indicative of different sources of infection in community acquired. PMID:24839441

  9. Risk factors for ESBL-producing Escherichia coli on pig farms: A longitudinal study in the context of reduced use of antimicrobials

    PubMed Central

    Bonten, Marc J. M.; Wagenaar, Jaap A.; Mevius, Dik; Heederik, Dick J. J.

    2017-01-01

    The presence of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) in food animals is a public health concern. This study aimed to determine prevalence of ESBL-E. coli on pig farms and to assess the effect of reducing veterinary antimicrobial use (AMU) and farm management practices on ESBL-E. coli occurrence on pig farms. During 2011–2013, 36 Dutch conventional pig farms participated in a longitudinal study (4 sampling times in 18 months). Rectal swabs were taken from 60 pigs per farm and pooled per 6 pigs within the same age category. Presence of ESBL-E. coli was determined by selective plating and ESBL genes were characterized by microarray, PCR and gene sequencing. An extensive questionnaire on farm characteristics and AMU as Defined Daily Dosages per Animal Year (DDDA/Y) was available for the 6-month periods before each sampling moment. Associations between the presence of ESBL-E. coli-positive pigs and farm management practices were modelled with logistic regression. The number of farms with ESBL-E. coli carrying pigs decreased from 16 to 10 and the prevalence of ESBL-E. coli-positive pooled pig samples halved from 27% to 13%. Overall, the most detected ESBL genes were blaCTX-M-1, blaTEM-52 and blaCTX-M-14. The presence of ESBL-E. coli carrying pigs was not related to total AMU, but it was strongly determined by the presence or absence of cephalosporin use at the farm (OR = 46.4, p = 0.006). Other farm management factors, related with improved biosecurity, were also plausibly related to lower probabilities for ESBL-E. coli-positive farms (e.g. presence of a hygiene lock, pest control delivered by a professional). In conclusion, ESBL-E. coli prevalence decreased in pigs during 2011 and 2013 in the Netherlands. On pig farms, the use of cephalosporins was associated with the presence of ESBL-E. coli carrying pigs. PMID:28323856

  10. Unexpected common occurrence of transferable extended spectrum cephalosporinase-producing Escherichia coli in Swedish surface waters used for drinking water supply.

    PubMed

    Egervärn, Maria; Englund, Stina; Ljunge, Marianne; Wiberg, Christer; Finn, Maria; Lindblad, Mats; Börjesson, Stefan

    2017-06-01

    The presence of Enterobacteriaceae producing extended spectrum beta-lactamases (ESBL) or transferable AmpC beta-lactamases (pAmpC) is increasingly being reported in humans, food-producing animals and food world-wide. However, the occurrence and impact of these so-called extended spectrum cephalosporinase (ESC)-producing Enterobacteriaceae in aquatic environments are poorly documented. This study investigated the occurrence, concentrations and characteristics of ESC-producing E. coli (ESC-Ec) in samples of surface water collected at five Swedish water treatment plants that normally have relatively high prevalence and concentration of E. coli in surface water. ESC-Ec was found in 27 of 98 surface water samples analysed. All but two positive samples were collected at two of the water treatment plants studied. The ESC-Ec concentration, 1-3cfu/100mL, represented approximately 4% of the total amount of E. coli in the respective surface water sample. In total, 74% of the isolates were multi-resistant, but no isolate was resistant to carbapenems. Six types of ESBL/pAmpC genes were found in the 27 E. coli isolates obtained from the positive samples, of which four (blaCTX-M-15, blaCMY-2, blaCTX-M-1 and blaCTX-M-14) were found during the whole sampling period, in samples taken at more than one water treatment plant. In addition, the genes were situated on various types of plasmids and most E. coli isolates were not closely related with regard to MLST types. The combinations of ESBL/pAmpC genes, plasmids and E. coli isolates were generally similar to those found previously in healthy and sick individuals in Sweden. In conclusion, the occurrence of ESC-Ec in Swedish surface water shows that resistant bacteria of clinical concern are present in aquatic environments even in a low-prevalence country such as Sweden.

  11. Acid Resistance and Molecular Characterization of Escherichia coli O157:H7 and Different Non-O157 Shiga Toxin-Producing E. coli Serogroups.

    PubMed

    Kim, Gwang-Hee; Breidt, Frederick; Fratamico, Pina; Oh, Deog-Hwan

    2015-10-01

    The objective of this study was to compare the acid resistance (AR) of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroups O26, O45, O103, O104, O111, O121, and O145 with O157:H7 STEC isolated from various sources in 400 mM acetic acid solutions (AAS) at pH 3.2 and 30 °C for 25 min with or without glutamic acid. Furthermore, the molecular subgrouping of the STEC strains was analyzed with the repetitive sequence-based PCR (rep-PCR) method using a DiversiLab(TM) system. Results for a total of 52 strains ranged from 0.31 to 5.45 log reduction CFU/mL in the absence of glutamic acid and 0.02 to 0.33 CFU/mL in the presence of glutamic acid except for B447 (O26:H11), B452 (O45:H2), and B466 (O104:H4) strains. Strains belonging to serogroups O111, O121, and O103 showed higher AR than serotype O157:H7 strains in the absence of glutamic acid. All STEC O157:H7 strains exhibited a comparable DNA pattern with more than 95% similarity in the rep-PCR results, as did the strains belonging to serogroups O111 and O121. Surprisingly, the DNA pattern of B458 (O103:H2) was similar to that of O157:H7 strains with 82% similarity, and strain B458 strain showed the highest AR to AAS among the O103 strains with 0.44 log reduction CFU/mL without glutamic acid. In conclusion, STEC serotypes isolated from different sources exhibited diverse AR and genetic subtyping patterns. Results indicated that some non-O157 STEC strains may have higher AR than STEC O157:H7 strains under specific acidic conditions, and the addition of glutamic acid provided enhanced protection against exposure to AAS.

  12. Characterization of fecal extended-spectrum-β-lactamase-producing Escherichia coli in a remote community during a long time period.

    PubMed

    Woerther, Paul-Louis; Angebault, Cécile; Jacquier, Hervé; Clermont, Olivier; El Mniai, Assyia; Moreau, Brigitte; Djossou, Félix; Peroz, Gilles; Catzeflis, François; Denamur, Erick; Andremont, Antoine

    2013-10-01

    Carriage of extended-spectrum beta-lactamase-producing enterobacteria (ESBL-E) has increased in community settings. Little is known about their long-term evolution. French Guiana Amerindians living in a very remote village, already sampled in 2001 and 2006 for ESBL-E fecal carriage, were screened again in October 2010. Sociodemographic data and antibiotic intake data were collected during the previous year. ESBL-E strains collected in 2010 and their plasmid contents were typed. The results were compared to those of the previous campaigns. The prevalence of ESBL-E carriage in 2010 was 5.3%, whereas it was 8.0% and 3.2% in 2006 and 2001, respectively. As previously determined, no individual factor was associated with carriage, including personal antibiotic exposure. However, overall antibiotic use had decreased to a 0.67 treatments/subject/year in 2010 versus 1.09 in 2006 (P < 0.001), which supports the idea that population exposure to antibiotics impacts on ESBL-E community carriage rates. A wide diversity of ESBL Escherichia coli strains belonging to the A0, A1, B1, and D2 phylogroups and producing the CTX-M-1, CTX-M-2, and CTX-M-8 enzymes were isolated. Despite the overall genetic diversity of the strains evaluated by repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing, two CTX-M-1-producing clones were found to have spread. In contrast, similar ESBL-bearing I1/Iγ plasmids were present in various strains both within and between carriers, suggesting high rates of plasmid transfer. Our results suggest that overall antibiotic exposure affects ESBL-E fecal carriage in the community. ESBL-E spread may be the result of both strain dissemination and the transfer of plasmids in intestinal microbiota.

  13. Characterization of Fecal Extended-Spectrum-β-Lactamase-Producing Escherichia coli in a Remote Community during a Long Time Period

    PubMed Central

    Angebault, Cécile; Jacquier, Hervé; Clermont, Olivier; El Mniai, Assyia; Moreau, Brigitte; Djossou, Félix; Peroz, Gilles; Catzeflis, François; Denamur, Erick; Andremont, Antoine

    2013-01-01

    Carriage of extended-spectrum beta-lactamase-producing enterobacteria (ESBL-E) has increased in community settings. Little is known about their long-term evolution. French Guiana Amerindians living in a very remote village, already sampled in 2001 and 2006 for ESBL-E fecal carriage, were screened again in October 2010. Sociodemographic data and antibiotic intake data were collected during the previous year. ESBL-E strains collected in 2010 and their plasmid contents were typed. The results were compared to those of the previous campaigns. The prevalence of ESBL-E carriage in 2010 was 5.3%, whereas it was 8.0% and 3.2% in 2006 and 2001, respectively. As previously determined, no individual factor was associated with carriage, including personal antibiotic exposure. However, overall antibiotic use had decreased to a 0.67 treatments/subject/year in 2010 versus 1.09 in 2006 (P < 0.001), which supports the idea that population exposure to antibiotics impacts on ESBL-E community carriage rates. A wide diversity of ESBL Escherichia coli strains belonging to the A0, A1, B1, and D2 phylogroups and producing the CTX-M-1, CTX-M-2, and CTX-M-8 enzymes were isolated. Despite the overall genetic diversity of the strains evaluated by repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing, two CTX-M-1-producing clones were found to have spread. In contrast, similar ESBL-bearing I1/Iγ plasmids were present in various strains both within and between carriers, suggesting high rates of plasmid transfer. Our results suggest that overall antibiotic exposure affects ESBL-E fecal carriage in the community. ESBL-E spread may be the result of both strain dissemination and the transfer of plasmids in intestinal microbiota. PMID:23917313

  14. Interactive effects of temperature, pH, and water activity on the growth kinetics of Shiga toxin-producing Escherichia coli O104:H4 3.

    PubMed

    Juneja, Vijay K; Mukhopadhyay, Sudarsan; Ukuku, Dike; Hwang, Cheng-An; Wu, Vivian C H; Thippareddi, Harshavardhan

    2014-05-01

    The risk of non-O157 Shiga toxin-producing Escherichia coli strains has become a growing public health concern. Several studies characterized the behavior of E. coli O157:H7; however, no reports on the influence of multiple factors on E. coli O104:H4 are available. This study examined the effects and interactions of temperature (7 to 46°C), pH (4.5 to 8.5), and water activity (aw ; 0.95 to 0.99) on the growth kinetics of E. coli O104:H4 and developed predictive models to estimate its growth potential in foods. Growth kinetics studies for each of the 23 variable combinations from a central composite design were performed. Growth data were used to obtain the lag phase duration (LPD), exponential growth rate, generation time, and maximum population density (MPD). These growth parameters as a function of temperature, pH, and aw as controlling factors were analyzed to generate second-order response surface models. The results indicate that the observed MPD was dependent on the pH, aw, and temperature of the growth medium. Increasing temperature resulted in a concomitant decrease in LPD. Regression analysis suggests that temperature, pH, and aw significantly affect the LPD, exponential growth rate, generation time, and MPD of E. coli O104:H4. A comparison between the observed values and those of E. coli O157:H7 predictions obtained by using the U. S. Department of Agriculture Pathogen Modeling Program indicated that E. coli O104:H4 grows faster than E. coli O157:H7. The developed models were validated with alfalfa and broccoli sprouts. These models will provide risk assessors and food safety managers a rapid means of estimating the likelihood that the pathogen, if present, would grow in response to the interaction of the three variables assessed.

  15. Wild Birds, Frequent Carriers of Extended-Spectrum β-Lactamase (ESBL) Producing Escherichia coli of CTX-M and SHV-12 Types.

    PubMed

    Alcalá, Leticia; Alonso, Carla Andrea; Simón, Carmen; González-Esteban, Chabier; Orós, Jesús; Rezusta, Antonio; Ortega, Carmelo; Torres, Carmen

    2016-11-01

    To get a better insight into the role of birds as reservoirs of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC β-lactamase (pAmpC) Escherichia coli producers, 100 fecal samples belonging to 15 different wild avian species from Northern Spain were analyzed. Cefotaxime-resistant (CTX(R)) E. coli isolates were identified in 16 of the 100 tested birds, which corresponded to 9 animal species (Gyps fulvus-griffon vulture, Larus michahellis-yellow-legged gull, Milvus migrans-black kite, Milvus milvus-red kite, Ciconia ciconia-white stork, Sturnus unicolor-spotless starling, Aquila chrysaetos-golden eagle, Cuculus canorus-common cuckoo, Tyto alba-barn owl). Fifteen isolates harbored ESBL or pAmpC-encoding genes (number of isolates): bla SHV-12 (9), bla CTX-M-1 (3), bla CTX-M-14 (2), and bla CMY-2 (1). The last CTX(R) isolate presented a -42-point-mutation in the chromosomal ampC promoter. Eleven out of 15 ESBL/pAmpC E. coli isolates were multiresistant (most common resistance phenotype: β-lactams-quinolones-tetracycline-sulfamethoxazole/trimethoprim). A plasmid-mediated quinolone resistance determinant (qnrS1) was identified in one E. coli from a barn owl. High genetic diversity was observed among ESBL/pAmpC E. coli isolates, with 12 different sequence types (STs), including several strains of STs frequently detected among human clinical isolates (ST38/D, ST131/B2, ST155/B1, ST10/A). The ST131 isolate belonged to the emergent ciprofloxacin-resistant H30R subclone. This study reveals a high percentage of bird as carriers of ESBL/pAmpC E. coli isolates in Spain, highlighting the elevated rate among storks, kites, and vultures. Wild birds can contribute to the global spread of ESBL/pAmpC-producing E. coli in natural ecosystems.

  16. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

  17. Risk Factors for Salmonella, Shiga Toxin-Producing Escherichia coli and Campylobacter Occurrence in Primary Production of Leafy Greens and Strawberries

    PubMed Central

    Ceuppens, Siele; Johannessen, Gro S.; Allende, Ana; Tondo, Eduardo César; El-Tahan, Fouad; Sampers, Imca; Jacxsens, Liesbeth; Uyttendaele, Mieke

    2015-01-01

    The microbiological sanitary quality and safety of leafy greens and strawberries were assessed in the primary production in Belgium, Brazil, Egypt, Norway and Spain by enumeration of Escherichia coli and detection of Salmonella, Shiga toxin-producing E. coli (STEC) and Campylobacter. Water samples were more prone to containing pathogens (54 positives out of 950 analyses) than soil (16/1186) and produce on the field (18/977 for leafy greens and 5/402 for strawberries). The prevalence of pathogens also varied markedly according to the sampling region. Flooding of fields increased the risk considerably, with odds ratio (OR) 10.9 for Salmonella and 7.0 for STEC. A significant association between elevated numbers of generic E. coli and detection of pathogens (OR of 2.3 for STEC and 2.7 for Salmonella) was established. Generic E. coli was found to be a suitable index organism for Salmonella and STEC, but to a lesser extent for Campylobacter. Guidelines on frequency of sampling and threshold values for E. coli in irrigation water may differ from region to region. PMID:26295251

  18. Mixed biofilm formation by Shiga toxin-producing Escherichia coli and Salmonella enterica serovar typhimurium enhanced bacterial resistance to sanitization due to extracellular polymeric substances

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin–producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their pla...

  19. Whole-Genome Sequencing Applied to the Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli O157:H7 in Argentina.

    PubMed

    Carbonari, Claudia Carolina; Fittipaldi, Nahuel; Teatero, Sarah; Athey, Taryn B T; Pianciola, Luis; Masana, Marcelo; Melano, Roberto G; Rivas, Marta; Chinen, Isabel

    2016-12-01

    Shiga toxin-producing Escherichia coli strains are worldwide associated with sporadic human infections and outbreaks. In this work, we report the availability of high-quality draft whole-genome sequences for 19 O157:H7 strains isolated in Argentina.

  20. Recovery of Shiga toxin-producing Escherichia coli 0157:H7 in tenderized veal cordon bleu following cooking on an electric skillet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The implication of veal products in several recalls due to contamination with Shiga toxin-producing Escherichia coli (STEC) and USDA FSIS verification sampling results revealing a higher percent positive rate of STEC in veal than in beef products provides justification for validating cooking practic...

  1. Occurrence of the Plasmid-Mediated Fluoroquinolone Resistance qepA1 Gene in Two Clonal Clinical Isolates of CTX-M-15-Producing Escherichia coli from Algeria.

    PubMed

    Yanat, Betitera; Dali Yahia, Radia; Yazi, Leila; Machuca, Jesús; Díaz-De-Alba, Paula; Touati, Abdelaziz; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2016-10-13

    QepA is a plasmid-mediated quinolone resistance determinant of low prevalence described worldwide, mainly in Enterobacteriaceae. This study describes, for the first time in Algeria, two clonally related, QepA-producing Escherichia coli clinical isolates positive for CTX-M-15. The clonal spread of these multidrug-resistant isolates is a major public health concern.

  2. Evaluating the efficacy of three USDA-approved antimicrobial sprays for reducing surrogate Shiga toxin-producing cells of "Escherichia coli on bob veal carcasses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) have recently been recognized as a problem for the veal industry, suggesting the need for effective antimicrobial intervention strategies throughout processing. Therefore, we evaluated the efficacy of lactic acid (4.5%), Citrilow™ (pH 1.2), and Beefxide®...

  3. Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS-PSD

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to severe outbreaks of foodborne illness throughout the world, e.g. Germany and France in 2011. STEC infections can result in bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. New analytical techniques are ne...

  4. Genetically marked strains of Shiga toxin-producing O157:H7 and non-O157 Escherichia coli: Tools for detection and modelling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing E. coli (STEC) are among the most important foodborne pathogens in the United States and worldwide. Nearly half of all STEC-induced diarrheal disease in the United States is caused by STEC O157:H7 while non-O157 STEC account for the remaining illnesses. Thus, the USDA Food Safe...

  5. Genotypic analyses of Shiga toxin-producing Escherichia coli O157 and non-O157 recovered from feces of domestic animals in rural farms in Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an...

  6. An environmental shiga toxin-producing Escherichia coli O145 clonal population exhibits high-level phenotypic variation that includes virulence traits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antibiotic resistance profile of environmental O145 strains isolated from a ...

  7. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  8. Growth characteristics of Shiga toxin-producing Escherichia coli (STEC) stressed by chlorine, sodium chloride, acid, and starvation on lettuce and cantaloupe

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin producing Escherichia coli (STEC) is one of the major foodborne pathogens causing serious illnesses, leading to hospitalizations in the United States. Bacteria that are exposed to environmental stresses during food processing may exhibit different growth patterns in subsequent growth env...

  9. Whole-Genome Sequencing Applied to the Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli O157:H7 in Argentina

    PubMed Central

    Fittipaldi, Nahuel; Teatero, Sarah; Athey, Taryn B. T.; Pianciola, Luis; Masana, Marcelo; Melano, Roberto G.; Rivas, Marta; Chinen, Isabel

    2016-01-01

    Shiga toxin-producing Escherichia coli strains are worldwide associated with sporadic human infections and outbreaks. In this work, we report the availability of high-quality draft whole-genome sequences for 19 O157:H7 strains isolated in Argentina. PMID:27908998

  10. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: The aim of this research was to examine relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA, in O157:H7 and non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes after adaptation to pineapple juice (PJ) and subsequently to determine survival with e...

  11. Hyperspectral imaging of shiga toxin-producing escherichia coli serogroups O26, O45, O103, O111, O121, and O145 on Rainbow Agar

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The U.S. Department of Agriculture, Food Safety Inspection Service has determined that six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) are adulterants in raw beef. Isolate and phenotypic discrimination of non-O157 STEC is problematic due ...

  12. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga tox...

  13. Comparison of antibody- versus pcr-based assays for serotyping shiga toxin-producing escherichia coli recovered from various cattle operations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serotyping of Shiga toxin-producing Escherichia coli (STEC) strains is contingent upon the availability of quality antisera. In this study, the serogroup of 161 STEC strains typed by conventional antisera and isolated from the fecal samples of California cattle were compared to two newly developed ...

  14. Disinfectant and antimicrobial susceptibility profiles of the big six non-O157 Shiga toxin-producing Escherichia coli strains from food animals and humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The disinfectant and antimicrobial susceptibility profiles of 144 non-O157 Shiga toxin-producing Escherichia coli (STECs) from food animals and humans were determined. An overall moderate prevalence of 38.9% antimicrobial resistance (AMR) was observed in these strains. Animal strains had a lower p...

  15. The effect of deep frying or conventional oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We investigated the effects deep frying or oven cooking on inactivation of Shiga toxin-producing cells of Escherichia coli (STEC) in meatballs. A finely-ground veal and/or a beef-pork-veal mixture were inoculated (ca. 7.0 log CFU/g) with an eight-strain, genetically-marked cocktail of rifampicin-res...

  16. Short-term evolution of Shiga toxin-producing Escherichia coli O157:H7 between two food-borne outbreaks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Shiga toxin-producing Escherichia coli (STEC) O157 is a public health threat and outbreaks occur worldwide. STEC O157 has a mosaic genome with extensive prophage integration, including bacteriophage-encoded Shiga toxins. Here, we investigate genomic differences in a strain of STEC O157 t...

  17. Genome sequencing and comparative genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-Types of Shiga toxin-producing Escherichia coli O104

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Various Shiga toxin-producing Escherichia coli (STEC) O104 H-types including H4, H7, H21, and H¯ have been associated with sporadic cases of illness and have caused outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. The aim of this work was to conduct a...

  18. Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104 and other STEC serogroups of public health concern

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens that cause outbreaks and serious cases of food-borne illness. Methods for detection and isolation of STEC, particularly the non-O157 STEC, are needed to prevent their transmission through contaminated fo...

  19. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  20. Canonical single nucleotide polymorphisms (SNPs) for high-resolution subtyping of Shiga-toxin producing Escherichia coli (STEC) O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to develop a canonical SNP panel for subtyping of Shiga-toxin producing Escherichia coli (STEC). To this purpose, 906 putative SNPs were identified using resequencing tiling arrays. A subset of 391 SNPs was further screened using high-throughput TaqMan PCR against a d...

  1. Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase.

    PubMed

    Alonso, C A; González-Barrio, D; Tenorio, Carmen; Ruiz-Fons, F; Torres, C

    2016-04-01

    Eighty-nine Escherichia coli isolates recovered from faeces of red deer and small mammals, cohabiting the same area, were analyzed to determine the prevalence and mechanisms of antimicrobial resistance and molecular typing. Antimicrobial resistance was detected in 6.7% of isolates, with resistances to tetracycline and quinolones being the most common. An E. coli strain carrying blaCTX-M-1 as well as other antibiotic resistant genes included in an unusual class 1 integron (Intl1-dfrA16-blaPSE-1-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26) was isolated from a deer. The blaCTX-M-1 gene was transferred by conjugation and transconjugants also acquired an IncN plasmid. This strain was typed as ST224, which seems to be well adapted to both clinical and environmental settings. The phylogenetic distribution of the 89 strains varied depending on the animal host. This work reveals low antimicrobial resistance levels among faecal E. coli from wild mammals, which reflects a lower selective pressure affecting these bacteria, compared to livestock. However, it is remarkable the detection of a multi-resistant ESBL-E. coli with an integron carrying clinically relevant antibiotic-resistance genes, which can contribute to the dissemination of resistance determinants among different ecosystems.

  2. Effect of competitive exclusion in reducing the occurrence of Escherichia coli producing extended-spectrum β-lactamases in the ceca of broiler chicks.

    PubMed

    Nuotio, L; Schneitz, C; Nilsson, O

    2013-01-01

    Extended-spectrum β-lactamases (ESBL) and class C serine β-lactamases (pAmpC) able to hydrolyze third-generation cephalosporins are a recognized threat to the efficacy of these drugs in treating serious infections. Broiler chicks are a known source of Escherichia coli harboring genes for these enzymes. Competitive exclusion (CE) has been used for decades in Finland to prevent the colonization of broiler ceca by Salmonella, but has not been widely used in Sweden. The markedly different prevalences of ESBL- or pAmpC-producing E. coli at slaughter in broilers produced in the 2 countries suggest a potential role for CE. The present study was undertaken to determine the efficacy of a commercial CE product in reducing the colonization of broiler ceca by ESBL- or pAmpC-producing E. coli. The challenge organisms were isolated from healthy broilers in Sweden. Each E. coli strain (1 ESBL and 2 pAmpC types) was subjected to 4 replicate trials. In each trial, a group of 20 newly hatched Ross breed chicks were treated by gavage with the CE product, whereas another group of 20 was left untreated. The next day, all 40 chicks were inoculated by gavage with the E. coli strain. The chicks were reared in cardboard boxes and received feed and water ad libitum. After a week the chicks were asphyxiated with CO(2), and their ceca removed and examined for the presence of the E. coli strains. The median and quartiles of the challenge E. coli estimates in the groups were determined, and the treated and control groups were compared with the Wilcoxon 2-sample test. In each trial, a substantial and statistically significant or highly significant reduction was observed in the colonization of the ceca of CE-treated chicks by E. coli strains, compared with that of untreated control. Referring to an arbitrary criterion for high shedders presented in the literature, it was concluded that at least for the ESBL E. coli, the results were also of epidemiological relevance.

  3. Reduction of Escherichia coli O157:H7 and Salmonella on fresh-cut produce by caprylic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caprylic acid (CA) was evaluated for reducing E. coli O157: H7 and Salmonella on spinach and lettuce leaves. Spinach, Romaine lettuce and Iceberg lettuce leaves were inoculated with a cocktail of five E. coli O157: H7 or Salmonella strains, air dried for 30 min, and then dipped in caprylic acid (10,...

  4. Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producing Escherichia coli isolated from Korean beef cattle

    PubMed Central

    Shin, Seung Won; Jung, Myunghwan; Shin, Min-Kyung

    2015-01-01

    In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum β-lactamase (ESBL) and/or AmpC β-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC β-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type β-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type β-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on β-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of β-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established. PMID:26119172

  5. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber

    PubMed Central

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L.; Lee, Sang Yup

    2010-01-01

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250–320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  6. Shiga toxin-producing Escherichia coli O157 associated with human infections in Switzerland, 2000-2009.

    PubMed

    Käppeli, U; Hächler, H; Giezendanner, N; Cheasty, T; Stephan, R

    2011-07-01

    Shiga toxin-producing Escherichia coli (STEC), an important foodborne pathogen, can cause mild to severe bloody diarrhoea (BD), sometimes followed by life-threatening complications such as haemolytic uraemic syndrome (HUS). A total of 44 O157 strains isolated from different patients from 2000 through 2009 in Switzerland were further characterized and linked to medical history data. Non-bloody diarrhoea was experienced by 15.9%, BD by 61.4% of the patients, and 29.5% developed HUS. All strains belonged to MLST type 11, were positive for stx2 variants (stx2 and/or stx2c), eae and ehxA, and only two strains showed antibiotic resistance. Of the 44 strains, nine phage types (PTs) were detected the most frequent being PT32 (43.2%) and PT8 (18.2%). By PFGE, 39 different patterns were found. This high genetic diversity within the strains leads to the conclusion that STEC O157 infections in Switzerland most often occur as sporadic cases.

  7. Salmonella spp. and Shiga toxin-producing Escherichia coli prevalence in an ocellated lizard (Timon lepidus) research center in Spain.

    PubMed

    Martínez, Remigio; Sánchez, Sergio; Alonso, Juan Manuel; Herrera-León, Silvia; Rey, Joaquín; Echeita, Maria Aurora; Morán, Jose María; García-Sánchez, Alfredo

    2011-12-01

    The aim of this work was to study the epidemiological status of Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) in an ocellated lizard research center focusing on the risk and hygiene aspects. Fecal and environmental samples were collected and examined for Salmonella spp. and STEC. Isolates were detected using real-time polymerase chain reaction (RT-PCR) and characterized using serotyping and pulsed-field gel electrophoresis (PFGE). Overall, 52% of samples were positive for Salmonella spp. using RT-PCR and seven isolates were obtained from samples from ocellated lizards and their environment, whereas no samples were positive for STEC. Salmonella isolates belonged to S. enterica subsp. enterica serovar Kibusi and S. enterica subsp. salamae serovars 41:z10:z6 and 18:z10:z6, some of which have previously been isolated from human sources. Indistinguishable and closely related PFGE types were found, which supported the existence of horizontal transmission between animals due to crowding of animals and the persistence of Salmonella in the environment. The results of the current study emphasize the need for improved prevention efforts and good hygiene practices in research centers, recuperation centers, and zoos with reptiles to minimize the exposure of personnel and visitors to this pathogen.

  8. Risk of haemolytic uraemic syndrome caused by shiga-toxin-producing Escherichia coli infection in adult women in Japan.

    PubMed

    Fujii, J; Mizoue, T; Kita, T; Kishimoto, H; Joh, K; Nakada, Y; Ugajin, S; Naya, Y; Nakamura, T; Tada, Y; Okabe, N; Maruyama, Y; Saitoh, K; Kurozawa, Y

    2016-04-01

    Shiga-toxin-producing Escherichia coli (STEC) infections usually cause haemolytic uraemic syndrome (HUS) equally in male and female children. This study investigated the localization of globotriaosylceramide (Gb3) in human brain and kidney tissues removed from forensic autopsy cases in Japan. A fatal case was used as a positive control in an outbreak of diarrhoeal disease caused by STEC O157:H7 in a kindergarten in Urawa in 1990. Positive immunodetection of Gb3 was significantly more frequent in female than in male distal and collecting renal tubules. To correlate this finding with a clinical outcome, a retrospective analysis of the predictors of renal failure in the 162 patients of two outbreaks in Japan was performed: one in Tochigi in 2002 and the other in Kagawa Prefecture in 2005. This study concludes renal failure, including HUS, was significantly associated with female sex, and the odds ratio was 4·06 compared to male patients in the two outbreaks. From 2006 to 2009 in Japan, the risk factor of HUS associated with STEC infection was analysed. The number of males and females and the proportion of females who developed HUS were calculated by age and year from 2006 to 2009. In 2006, 2007 and 2009 in adults aged >20 years, adult women were significantly more at risk of developing HUS in Japan.

  9. An In Vitro Combined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Skinner, Craig; Zhang, Guodong; Patfield, Stephanie

    2015-01-01

    Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the “big six” serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments. PMID:26100707

  10. An outbreak of Shiga toxin-producing Escherichia coli serogroup O157 linked to a lamb-feeding event.

    PubMed

    Rowell, S; King, C; Jenkins, C; Dallman, T J; Decraene, V; Lamden, K; Howard, A; Featherstone, C A; Cleary, P

    2016-09-01

    Fifteen confirmed cases and 15 possible cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 21/28 were linked to direct contact with lambs at a 'Lambing Live' event in the North West of England between 29 March and 21 April 2014. Twenty-one (70%) of the cases were female, 23 (77%) were children aged <16 years, of whom 14 (46%) were in the 0-5 years age group. Five children developed haemolytic uraemic syndrome. Multilocus variable number tandem repeat analysis (MLVA) profiles on 14 human cases were indistinguishable, and 6/10 animal isolates had a MLVA profile identical to the outbreak profile. Whole-genome sequencing analysis revealed that all isolates, both human and animal, fell within a 5-single nucleotide polymorphism cluster indicating the isolates belonged to the same point source. On inspection of the premises, extensive and uncontrolled physical contact between visitors and animals was occuring within the animal pens and during bottle-feeding. Public areas were visibly contaminated with animal faeces. Information to visitors, and the infection control awareness demonstrated by staff, was inadequate. Managing the risk to visitors of STEC O157 infection at animal petting events and open farms requires implementation of stringent control measures by the operator, as outlined in the industry code of practice. Enforcement action is sometimes required to prevent high-risk activities taking place at both permanent and temporary attractions.

  11. Factors associated with recovery of meat products following recalls due to Shiga toxin-producing Escherichia coli.

    PubMed

    Seys, S A; Sampedro, F; Hedberg, C W

    2016-10-01

    Food-product recall data for recalls due to Shiga toxin-producing Escherichia coli (STEC) from 2000 to 2012 were obtained for establishments regulated by the United States Department of Agriculture, Food Safety and Inspection Service (FSIS). Statistical tests were used to assess the factors associated with recovery of product following STEC recalls along with the relationship between cluster detection and jurisdictions. Our results indicated that the percentage of recalled product recovered following a recall action due to STEC was dependent on the complexity of distribution, type of distribution, amount of time between production and recall dates, and the number of pounds of product recalled. Illness-related STEC recalls were associated with a lower percentage of product recovery which was probably impacted by larger amounts of product recalled, broader production scope, and delays from epidemiological and traceback investigations. Further, detection of illnesses related to STEC recalls seemed to be enhanced in states with additional resources and a history of successful foodborne investigations. This makes an argument for additional resources dedicated to public health agencies specifically for the surveillance of foodborne illnesses.

  12. Geographic Divergence of Bovine and Human Shiga Toxin–Producing Escherichia coli O157:H7 Genotypes, New Zealand1

    PubMed Central

    Cookson, Adrian L.; Campbell, Donald M.; Duncan, Gail E.; Prattley, Deborah; Carter, Philip; Besser, Thomas E.; Shringi, Smriti; Hathaway, Steve; Marshall, Jonathan C.; French, Nigel P.

    2014-01-01

    Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a zoonotic pathogen of public health concern worldwide. To compare the local and large-scale geographic distributions of genotypes of STEC O157:H7 isolates obtained from various bovine and human sources during 2008–2011, we used pulsed-field gel electrophoresis and Shiga toxin–encoding bacteriophage insertion (SBI) typing. Using multivariate methods, we compared isolates from the North and South Islands of New Zealand with isolates from Australia and the United States. The STEC O157:H7 population structure differed substantially between the 2 islands and showed evidence of finer scale spatial structuring, which is consistent with highly localized transmission rather than disseminated foodborne outbreaks. The distribution of SBI types differed markedly among isolates from New Zealand, Australia, and the United States. Our findings also provide evidence for the historic introduction into New Zealand of a subset of globally circulating STEC O157:H7 strains that have continued to evolve and be transmitted locally between cattle and humans. PMID:25568924

  13. [Status of emerging drug resistance in Shiga toxin-producing Escherichia coli in Japan during 1996: a minireview].

    PubMed

    Yamamoto, T; Wakisaka, N

    1998-10-01

    A total of 192 Shiga toxin-producing Escherichia coli (STEC) strains isolated from the 1996 episodes in Japan were tested for their in vitro susceptibilities to 41 antimicrobial agents. Drug resistance was found with kanamycin, tetracycline, nalidixic acid, ampicillin, streptomycin, sulfamethoxazole, and fosfomycin. The expression of fosfomycin resistance was greatly dependent on culture conditions and resistance was detected (e.g.) when Mueller-Hinton agar or nutrient agar supplemented with horse blood (or glucose-6-phosphate) was used as test media. All the STEC strains belonging to serotype O26 exhibited fosfomycin resistance. Multiple drug-resistant strains spread 8 of 18 prefectures examined. Out of eleven O157: H7 outbreaks, only one outbreak revealed infections due to multiple drug-resistant strains which carried an R plasmid. Tetracycline, streptomycin, and sulfamethoxazole resistance, which was previously described with O157: H7 strains isolated from a large outbreak as well as sporadic cases in the United States, were also found in Japan with human and bovine isolates (but not with porcine isolates). In contrast, the STEC strains were highly susceptible to newer quinolones, cephems, trimethoprim, gentamicin, and azithromycin. No drug resistance was observed with dibekacin and minocycline.

  14. N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli

    PubMed Central

    Eitzinger, Christian; Ehrlenbach, Silvia; Lindner, Herbert; Kremser, Leopold; Gottardi, Waldemar; Debabov, Dmitri; Anderson, Mark

    2012-01-01

    N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives. PMID:23139739

  15. Inactivation of a diverse set of shiga toxin-producing Escherichia coli in ground beef by high pressure processing.

    PubMed

    Sheen, Shiowshuh; Cassidy, Jennifer; Scullen, Butch; Sommers, Christopher

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are regularly implicated in foodborne illness outbreaks and recalls of ground beef. In this study we determined the High Pressure Processing (HPP) D10 value (the processing conditions needed to reduce the microbial population by 1 log) of 39 STEC isolates, including the "big six" serovars, O104 and O157:H7. STEC isolates included those isolated from animals and environmental sources in addition to those associated with illness in humans. Individual STEC were inoculated into 80% lean ground beef and treated with HPP (350 MPa, 4 °C, up to 40 min). The mean D10 was 9.74 min, with a range of 0.89-25.70 min. The D10 of the STEC involved in human illness was 9.25 vs. 10.40 min for those not involved in human illness (p > 0.05). The presence or absence of genes encoding virulence factors (e.g. Shiga toxin 1 or 2, intimin, or enterohemolysin) had no effect on the HPP D10 (p > 0.05). The high D10 of some STEC involved in human illness should be considered in selecting HPP processing parameters for ground beef. This study demonstrates the heterogeneity of STEC resistance to HPP. Risk assessors and the food industry can use this information to provide safer meat products to consumers.