Molecular characteristics, biofilm-forming abilities, and quorum sensing molecules in Vibrio parahaemolyticus strains isolated from marine and clinical environments in Korea.

PubMed

Mizan, Md Furkanur Rahaman; Bang, Hyeon-Jo; Sadekuzzaman, Mohammad; Lee, Nari; Kim, Tae-Jo; Ha, Sang-Do

2017-05-01

Vibrio parahaemolyticus is an inhabitant of marine and estuarine environments and causes seafood-borne gastroenteritis in humans. In this study, an UltraFast LabChip Real-Time PCR assay was evaluated for rapid detection and quantification of pathogenic V. parahaemolyticus isolates. Escherichia coli and Vibrio harveyi were used as negative controls. Twenty-six tdh-positive, biofilm-producing V. parahaemolyticus isolates were analyzed by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). REP-PCR analysis showed that the majority of the V. parahaemolyticus isolates originated from seafood and that clinical specimens formed two major clusters at 92.8% and 32% similarity levels. The presence and quantification of Autoinducer-2 was carried out using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after derivatization of Autoinducer-2 with 2, 3-diaminonaphthalene. The presence of tdh-positive V. parahaemolyticus in marine samples highlights the need for constant environmental monitoring to protect public health.

Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays

PubMed Central

2017-01-01

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C2-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers. PMID:28486805

Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays.

PubMed

Li, Ping; Dou, Xiaoqiu; Feng, Chuanliang; Müller, Mareike; Chang, Matthew Wook; Frettlöh, Martin; Schönherr, Holger

2017-08-08

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C 2 -phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers.

DNA Microarray-Based Identification of Genes Controlled by Autoinducer 2-Stimulated Quorum Sensing in Escherichia Coli

DTIC Science & Technology

2001-09-01

and pathogenicity in Erwinia carotovora (rsmA) (12). Additionally, csrA has been documented to affect cell size and surface properties, which is in...machinery to cell wall 13.1 b1502 Putative adhesin; similar to FimH protein 13.0 tap Methyl-accepting chemotaxis protein IV, peptide sensor receptor...oxohexanoyl)-L-homoserine lactone 5246 DELISA ET AL. J. BACTERIOL. regulates carbapenem antibiotic production in Erwinia carotovora . Biochem. J. 288:997

Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene

PubMed Central

How, Kah Yan; Hong, Kar-Wai; Sam, Choon-Kook; Koh, Chong-Lek; Yin, Wai-Fong; Chan, Kok-Gan

2015-01-01

Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production, and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homologue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was ∼20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high-resolution liquid chromatography–mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl-homoserine lactone and 3-hydroxy-dodecanoyl-homoserine lactone from induced E. coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium. PMID:25926817

A Host-Produced Autoinducer-2 Mimic Activates Bacterial Quorum Sensing.

PubMed

Ismail, Anisa S; Valastyan, Julie S; Bassler, Bonnie L

2016-04-13

Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses. Copyright © 2016 Elsevier Inc. All rights reserved.

Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species.

PubMed

Kimura, Soichiro; Tateda, Kazuhiro; Ishii, Yoshikazu; Horikawa, Manabu; Miyairi, Shinichi; Gotoh, Naomasa; Ishiguro, Masaji; Yamaguchi, Keizo

2009-06-01

Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12)-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C(12)-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C(12)-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C(12)-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 microM 3-oxo-C(12)-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C(12)-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C(12)-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.

The influences of LuxX in Escherichia coli biofilm formation and improving teacher quality through the Bio-Bus Program

NASA Astrophysics Data System (ADS)

Robbins, Chandan Morris

The objectives of this work are: (1) to agarose-stabilize fragile biofilms for quantitative structure analysis; (2) to understand the influences of LuxS on biofilm formation; (3) to improve teacher quality by preparing Georgia's middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that QS, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and Georgia's students are better equipped to become tomorrow's leaders. INDEX WORDS: Biofilm, Escherichia coli, Quorum sensing, LuxS, Autoinducer-2, Microbial ecology

Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats ▿ †

PubMed Central

Lee, Jin-Hyung; Regmi, Sushil Chandra; Kim, Jung-Ae; Cho, Moo Hwan; Yun, Hyungdon; Lee, Chang-Soo; Lee, Jintae

2011-01-01

Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagic Escherichia coli O157:H7. The antioxidant phloretin, which is abundant in apples, markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgA and csgB), and dozens of prophage genes in E. coli O157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production in E. coli O157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor of E. coli O157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensal E. coli biofilms. PMID:21930760

Apple flavonoid phloretin inhibits Escherichia coli O157:H7 biofilm formation and ameliorates colon inflammation in rats.

PubMed

Lee, Jin-Hyung; Regmi, Sushil Chandra; Kim, Jung-Ae; Cho, Moo Hwan; Yun, Hyungdon; Lee, Chang-Soo; Lee, Jintae

2011-12-01

Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagic Escherichia coli O157:H7. The antioxidant phloretin, which is abundant in apples, markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx(2)), autoinducer-2 importer genes (lsrACDBF), curli genes (csgA and csgB), and dozens of prophage genes in E. coli O157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production in E. coli O157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor of E. coli O157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensal E. coli biofilms.

Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

PubMed

Bai, Yunpeng; Patil, Santoshkumar N; Bowden, Steven D; Poulter, Simon; Pan, Jie; Salmond, George P C; Welch, Martin; Huck, Wilhelm T S; Abell, Chris

2013-05-21

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Nucleotide, c-di-GMP, c-di-AMP, cGMP, cAMP, (p)ppGpp signaling in bacteria and implications in pathogenesis.

PubMed

Kalia, Dimpy; Merey, Gökçe; Nakayama, Shizuka; Zheng, Yue; Zhou, Jie; Luo, Yiling; Guo, Min; Roembke, Benjamin T; Sintim, Herman O

2013-01-07

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022).

PubMed

González Barrios, Andrés F; Zuo, Rongjun; Hashimoto, Yoshifumi; Yang, Li; Bentley, William E; Wood, Thomas K

2006-01-01

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-mum) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 muM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).

Cationic Conjugated Polymers-Induced Quorum Sensing of Bacteria Cells.

PubMed

Zhang, Pengbo; Lu, Huan; Chen, Hui; Zhang, Jiangyan; Liu, Libing; Lv, Fengting; Wang, Shu

2016-03-15

Bacteria quorum sensing (QS) has attracted significant interest for understanding cell-cell communication and regulating biological functions. In this work, we demonstrate that water-soluble cationic conjugated polymers (PFP-G2) can interact with bacteria to form aggregates through electrostatic interactions. With bacteria coated in the aggregate, PFP-G2 can induce the bacteria QS system and prolong the time duration of QS signal molecules (autoinducer-2 (AI-2)) production. The prolonged AI-2 can bind with specific protein and continuously regulate downstream gene expression. Consequently, the bacteria show a higher survival rate against antibiotics, resulting in decreased antimicrobial susceptibility. Also, AI-2 induced by PFP-G2 can stimulate 55.54 ± 12.03% more biofilm in E. coli. This method can be used to understand cell-cell communication and regulate biological functions, such as the production of signaling molecules, antibiotics, other microbial metabolites, and even virulence.

Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

PubMed Central

Crépin, Alexandre; Barbey, Corinne; Beury-Cirou, Amélie; Hélias, Valérie; Taupin, Laure; Reverchon, Sylvie; Nasser, William; Faure, Denis; Dufour, Alain; Orange, Nicole; Feuilloley, Marc; Heurlier, Karin; Burini, Jean-François; Latour, Xavier

2012-01-01

Background Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. Methodology/Principal Findings Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. Conclusions/Significance Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates. PMID:22539957

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone.

PubMed

Ji, Guangyong; Pei, Wuhong; Zhang, Linsheng; Qiu, Rongde; Lin, Jianqun; Benito, Yvonne; Lina, Gerard; Novick, Richard P

2005-05-01

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.

Ecological feedback in quorum-sensing microbial populations can induce heterogeneous production of autoinducers

PubMed Central

Bauer, Matthias; Knebel, Johannes; Lechner, Matthias; Pickl, Peter; Frey, Erwin

2017-01-01

Autoinducers are small signaling molecules that mediate intercellular communication in microbial populations and trigger coordinated gene expression via ‘quorum sensing’. Elucidating the mechanisms that control autoinducer production is, thus, pertinent to understanding collective microbial behavior, such as virulence and bioluminescence. Recent experiments have shown a heterogeneous promoter activity of autoinducer synthase genes, suggesting that some of the isogenic cells in a population might produce autoinducers, whereas others might not. However, the mechanism underlying this phenotypic heterogeneity in quorum-sensing microbial populations has remained elusive. In our theoretical model, cells synthesize and secrete autoinducers into the environment, up-regulate their production in this self-shaped environment, and non-producers replicate faster than producers. We show that the coupling between ecological and population dynamics through quorum sensing can induce phenotypic heterogeneity in microbial populations, suggesting an alternative mechanism to stochastic gene expression in bistable gene regulatory circuits. DOI: http://dx.doi.org/10.7554/eLife.25773.001 PMID:28741470

The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

PubMed

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-12-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

The Staphylococcus aureus Autoinducer-2 Synthase LuxS Is Regulated by Ser/Thr Phosphorylation▿

PubMed Central

Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J.; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

2010-01-01

The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis. PMID:20870760

Quorum-Quenching Human Designer Cells for Closed-Loop Control of Pseudomonas aeruginosa Biofilms.

PubMed

Sedlmayer, Ferdinand; Jaeger, Tina; Jenal, Urs; Fussenegger, Martin

2017-08-09

Current antibiotics gradually lose their efficacy against chronic Pseudomonas aeruginosa infections due to development of increased resistance mediated by biofilm formation, as well as the large arsenal of microbial virulence factors that are coordinated by the cell density-dependent phenomenon of quorum sensing. Here, we address this issue by using synthetic biology principles to rationally engineer quorum-quencher cells with closed-loop control to autonomously dampen virulence and interfere with biofilm integrity. Pathogen-derived signals dynamically activate a synthetic mammalian autoinducer sensor driving downstream expression of next-generation anti-infectives. Engineered cells were able to sensitively score autoinducer levels from P. aeruginosa clinical isolates and mount a 2-fold defense consisting of an autoinducer-inactivating enzyme to silence bacterial quorum sensing and a bipartite antibiofilm effector to dissolve the biofilm matrix. The self-guided cellular device fully cleared autoinducers, potentiated bacterial antibiotic susceptibility, substantially reduced biofilms, and alleviated cytotoxicity to lung epithelial cells. We believe this strategy of dividing otherwise coordinated pathogens and breaking up their shielded stronghold represents a blueprint for cellular anti-infectives in the postantibiotic era.

Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

PubMed

Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

2013-09-03

Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

Detection and quantification of Vibrio fischeri autoinducer from symbiotic squid light organs.

PubMed

Boettcher, K J; Ruby, E G

1995-02-01

Vibrio fischeri is the specific light organ symbiont of the sepiolid squid species Euprymna scolopes and Euprymna morsei. Both species of squid are luminescent by virtue of their bacterial symbionts, but the natural symbionts of E. scolopes do not produce visible luminescence in laboratory culture. The primary cause of this depressed luminescence by E. scolopes symbionts in culture was found to be the production of relatively low levels of V. fischeri autoinducer, a positive transcriptional coregulator of the lux regulon, identified as N-(3-oxohexanoyl) homoserine lactone. Concentrations of autoinducer activity produced by these symbionts in culture were quantified and found to be at least 10-fold lower than those produced by E. morsei isolates (which are visibly luminous outside the association) and perhaps 10,000-fold lower than those of the brightest V. fischeri strains. Despite the differences in their symbiont strains, the intact light organs of the two species of squid contained comparable amounts of extractable autoinducer activity (between 100 and 200 pg per adult animal). The chromatographic behavior of this autoinducer activity on reverse-phase high-performance liquid chromatography was consistent with its presumptive identification as V. fischeri autoinducer. Within the 5-microliter volume of the epithelial core of the light organ in which the symbiotic V. fischeri strains are housed, these amounts would result in an effective autoinducer concentration of at least 100 nM. Because these levels are over 40-fold higher than the concentration needed for the induction of luminescence of bacteria in culture, we conclude that the inherent degree of autoinducer production by strains of V. fischeri may not influence their effectiveness as light organ symbionts. Furthermore, this study provides the first direct evidence that the phenomenon of cell density-dependent autoinduction, discovered and described first for laboratory cultures of V. fischeri but believed to be a general phenomenon in many species of host-associated symbionts and pathogens, is in fact a consequence of bacterial colonizations of host tissues.

The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, R.; Bolitho, M; Higgins, D

2009-01-01

Vibrio cholerae, the bacterium that causes the disease cholera, controls virulence factor production and biofilm development in response to two extracellular quorum-sensing molecules, called autoinducers. The strongest autoinducer, called CAI-1 (for cholera autoinducer-1), was previously identified as (S)-3-hydroxytridecan-4-one. Biosynthesis of CAI-1 requires the enzyme CqsA. Here, we determine the CqsA reaction mechanism, identify the CqsA substrates as (S)-2-aminobutyrate and decanoyl coenzyme A, and demonstrate that the product of the reaction is 3-aminotridecan-4-one, dubbed amino-CAI-1. CqsA produces amino-CAI-1 by a pyridoxal phosphate-dependent acyl-CoA transferase reaction. Amino-CAI-1 is converted to CAI-1 in a subsequent step via a CqsA-independent mechanism. Consistent with this,more » we find cells release {ge}100 times more CAI-1 than amino-CAI-1. Nonetheless, V. cholerae responds to amino-CAI-1 as well as CAI-1, whereas other CAI-1 variants do not elicit a quorum-sensing response. Thus, both CAI-1 and amino-CAI-1 have potential as lead molecules in the development of an anticholera treatment.« less

Processing the Interspecies Quorum-sensing Signal Autoinducer-2 (AI-2)

PubMed Central

Marques, João C.; Lamosa, Pedro; Russell, Caitlin; Ventura, Rita; Maycock, Christopher; Semmelhack, Martin F.; Miller, Stephen T.; Xavier, Karina B.

2011-01-01

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior. PMID:21454635

Processing the Interspecies Quorum-sensing Signal Autoinducer-2 (AI-2)

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Marques; P Lamosa; C Russell

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsiblemore » for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.« less

Small Talk: Cell-to-Cell Communication in Bacteria

ScienceCinema

Bassler, Bonnie [Princeton University, Princeton, New Jersey, United States

2017-12-09

Cell-cell communication in bacteria involves the production, release, and subsequent detection of chemical signaling molecules called autoinducers. This process, called quorum sensing, allows bacteria to regulate gene expression on a population-wide scale. Processes controlled by quorum sensing are usually ones that are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. For example, quorum sensing controls bioluminescence, secretion of virulence factors, biofilm formation, sporulation, and the exchange of DNA. Thus, quorum sensing is a mechanism that allows bacteria to function as multi-cellular organisms. Bacteria make, detect, and integrate information from multiple autoinducers, some of which are used exclusively for intra-species communication while others enable communication between species. Research is now focused on the development of therapies that interfere with quorum sensing to control bacterial virulence.

Establishing bacterial communities by 'word of mouth': LuxS and autoinducer 2 in biofilm development.

PubMed

Hardie, Kim Rachael; Heurlier, Karin

2008-08-01

Multicellular bacterial communities (biofilms) abound in nature, and their successful formation and survival is likely to require cell-cell communication--including quorum sensing--to co-ordinate appropriate gene expression. The only mode of quorum sensing that is shared by both Gram-positive and Gram-negative bacteria involves the production of the signalling molecule autoinducer 2 by LuxS. A survey of the current literature reveals that luxS contributes to biofilm development in some bacteria. However, inconsistencies prevent biofilm development being attributed to the production of AI2 in all cases.

N-acyl homoserine lactone-degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures.

PubMed

Tinh, Nguyen Thi Ngoc; Asanka Gunasekara, R A Y S; Boon, Nico; Dierckens, Kristof; Sorgeloos, Patrick; Bossier, Peter

2007-10-01

Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of Pacific white shrimp Penaeus vannamei, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules. The ECs, characterized by denaturing gradient gel electrophoresis analysis and subsequent rRNA sequencing, degraded AHL molecules in the degradation assays. Apparently, the resting cells of the ECs also degraded one of the three types of quorum-sensing signal molecules produced by Vibrio harveyi in vitro [i.e. harveyi autoinducer 1 (HAI-1)]. The most efficient AHL-degrading ECs, EC5, was tested in Brachionus experiments. EC5 degraded the V. harveyi HAI-1 autoinducer in vivo, neutralizing the negative effect of V. harveyi autoinducer 2 (AI-2) mutant, in which only the HAI-1- and CAI-1-mediated components of the quorum-sensing system are functional on the growth of Brachionus. This suggests that EC5 interferes with HAI-1-regulated metabolism in V. harveyi. These AHL-degrading ECs need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria.

Recent progresses on AI-2 bacterial quorum sensing inhibitors.

PubMed

Zhu, Peng; Li, Minyong

2012-01-01

Quorum sensing (QS) is a communication procedure that predominates gene expression in response to cell density and fluctuations in the neighboring environment as a result of discerning molecules termed autoinducers (AIs). It has been embroiled that QS can govern bacterial behaviors such as the secretion of virulence factors, biofilm formation, bioluminescence production, conjugation, sporulation and swarming motility. Autoinducer 2 (AI-2), a QS signaling molecule brought up to be involved in interspecies communication, exists in both gram-negative and -positive bacteria. Therefore, novel approaches to interrupt AI-2 quorum sensing are being recognized as next generation antimicrobials. In the present review article, we summarized recent progresses on AI-2 bacterial quorum sensing inhibitors and discussed their potential as the antibacterial agents.

[Resting forms of gram negative chemolithoautotrophic bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum].

PubMed

Loĭko, N G; Soina, V S; Sorokin, D Iu; Mitiushina, L L; El'-Registan, G I

2003-01-01

The haloalkaliphilic chemoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cyst-like refractile cells (CRC), whose production was controlled by the level of the d1 extracellular factors, exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promoted the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed with taking into account the effect of the d1 autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.

Effect of the synthetic cannabinoid HU-210 on quorum sensing and on the production of quorum sensing-mediated virulence factors by Vibrio harveyi.

PubMed

Soni, Divya; Smoum, Reem; Breuer, Aviva; Mechoulam, Raphael; Steinberg, Doron

2015-08-12

Bacterial populations communicate through the cell density-dependent mechanism of quorum sensing (QS). Vibrio harveyi, one of the best studied model organisms for QS, was used to explore effects of the synthetic cannabinoid HU-210 on QS and different QS-regulated physiological processes in bacteria. Analysis of QS-regulated bioluminescence in wild-type and mutant strains of V. harveyi revealed that HU-210 affects the autoinducer-2 (AI-2) pathway, one of three known QS cascades of V. harveyi. Furthermore, QS-mediated biofilm formation and swimming motility in the mutant strain BB152 (AI-1(-), AI-2(+)) were significantly reduced in the presence of HU-210. HU-210 inhibited QS-mediated virulence factor production without any inhibitory effect on bacterial growth. It also alters the expression of several genes, which are regulated by QS, specifically downregulating the genes of the AI-2 QS cascade. First evidence is being provided for interference of bacterial signal-transduction systems by a synthetic cannabinoid. The effect of HU-210 was specific to the AI-2 cascade in V. harveyi. AI-2 is known as a "universal autoinducer" and interference with its activity opens a broad spectrum of applications for synthetic cannabinoids in future research as a potential anti-QS agent.

Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

PubMed

Dunlap, P V

1992-07-01

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

Autoinducer-2-like activity associated with foods and its interaction with food additives.

PubMed

Lu, Lingeng; Hume, Michael E; Pillai, Suresh D

2004-07-01

The autoinducer-2 (AI-2) molecule produced by bacteria as part of quorum sensing is considered to be a universal inducer signal in bacteria because it reportedly influences gene expression in a variety of both gram-negative and gram-positive bacteria. The objective of this study was to determine whether selected fresh produce and processed foods have AI-2-like activity and whether specific food additives can act as AI-2 mimics and result in AI-2-like activity. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used as the basis for determining AI-2 activity in the selected foods and food ingredients. Maximum AI-2 activity was seen on the frozen fish sample (203-fold, compared with the negative control) followed by tomato, cantaloupe, carrots, tofu, and milk samples. Interestingly, some samples were capable of inhibiting AI-2 activity. Turkey patties showed the highest inhibition (99.8% compared with the positive control) followed by chicken breast (97.5%), homemade cheeses (93.7%), beef steak (90.6%), and beef patties (84.4%). AI-2 activity was almost totally inhibited by sodium propionate, whereas sodium benzoate caused 93.3% inhibition, compared with 75% inhibition by sodium acetate. Sodium nitrate did not have any appreciable effect, even at 200 ppm. Understanding the relationships that exist between AI-2 activity on foods and the ecology of pathogens and food spoilage bacteria on foods could yield clues about factors controlling food spoilage and pathogen virulence.

Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens▿

PubMed Central

Chalabaev, Sabina; Turlin, Evelyne; Bay, Sylvie; Ganneau, Christelle; Brito-Fravallo, Emma; Charles, Jean-François; Danchin, Antoine; Biville, Francis

2008-01-01

Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes. PMID:18245247

Design and Synthesis of Potent “Sulfur-free” Transition State Analogue Inhibitors of 5′-Methylthioadenosine Nucleosidase and 5′-Methylthioadenosine Phosphorylase

PubMed Central

Longshaw, Alistair I.; Adanitsch, Florian; Gutierrez, Jemy A.; Evans, Gary B.; Tyler, Peter C.; Schramm, Vern L.

2013-01-01

5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is a dual substrate bacterial enzyme involved in S-adenosylmethionine (SAM)-related quorum sensing pathways that regulates virulence in many bacterial species. MTANs from many bacteria are directly involved in the quorum sensing mechanism by regulating the synthesis of autoinducer molecules that are used by bacterial communities to communicate. In humans, 5′-methylthioadenosine phosphorylase (MTAP) is involved in polyamine biosynthesis as well as in purine and SAM salvage pathways and thus has been identified as an anticancer target. Previously we have described the synthesis and biological activity of several aza-C-nucleoside mimics with a sulfur atom at the 5′ position that are potent E. coli MTAN and human MTAP inhibitors. Because of the possibility that the sulfur may affect bioavailability we were interested in synthesizing “sulfur-free” analogues. Herein we describe the preparation of a series of “sulfur-free” transition state analogues inhibitors, of E. coli MTAN and human MTAP that have low nano- to pico-molar dissociation constants and are potentially novel bacterial anti-infective and anti-cancer drug candidates. PMID:20718423

Self-sensing in Bacillus subtilis quorum-sensing systems

PubMed Central

Bareia, Tasneem; Pollak, Shaul; Eldar, Avigdor

2017-01-01

Bacterial cell-cell signaling, or quorum sensing, is characterized by the secretion and group-wide detection of small diffusible signal molecules called autoinducers. This mechanism allows cells to coordinate their behavior in a density-dependent manner. A quorum-sensing cell may directly respond to the autoinducers it produces in a cell-autonomous and quorum-independent manner, but the strength of such self-sensing effect and its impact on bacterial physiology are unclear. Here, we explored the existence and impact of self-sensing in the Bacillus subtilis ComQXP and Rap-Phr quorum-sensing systems. By comparing the quorum-sensing response of autoinducer-secreting and non-secreting cells in co-culture, we found that secreting cells consistently showed a stronger response than non-secreting cells. Combining genetic and quantitative analyses, we demonstrated this effect to be a direct result of self-sensing and ruled out an indirect regulatory effect of the autoinducer production genes on response sensitivity. In addition, self-sensing in the ComQXP system affected persistence to antibiotic treatment. Together, these findings indicate the existence of self-sensing in the two most common designs of quorum-sensing systems of Gram-positive bacteria. PMID:29038467

Growth-mediated autochemotactic pattern formation in self-propelling bacteria

NASA Astrophysics Data System (ADS)

Mukherjee, Mrinmoy; Ghosh, Pushpita

2018-01-01

Bacteria, while developing a multicellular colony or biofilm, can undergo pattern formation by diverse intricate mechanisms. One such route is directional movement or chemotaxis toward or away from self-secreted or externally employed chemicals. In some bacteria, the self-produced signaling chemicals or autoinducers themselves act as chemoattractants or chemorepellents and thereby regulate the directional movements of the cells in the colony. In addition, bacteria follow a certain growth kinetics which is integrated in the process of colony development. Here, we study the interplay of bacterial growth dynamics, cell motility, and autochemotactic motion with respect to the self-secreted diffusive signaling chemicals in spatial pattern formation. Using a continuum model of motile bacteria, we show growth can act as a crucial tuning parameter in determining the spatiotemporal dynamics of a colony. In action of growth dynamics, while chemoattraction toward autoinducers creates arrested phase separation, pattern transitions and suppression can occur for a fixed chemorepulsive strength.

Exploring the selectivity of auto-inducer complex with LuxR using molecular docking, mutational studies and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Rajamanikandan, Sundaraj; Srinivasan, Pappu

2017-03-01

Bacteria communicate with one another using extracellular signaling molecules called auto-inducers (AHLs), a process termed as quorum sensing. The quorum sensing process allows bacteria to regulate various physiological activities. In this regard, quorum sensing master regulator LuxR from Vibrio harveyi represents an attractive therapeutic target for the development of novel anti-quorum sensing agents. Eventhough the binding of AHL complex with LuxR is evidenced in earlier reports, but their mode of binding is not clearly determined. Therefore, in the present work, molecular docking, in silico mutational studies, molecular dynamics simulations and free energy calculations were performed to understand the selectivity of AHL into the binding site of LuxR. The results revealed that Asn133 and Gln137 residues play a crucial role in recognizing AHL more effectively into the binding site of LuxR with good binding free energy. In addition to that, the carbonyl group presents in the lactone ring and amide group of AHL plays a vital role in the formation of hydrogen bond interactions with the protein. Further, structure based virtual screening was performed using ChemBridge database to screen potent lead molecules against LuxR. 4-benzyl-2-pyrrolidinone and N-[2(1-cyclohexen-1-yl) enthyl]-N'(2-ethoxyphenyl) were selected based on dock score, binding affinity and mode of interactions with the receptor. Furthermore, binding free energy, density functional theory and ADME prediction were performed to rank the lead molecules. Thus, the identified lead molecules can be used for the development of anti-quorum sensing drugs.

Inactivation of ferric uptake regulator (Fur) attenuates Helicobacter pylori J99 motility by disturbing the flagellar motor switch and autoinducer-2 production.

PubMed

Lee, Ai-Yun; Kao, Cheng-Yen; Wang, Yao-Kuan; Lin, Ssu-Yuan; Lai, Tze-Ying; Sheu, Bor-Shyang; Lo, Chien-Jung; Wu, Jiunn-Jong

2017-08-01

Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 (AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching. © 2017 John Wiley & Sons Ltd.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

PubMed

Quan, Yufen; Meng, Fankang; Ma, Xinyu; Song, Xinhao; Liu, Xiao; Gao, Weixia; Dang, Yulei; Meng, Yao; Cao, Mingfeng; Song, Cunjiang

2017-09-19

Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Suppression of bacterial cell-cell signalling, biofilm formation and type III secretion system by citrus flavonoids.

PubMed

Vikram, A; Jayaprakasha, G K; Jesudhasan, P R; Pillai, S D; Patil, B S

2010-08-01

This study investigated the quorum sensing, biofilm and type three secretion system (TTSS) inhibitory properties of citrus flavonoids. Flavonoids were tested for their ability to inhibit quorum sensing using Vibrio harveyi reporter assay. Biofilm assays were carried out in 96-well plates. Inhibition of biofilm formation in Escherichia coli O157:H7 and V. harveyi by citrus flavonoids was measured. Furthermore, effect of naringenin on expression of V. harveyi TTSS was investigated by semi-quantitative PCR. Differential responses for different flavonoids were observed for different cell-cell signalling systems. Among the tested flavonoids, naringenin, kaempferol, quercetin and apigenin were effective antagonists of cell-cell signalling. Furthermore, these flavonoids suppressed the biofilm formation in V. harveyi and E. coli O157:H7. In addition, naringenin altered the expression of genes encoding TTSS in V. harveyi. The results of the study indicate a potential modulation of bacterial cell-cell communication, E. coli O157:H7 biofilm and V. harveyi virulence, by flavonoids especially naringenin, quercetin, sinensetin and apigenin. Among the tested flavonoids, naringenin emerged as potent and possibly a nonspecific inhibitor of autoinducer-mediated cell-cell signalling. Naringenin and other flavonoids are prominent secondary metabolites present in citrus species. Therefore, citrus, being a major source of some of these flavonoids and by virtue of widely consumed fruit, may modulate the intestinal microflora. Currently, a limited number of naturally occurring compounds have demonstrated their potential in inhibition of cell-cell communications; therefore, citrus flavonoids may be useful as lead compounds for the development of antipathogenic agents.

Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils.

PubMed

Cheng, Hsiao-Ying; Masiello, Caroline A; Del Valle, Ilenne; Gao, Xiaodong; Bennett, George N; Silberg, Jonathan J

2018-03-16

Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, C 2 H 4 is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CH 3 Br is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

Autoinducer-2 Production in Campylobacter jejuni Contributes to Chicken Colonization ▿

PubMed Central

Quiñones, Beatriz; Miller, William G.; Bates, Anna H.; Mandrell, Robert E.

2009-01-01

Inactivation of luxS, encoding an AI-2 biosynthesis enzyme, in Campylobacter jejuni strain 81-176 significantly reduced colonization of the chick lower gastrointestinal tract, chemotaxis toward organic acids, and in vitro adherence to LMH chicken hepatoma cells. Thus, AI-2 production in C. jejuni contributes to host colonization and interactions with epithelial cells. PMID:19011073

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

PubMed Central

See-Too, Wah Seng; Ee, Robson; Lim, Yan-Lue; Convey, Peter; Pearce, David A.; Yin, Wai-Fong; Chan, Kok-Gan

2017-01-01

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions. PMID:28225085

A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors And Controls Bacterial Pathogenicity

PubMed Central

Swem, Lee R.; Swem, Danielle L.; O’Loughlin, Colleen T.; Gatmaitan, Raleene; Zhao, Bixiao; Ulrich, Scott M.; Bassler, Bonnie L.

2009-01-01

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development. PMID:19647512

Deducing receptor signaling parameters from in vivo analysis: LuxN/AI-1 quorum sensing in Vibrio harveyi

PubMed Central

Swem, Lee R.; Swem, Danielle L.; Wingreen, Ned S.; Bassler, Bonnie L.

2008-01-01

Summary Quorum sensing, a process of bacterial cell-cell communication, relies on production, detection, and response to autoinducer signaling molecules. Here we focus on LuxN, a nine transmembrane domain protein from Vibrio harveyi, and the founding example of membrane-bound receptors for acyl-homoserine lactone (AHL) autoinducers. Previously, nothing was known about signal recognition by membrane-bound AHL receptors. We used mutagenesis and suppressor analyses to identify the AHL-binding domain of LuxN, and discovered LuxN mutants that confer decreased and increased AHL sensitivity. Our analysis of dose-response curves of multiple LuxN mutants pins these inverse phenotypes on quantifiable opposing shifts in the free-energy bias of LuxN for its kinase and phosphatase states. To extract signaling parameters, we exploited a strong LuxN antagonist, one of fifteen small-molecule antagonists we identified. We find that quorum-sensing-mediated communication can be manipulated positively and negatively to control bacterial behavior, and that signaling parameters can be deduced from in vivo data. PMID:18692469

Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4

PubMed Central

Hong, Kar Wai; Chan, Kok-Gan

2015-01-01

Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity. PMID:26290785

A Computational Study of Phenotype Switching in Bacillus Subtilis Biofilm

NASA Astrophysics Data System (ADS)

Smith, Howard; Wang, Xiaoling; Jiang, Yi

Bacillus Subtilis (B. Subtilis), is known to differentiate into three main phenotypes during biofilm growth. Novel techniques to track the spatial and temporal evolution of the three main phenotypes exhibited by B. Subtilis have been developed. However, the techniques do not explain the environmental causes of the phenotype switching and how this leads to the spatiotemporal organization of the biofilm. We hypothesize that cells switch their phenotype according to nutrients and autoinducer levels. We test the hypothesis using a hybrid agent-based and continuous model. The bacteria in our model are individual cells that can (i) grow and divide by the intake of nutrients, (ii) produce and secrete EPS, (iii) form spores and (iv) produce an auto inducer. Using a threshold for nutrient and thresholds for autoinducers, we were able to reproduce the experimental spatiotemporal dynamics. From our simulations we observed that in order to reproduce experimental results, two different autoinducers were necessary. The results also suggest that low-EPS producing biofilms generally obtained higher cell populations. Furthermore, most of the cells that become spore forming cells arise from matrix producing cells.

RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP

PubMed Central

Armbruster, Chelsie E.; Pang, Bing; Murrah, Kyle; Juneau, Richard A.; Perez, Antonia C.; Weimer, Kristin E.D.; Swords, W. Edward

2011-01-01

Summary Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signaling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle-ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2. PMID:21923771

RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP.

PubMed

Armbruster, Chelsie E; Pang, Bing; Murrah, Kyle; Juneau, Richard A; Perez, Antonia C; Weimer, Kristin E D; Swords, W Edward

2011-11-01

Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2. © 2011 Blackwell Publishing Ltd.

Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

PubMed Central

Kirkpatrick, Christopher; Maurer, Lisa M.; Oyelakin, Nikki E.; Yoncheva, Yuliya N.; Maurer, Russell; Slonczewski, Joan L.

2001-01-01

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins. PMID:11591692

Small Molecule Inhibitors of AI-2 Signaling in Bacteria: State-of-the-Art and Future Perspectives for Anti-Quorum Sensing Agents

PubMed Central

Guo, Min; Gamby, Sonja; Zheng, Yue; Sintim, Herman O.

2013-01-01

Bacteria respond to different small molecules that are produced by other neighboring bacteria. These molecules, called autoinducers, are classified as intraspecies (i.e., molecules produced and perceived by the same bacterial species) or interspecies (molecules that are produced and sensed between different bacterial species). AI-2 has been proposed as an interspecies autoinducer and has been shown to regulate different bacterial physiology as well as affect virulence factor production and biofilm formation in some bacteria, including bacteria of clinical relevance. Several groups have embarked on the development of small molecules that could be used to perturb AI-2 signaling in bacteria, with the ultimate goal that these molecules could be used to inhibit bacterial virulence and biofilm formation. Additionally, these molecules have the potential to be used in synthetic biology applications whereby these small molecules are used as inputs to switch on and off AI-2 receptors. In this review, we highlight the state-of-the-art in the development of small molecules that perturb AI-2 signaling in bacteria and offer our perspective on the future development and applications of these classes of molecules. PMID:23994835

Iron control of the Vibrio fischeri luminescence system in Escherichia coli.

PubMed

Dunlap, P V

1992-01-01

Iron influences luminescence in Vibrio fischeri; cultures iron-restricted for growth rate induce luminescence at a lower optical density (OD) than faster growing, iron-replete cultures. An iron restriction effect analogous to that in V. fischeri (slower growth, induction of luminescence at a lower OD) was established using Escherichia coli tonB and tonB+ strains transformed with recombinant plasmids containing the V. fischeri lux genes (luxR luxICDABEG) and grown in the presence and absence of the iron chelator ethylenediamine-di(o-hydroxylphenyl acetic acid) (EDDHA). This permitted the mechanism of iron control of luminescence to be examined. A fur mutant and its parent strain containing the intact lux genes exhibited no difference in the OD at induction of luminescence. Therefore, an iron-binding repressor protein apparently is not involved in iron control of luminescence. Furthermore, in the tonB and in tonB+ strains containing lux plasmids with Mu dI(lacZ) fusions in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the luxICDABEG promoter) both increased by a similar amount (8-9 fold each for tonB, 2-3 fold each for tonB+) in the presence of EDDHA. Similar results were obtained with the luxR gene present on a complementing plasmid. The previously identified regulatory factors that control the lux system (autoinducer-LuxR protein, cyclic AMP-cAMP receptor protein) differentially control expression from the luxR and luxICDABEG promoters, increasing expression from one while decreasing expression from the other. Consequently, these results suggest that the effect of iron on the V. fischeri luminescence system is indirect.

Isolation of the Autoinducer-Quenching Strain that Inhibits LasR in Pseudomonas aeruginosa

PubMed Central

Weng, Lixing; Zhang, Yuqian; Yang, Yuxiang; Wang, Lianhui

2014-01-01

Quorum sensing (QS) has been recognized as a general phenomenon in microorganisms and plays an important role in many pathogenic bacteria. In this report, we used the Agrobacterium tumefaciens biosensor strain NT1 to rapidly screen for autoinducer-quenching inhibitors from bacteria. After initial screening 5389 isolates obtained from land and beach soil, 53 putative positive strains were identified. A confirmatory bioassay was carried out after concentrating the putative positive culture supernatant, and 22 strains were confirmed to have anti-LasR activity. Finally, we determined the strain JM2, which could completely inhibit biofilm formation of Pseudomonas aeruginosa PAO1, belonged to the genus Pseudomonas by analysis of 16S rDNA. Partially purified inhibitor factor(s) F5 derived from culture supernatants specifically inhibited LasR-controlled elastase and protease in wild type P. aeruginosa PAO1 by 68% and 73%, respectively, without significantly affecting growth; the rhl-controlled pyocyanin and rhamnolipids were inhibited by 54% and 52% in the presence of 100 μg/mL of F5. The swarming motility and biofilm of PAO1 were also inhibited by F5. Real time RT-PCR on samples from 100 μg/mL F5-treated P. aeruginosa showed downregulation of autoinducer synthase (LasRI and rhlI) and cognate receptor (lasR and rhlR) genes by 50%, 28%, 48%, and 29%, respectively. These results provide compelling evidence that the F5 inhibitor(s) interferes with the las system and significantly inhibits biofilm formation. PMID:24736783

Antiquorum sensing, antibiofilm formation and cytotoxicity activity of commonly used medicinal plants by inhabitants of Borabu sub-county, Nyamira County, Kenya

PubMed Central

Omwenga, Eric Omori; Hensel, Andreas; Pereira, Susana; Shitandi, Alfred Anakalo

2017-01-01

Productions of various bacterial traits like production of virulence factors (e.g. toxins, enzymes), biofilm formation, luminescence among others, have been known to be controlled by quorum sensing (QS), a process that is dependent on chemical signals or autoinducers (AIs). Bacteria known to rely on such AIs are known to be virulent and tend to be resistant against various antimicrobial agents. Therefore, strategies aimed at the inhibition of QS pathways, are regarded as potential novel therapies in managing bacterial virulence hence reducing their ability to induce infections in humans. In the present study, a portfolio of 25 medicinal plant extracts (ethanol 50% v/v) used in southwestern Kenya were assayed against a transformed E. coli Top 10 reporter QS strain. This biosensor responds to the exogenous addition of 3-oxo-N-hexanoyl homoserine lactone (3OC6HSL) expressing green fluorescent protein (GFP). The large majority of the screened medicinal plants seemed to exhibit toxic effects and almost none of them induced antiquorum sensing (AQS) activity. This could be the consequence of the presence of mixed compounds in the extracts. Elaeodendron buchananii Loes and Acacia gerrardii Benth extracts that seemed to show AQS activity were further proved found to possess mild AQS but with defined antimicrobial activities, and no antibiofilm formation inhibition. As a control, an E. coli pBCA9145_jtk2828::sfGFP strain that produces constitutively GFP was used and confirmed that none of the two extracts quenched the fluorescence of sfGFP. Cytotoxicity assays with mammalian MDCK cells also did indicate that the selected extracts with putative AQS activity, also reduced the cell viability. Therefore, further studies will be needed to separate and re-test the individual compounds especially from the selected two promising plants. PMID:29091715

Quorum-Sensing Signal-Response Systems in Gram-Negative Bacteria

PubMed Central

Papenfort, Kai; Bassler, Bonnie

2016-01-01

Abstract / Preface Bacteria use quorum sensing to orchestrate gene expression programmes that underlie collective behaviours. Quorum sensing relies on the production, release, detection and group-level response to extracellular signalling molecules, which are called autoinducers. Recent work has discovered new autoinducers in Gram-negative bacteria, shown how these molecules are recognized by cognate receptors, revealed new regulatory components that are embedded in canonical signalling circuits and identified novel regulatory network designs. In this Review we examine how, together, these features of quorum sensing signal–response systems combine to control collective behaviours in Gram-negative bacteria and we discuss the implications for host–microbial associations and antibacterial therapy. PMID:27510864

Flavonoids Suppress Pseudomonas aeruginosa Virulence through Allosteric Inhibition of Quorum-sensing Receptors*

PubMed Central

Paczkowski, Jon E.; Mukherjee, Sampriti; McCready, Amelia R.; Cong, Jian-Ping; Aquino, Christopher J.; Kim, Hahn; Henke, Brad R.; Smith, Chari D.; Bassler, Bonnie L.

2017-01-01

Quorum sensing is a process of cell-cell communication that bacteria use to regulate collective behaviors. Quorum sensing depends on the production, detection, and group-wide response to extracellular signal molecules called autoinducers. In many bacterial species, quorum sensing controls virulence factor production. Thus, disrupting quorum sensing is considered a promising strategy to combat bacterial pathogenicity. Several members of a family of naturally produced plant metabolites called flavonoids inhibit Pseudomonas aeruginosa biofilm formation by an unknown mechanism. Here, we explore this family of molecules further, and we demonstrate that flavonoids specifically inhibit quorum sensing via antagonism of the autoinducer-binding receptors, LasR and RhlR. Structure-activity relationship analyses demonstrate that the presence of two hydroxyl moieties in the flavone A-ring backbone are essential for potent inhibition of LasR/RhlR. Biochemical analyses reveal that the flavonoids function non-competitively to prevent LasR/RhlR DNA binding. Administration of the flavonoids to P. aeruginosa alters transcription of quorum sensing-controlled target promoters and suppresses virulence factor production, confirming their potential as anti-infectives that do not function by traditional bacteriocidal or bacteriostatic mechanisms. PMID:28119451

A comparison of fluctuations of Campylobacter and Escherichia coli concentrations on broiler chicken carcasses during processing in two slaughterhouses.

PubMed

Pacholewicz, Ewa; Swart, Arno; Schipper, Maarten; Gortemaker, Betty G M; Wagenaar, Jaap A; Havelaar, Arie H; Lipman, Len J A

2015-07-16

The causes of differences in Campylobacter and Escherichia coli concentrations on broiler chicken carcasses after chilling between slaughterhouses are not fully identified. Therefore, it is a challenge for slaughterhouses to comply with Process Hygiene Criteria for broiler meat. The aim of the study was to identify which processing steps contribute to increases or decreases in Campylobacter and E. coli concentrations within and between two slaughterhouses. Identifying the processing steps with variable performance could explain the differences in bacterial concentrations after chilling between slaughterhouses. Thermotolerant Campylobacter and E. coli concentrations on carcasses during broiler processing were measured during the summer period in 21 trials after bleeding, scalding, defeathering, evisceration and chilling. In two slaughterhouses with comparable Campylobacter and E. coli concentrations in the incoming batches (after bleeding), the mean log10 concentrations are found to be significantly different after chilling. Campylobacter concentrations decreased by 1.40 log10 in Slaughterhouse 1 and by 1.86 log10 in Slaughterhouse 2, whereas E. coli decreased by 2.19 log10 in Slaughterhouse 1 and by 2.84 log10 in Slaughterhouse 2. Higher concentrations of Campylobacter and E. coli on carcasses after chilling were observed in Slaughterhouse 1 in which an increase in concentrations was observed after evisceration. The effect of processing on Campylobacter and E. coli concentrations in Slaughterhouse 1 did not differ between batches. In Slaughterhouse 2, the effect of processing on the concentrations of both bacteria varied over batches. Changes in E. coli concentration levels during processing were similar to Campylobacter except for defeathering. E. coli concentration significantly decreased after defeathering in both slaughterhouses, whereas Campylobacter increased in Slaughterhouse 2 and in Slaughterhouse 1 no significant changes were observed. The patterns of increases and decreases in bacterial concentrations during processing are specific for each slaughterhouse. Inhomogeneous patterns potentially explain the differences in concentrations after chilling between slaughterhouses. Critical processing steps should be validated in each slaughterhouse by longitudinal studies and potentially based on E. coli. E. coli has a potential to be used as an indicator of processing hygiene, because the impact of most of the studied processing steps was similar as for Campylobacter. Copyright © 2015 Elsevier B.V. All rights reserved.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli through processing in two broiler chicken slaughterhouses.

PubMed

Pacholewicz, Ewa; Liakopoulos, Apostolos; Swart, Arno; Gortemaker, Betty; Dierikx, Cindy; Havelaar, Arie; Schmitt, Heike

2015-12-23

Whilst broilers are recognised as a reservoir of extended-spectrum-β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC)-producing Escherichia coli, there is currently limited knowledge on the effect of slaughtering on its concentrations on poultry meat. The aim of this study was to establish the concentration of ESBL/AmpC producing E. coli on broiler chicken carcasses through processing. In addition the changes in ESBL/AmpC producing E. coli concentrations were compared with generic E. coli and Campylobacter. In two slaughterhouses, the surface of the whole carcasses was sampled after 5 processing steps: bleeding, scalding, defeathering, evisceration and chilling. In total, 17 batches were sampled in two different slaughterhouses during the summers of 2012 and 2013. ESBL/AmpC producing E. coli was enumerated on MacConkey agar with 1mg/l cefotaxime, and the ESBL/AmpC phenotypes and genotypes were characterised. The ESBL/AmpC producing E. coli concentrations varied significantly between the incoming batches in both slaughterhouses. The concentrations on broiler chicken carcasses were significantly reduced during processing. In Slaughterhouse 1, all subsequent processing steps reduced the concentrations except evisceration which led to a slight increase that was statistically not significant. The changes in concentration between processing steps were relatively similar for all sampled batches in this slaughterhouse. In contrast, changes varied between batches in Slaughterhouse 2, and the overall reduction through processing was higher in Slaughterhouse 2. Changes in ESBL/AmpC producing E. coli along the processing line were similar to changes in generic E. coli in both slaughterhouses. The effect of defeathering differed between ESBL/AmpC producing E. coli and Campylobacter. ESBL/AmpC producing E. coli decreased after defeathering, whereas Campylobacter concentrations increased. The genotypes of ESBL/AmpC producing E. coli (blaCTX-M-1, blaSHV-12, blaCMY-2, blaTEM-52c, blaTEM-52cvar) from both slaughterhouses match typical poultry genotypes. Their distribution differed between batches and changed throughout processing for some batches. The concentration levels found after chilling were between 10(2) and 10(5)CFU/carcass. To conclude, changes in ESBL/AmpC producing E. coli concentrations on broiler chicken carcasses during processing are influenced by batch and slaughterhouse, pointing to the role of both primary production and process control for reducing ESBL/AmpC producing E. coli levels in final products. Due to similar changes upon processing, E. coli can be used as a process indicator of ESBL/AmpC producing E. coli, because the processing steps had similar impact on both organisms. Cross contamination may potentially explain shifts in genotypes within some batches through the processing. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

Autoinducer-2 Plays a Crucial Role in Gut Colonization and Probiotic Functionality of Bifidobacterium breve UCC2003

PubMed Central

Bottacini, Francesca; Lanigan, Noreen; Casey, Pat G.; Huys, Geert; Nelis, Hans J.; van Sinderen, Douwe; Coenye, Tom

2014-01-01

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition. PMID:24871429

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

PubMed

Christiaen, Steven E A; O'Connell Motherway, Mary; Bottacini, Francesca; Lanigan, Noreen; Casey, Pat G; Huys, Geert; Nelis, Hans J; van Sinderen, Douwe; Coenye, Tom

2014-01-01

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media.

PubMed

Nieto-Peñalver, Carlos G; Bertini, Elisa V; de Figueroa, Lucía I C

2012-07-01

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.

Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7.

PubMed

Harris, Shaun M; Yue, Wan-Fu; Olsen, Sarena A; Hu, Jia; Means, Warrie J; McCormick, Richard J; Du, Min; Zhu, Mei-Jun

2012-10-15

Escherichia coli (E. coli) O157:H7 remains a major food safety concern associated with meat, especially beef products. Shiga toxins (Stx) are key virulence factors produced by E. coli O157:H7 that are responsible for hemorrhagic colitis and Hemolytic Uremic Syndrome. Stx are heat stable and can be absorbed after oral ingestion. Despite the extensive study of E. coli O157:H7 survival during meat processing, little attention is paid to the production of Stx during meat processing. The objective of this study was to elucidate the effect of salt, an essential additive to processed meat, at concentrations relevant to meat processing (0%, 1%, 2%, 3%, W/V) on Stx2 production and Stx2 prophage induction by E. coli O157:H7 strains. For both E. coli O157:H7 86-24 and EDL933 strains, including 2% salt in LB broth decreased (P<0.05) E. coli O157:H7 population, but increased (P<0.05) Stx2 production (as measured relative to Log(10)CFU) compared to that of the control (1% salt). Supplementing 3% salt decreased (P<0.05) both E. coli O157:H7 number and Stx2 production. Quantitative RT-PCR indicated that stx2 mRNA expression in culture media containing 2% salt was greatly increased (P<0.05) compared to other salt concentrations. Consistent with enhanced Stx2 production and stx2 expression, the 2% salt group had highest lambdoid phage titer and stx2 prophage induction among all salt treatments. RecA is a key mediator of bacterial response to stress, which mediates prophage activation. Quantitative RT-PCR further indicated that recA mRNA expression was higher in both 2% and 3% salt than that of 0% and 1% salt treatments, indicating that stress was involved in enhanced Stx2 production. In conclusion, salt at the concentration used for meat processing enhances Stx production, a process linked to bacterial stress response and lambdoid prophage induction. Published by Elsevier B.V.

Involvement of LuxS in Aeromonas salmonicida metabolism, virulence and infection in Atlantic salmon (Salmo salar L).

PubMed

Meng, Lingjie; Du, Yishuai; Liu, Pengfei; Li, Xian; Liu, Ying

2017-05-01

Quorum sensing is a bacterial density dependent communication system, which regarded to regulate co-operative behaviors of community and mediated by extracellular signal molecules named autoinducers (AI). Among various signals, autoinducer-2 (AI-2) is believed to be the messengers inter species and produced by LuxS. For Aeromonas salmonicida (A. salmonicida), an opportunistic pathogen to many cold-water teleost, little information has been known about the function of AI-2 and LuxS. Therefore, our aim was to preliminarily clarify the function of LuxS in A. salmonicida. The consequences demonstrated that wild type A. salmonicida exhibited AI-2 activity and luxS defective mutant strain fail to produce AI-2 signals. Furthermore, it was suggested that luxS deficiency could impact bacterial morphology, surface properties and virulence dramatically. Challenge experiment showed a tendency that immune factors expressed earlier when Atlantic salmon was infected with ΔluxS strain. Overall, we hypothesis that AI-2 quorum sensing could regulate the expression of A-layer protein coding gene vapA, and then influence bacterial survival ability when suffered from attack of the host immune system. Though additional studies are warranted, our study will supply a new thinking to control the damage caused by A. salmonicida. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Low-molecular-weight autoregulatory factors in bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum].

PubMed

Loĭko, N G; Kozlova, A N; Osipov, G A; El'-Registan, G I

2002-01-01

The haloalkaliphilic, lithoautotrophic, sulfur-oxidizing gram-negative bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum were found to possess a special system for the autoregulation of their growth. The system includes the extracellular autoinducers of anabiosis (the d1 factor) and autolysis (the d2 factor). The principal components of the d1 factor are alkylhydroxybenzenes. The principal components of the d2 factor are free unsaturated fatty acids dominated by oleic acid isomers. Like the respective autoregulators of neutrophilic bacteria, the d1 factor of haloalkaliphilic bacteria presumably controls their growth and transition to a anabiotic state, while the d2 factor controls autolytic processes. Alkylhydroxybenzenes of both microbial and chemical origin were found to influence bacterial respiration. The low-molecular-weight osmoprotectant glycine betaine enhanced the thermostability of trypsin. This suggests that glycine betaine, like the d1 factor, serves as a molecular chaperone.

Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal

PubMed Central

Anderson, Jeneva K.; Huang, Julie Y.; Wreden, Christopher; Sweeney, Emily Goers; Goers, John; Remington, S. James

2015-01-01

ABSTRACT The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal. PMID:26152582

Chemical communication in the gut: Effects of microbiota-generated metabolites on gastrointestinal bacterial pathogens.

PubMed

Vogt, Stefanie L; Peña-Díaz, Jorge; Finlay, B Brett

2015-08-01

Gastrointestinal pathogens must overcome many obstacles in order to successfully colonize a host, not the least of which is the presence of the gut microbiota, the trillions of commensal microorganisms inhabiting mammals' digestive tracts, and their products. It is well established that a healthy gut microbiota provides its host with protection from numerous pathogens, including Salmonella species, Clostridium difficile, diarrheagenic Escherichia coli, and Vibrio cholerae. Conversely, pathogenic bacteria have evolved mechanisms to establish an infection and thrive in the face of fierce competition from the microbiota for space and nutrients. Here, we review the evidence that gut microbiota-generated metabolites play a key role in determining the outcome of infection by bacterial pathogens. By consuming and transforming dietary and host-produced metabolites, as well as secreting primary and secondary metabolites of their own, the microbiota define the chemical environment of the gut and often determine specific host responses. Although most gut microbiota-produced metabolites are currently uncharacterized, several well-studied molecules made or modified by the microbiota are known to affect the growth and virulence of pathogens, including short-chain fatty acids, succinate, mucin O-glycans, molecular hydrogen, secondary bile acids, and the AI-2 quorum sensing autoinducer. We also discuss challenges and possible approaches to further study of the chemical interplay between microbiota and gastrointestinal pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modeling and validation of autoinducer-mediated bacterial gene expression in microfluidic environments

PubMed Central

Austin, Caitlin M.; Stoy, William; Su, Peter; Harber, Marie C.; Bardill, J. Patrick; Hammer, Brian K.; Forest, Craig R.

2014-01-01

Biosensors exploiting communication within genetically engineered bacteria are becoming increasingly important for monitoring environmental changes. Currently, there are a variety of mathematical models for understanding and predicting how genetically engineered bacteria respond to molecular stimuli in these environments, but as sensors have miniaturized towards microfluidics and are subjected to complex time-varying inputs, the shortcomings of these models have become apparent. The effects of microfluidic environments such as low oxygen concentration, increased biofilm encapsulation, diffusion limited molecular distribution, and higher population densities strongly affect rate constants for gene expression not accounted for in previous models. We report a mathematical model that accurately predicts the biological response of the autoinducer N-acyl homoserine lactone-mediated green fluorescent protein expression in reporter bacteria in microfluidic environments by accommodating these rate constants. This generalized mass action model considers a chain of biomolecular events from input autoinducer chemical to fluorescent protein expression through a series of six chemical species. We have validated this model against experimental data from our own apparatus as well as prior published experimental results. Results indicate accurate prediction of dynamics (e.g., 14% peak time error from a pulse input) and with reduced mean-squared error with pulse or step inputs for a range of concentrations (10 μM–30 μM). This model can help advance the design of genetically engineered bacteria sensors and molecular communication devices. PMID:25379076

Streptococcus gordonii LuxS/autoinducer-2 quorum-sensing system modulates the dual-species biofilm formation with Streptococcus mutans.

PubMed

Wang, Xiao; Li, Xiaolan; Ling, Junqi

2017-07-01

Dental plaques are mixed-species biofilms that are related to the development of dental caries. Streptococcus mutans (S. mutans) is an important cariogenic bacterium that forms mixed-species biofilms with Streptococcus gordonii (S. gordonii), an early colonizer of the tooth surface. The LuxS/autoinducer-2(AI-2) quorum sensing system is involved in the regulation of mixed-species biofilms, and AI-2 is proposed as a universal signal for the interaction between bacterial species. In this work, a S. gordonii luxS deficient strain was constructed to investigate the effect of the S. gordonii luxS gene on dual-species biofilm formed by S. mutans and S. gordonii. In addition, AI-2 was synthesized in vitro by incubating recombinant LuxS and Pfs enzymes of S. gordonii together. The effect of AI-2 on S. mutans single-species biofilm formation and cariogenic virulence gene expression were also assessed. The results showed that luxS disruption in S. gordonii altered dual-species biofilm formation, architecture, and composition, as well as the susceptibility to chlorhexidine. And the in vitro synthesized AI-2 had a concentration-dependent effect on S. mutans biofilm formation and virulence gene expression. These findings indicate that LuxS/AI-2 quorum-sensing system of S. gordonii plays a role in regulating the dual-species biofilm formation with S. mutans. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quorum Sensing in Marine Microbial Environments.

PubMed

Hmelo, Laura R

2017-01-03

Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surface-attached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures.

Quorum Sensing in Marine Microbial Environments

NASA Astrophysics Data System (ADS)

Hmelo, Laura R.

2017-01-01

Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surface-attached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures.

Atomic Force Microscopy Reveals a Morphological Differentiation of Chromobacterium violaceum Cells Associated with Biofilm Development and Directed by N-Hexanoyl-L-Homoserine Lactone

PubMed Central

Kamaeva, Anara A.; Vasilchenko, Alexey S.; Deryabin, Dmitry G.

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure. PMID:25111599

Atomic force microscopy reveals a morphological differentiation of chromobacterium violaceum cells associated with biofilm development and directed by N-hexanoyl-L-homoserine lactone.

PubMed

Kamaeva, Anara A; Vasilchenko, Alexey S; Deryabin, Dmitry G

2014-01-01

Chromobacterium violaceum abounds in soil and water ecosystems in tropical and subtropical regions and occasionally causes severe and often fatal human and animal infections. The quorum sensing (QS) system and biofilm formation are essential for C. violaceum's adaptability and pathogenicity, however, their interrelation is still unknown. C. violaceum's cell and biofilm morphology were examined by atomic force microscopy (AFM) in comparison with growth rates, QS-dependent violacein biosynthesis and biofilm biomass quantification. To evaluate QS regulation of these processes, the wild-type strain C. violaceum ATCC 31532 and its mini-Tn5 mutant C. violaceum NCTC 13274, cultivated with and without the QS autoinducer N-hexanoyl-L-homoserine lactone (C6-HSL), were used. We report for the first time the unusual morphological differentiation of C. violaceum cells, associated with biofilm development and directed by the QS autoinducer. AFM revealed numerous invaginations of the external cytoplasmic membrane of wild-type cells, which were repressed in the mutant strain and restored by exogenous C6-HSL. With increasing bacterial growth, polymer matrix extrusions formed in place of invaginations, whereas mutant cells were covered with a diffusely distributed extracellular substance. Thus, quorum sensing in C. violaceum involves a morphological differentiation that organises biofilm formation and leads to a highly differentiated matrix structure.

Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Kumagai, H

1992-11-30

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Construction of a Cell Based Sensor for the Detection of Autoinducer-2 (Reprint)

DTIC Science & Technology

2012-09-01

transporting pipelines, the distribution of species was found to be B. cereus ACE4 (30%), S . marcescens ACE2 (10%), and 10% of each species of B. subtilis...AR12, P. aeruginosa AI1, K. oxytoca ACP, P. stutzeri AP2, B. litoralis AN1, and Bacillus sp. AN5 [1]. Of these, B. cereus, S . marcescens , and B...NUMBER 6. AUTHOR( S ) Matthew D. Servinsky, Patrick C. Allen, Chen-Yu Tsao, Christopher M. Byrd, Christian J. Sund, and William E. Bentley 5d

Transcriptional Regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK Operons and Their Role in Biofilm Formation

PubMed Central

Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Lamont, Richard J.

2013-01-01

Autoinducer-2 (AI-2) is required for biofilm formation and virulence of the oral pathogen Aggregatibacter actinomycetemcomitans, and we previously showed that lsrB codes for a receptor for AI-2. The lsrB gene is expressed as part of the lsrACDBFG operon, which is divergently transcribed from an adjacent lsrRK operon. In Escherichia coli, lsrRK encodes a repressor and AI-2 kinase that function to regulate lsrACDBFG. To determine if lsrRK controls lsrACDBFG expression and influences biofilm growth of A. actinomycetemcomitans, we first defined the promoters for each operon. Transcriptional reporter plasmids containing the 255-bp lsrACDBFG-lsrRK intergenic region (IGR) fused to lacZ showed that essential elements of lsrR promoter reside 89 to 255 bp upstream from the lsrR start codon. Two inverted repeat sequences that represent potential binding sites for LsrR and two sequences resembling the consensus cyclic AMP receptor protein (CRP) binding site were identified in this region. Using electrophoretic mobility shift assay (EMSA), purified LsrR and CRP proteins were shown to bind probes containing these sequences. Surprisingly, the 255-bp IGR did not contain the lsrA promoter. Instead, a fragment encompassing nucleotides +1 to +159 of lsrA together with the 255-bp IGR was required to promote lsrA transcription. This suggests that a region within the lsrA coding sequence influences transcription, or alternatively that the start codon of A. actinomycetemcomitans lsrA has been incorrectly annotated. Transformation of ΔlsrR, ΔlsrK, ΔlsrRK, and Δcrp deletion mutants with lacZ reporters containing the lsrA or lsrR promoter showed that LsrR negatively regulates and CRP positively regulates both lsrACDBFG and lsrRK. However, in contrast to what occurs in E. coli, deletion of lsrK had no effect on the transcriptional activity of the lsrA or lsrR promoters, suggesting that another kinase may be capable of phosphorylating AI-2 in A. actinomycetemcomitans. Finally, biofilm formation of the ΔlsrR, ΔlsrRK, and Δcrp mutants was significantly reduced relative to that of the wild type, indicating that proper regulation of the lsr locus is required for optimal biofilm growth by A. actinomycetemcomitans. PMID:23104800

Determining the relative contribution and hierarchy of hha and qseBC in the regulation of flagellar motility of Escherichia coli O157:H7.

PubMed

Sharma, Vijay K; Casey, Thomas A

2014-01-01

In recent studies, we demonstrated that a deletion of hha caused increased secretion of locus of enterocyte encoded adherence proteins and reduced motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7. In addition to the importance of hha in positive regulation of motility, a two-component quorum sensing pathway encoded by the qseBC genes has been shown to activate bacterial motility in response to mammalian stress hormones epinephrine and norepinephrine as well as bacterially produced autoinducer-3. In this study, we compared regulatory contribution and hierarchy of hha, a member of the Hha/YmoA family of nucleoid-associated proteins, to that of qseBC in the expression of EHEC O157:H7 motility. Since norepinephrine affects motility of EHEC O157:H7 through a qseBC-encoded two-component quorum sensing signaling, we also determined whether the hha-mediated regulation of motility is affected by norepinephrine and whether this effect is qseBC dependent. We used single (Δhha or ΔqseC) and double (Δhha ΔqseC) deletion mutants to show that hha exerts a greater positive regulatory effect in comparison to qseBC on the expression of motility by EHEC O157:H7. We also show that Hha is hierarchically superior in transcriptional regulation of motility than QseBC because transcription of qseC was significantly reduced in the hha deletion mutant compared to that in the parental and the hha-complemented mutant strains. These results suggest that hha regulates motility of EHEC O157:H7 directly as well as indirectly by controlling the transcription of qseBC.

Determining the Relative Contribution and Hierarchy of hha and qseBC in the Regulation of Flagellar Motility of Escherichia coli O157:H7

PubMed Central

Sharma, Vijay K.; Casey, Thomas A.

2014-01-01

In recent studies, we demonstrated that a deletion of hha caused increased secretion of locus of enterocyte encoded adherence proteins and reduced motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7. In addition to the importance of hha in positive regulation of motility, a two-component quorum sensing pathway encoded by the qseBC genes has been shown to activate bacterial motility in response to mammalian stress hormones epinephrine and norepinephrine as well as bacterially produced autoinducer-3. In this study, we compared regulatory contribution and hierarchy of hha, a member of the Hha/YmoA family of nucleoid-associated proteins, to that of qseBC in the expression of EHEC O157:H7 motility. Since norepinephrine affects motility of EHEC O157:H7 through a qseBC-encoded two-component quorum sensing signaling, we also determined whether the hha-mediated regulation of motility is affected by norepinephrine and whether this effect is qseBC dependent. We used single (Δhha or ΔqseC) and double (Δhha ΔqseC) deletion mutants to show that hha exerts a greater positive regulatory effect in comparison to qseBC on the expression of motility by EHEC O157:H7. We also show that Hha is hierarchically superior in transcriptional regulation of motility than QseBC because transcription of qseC was significantly reduced in the hha deletion mutant compared to that in the parental and the hha-complemented mutant strains. These results suggest that hha regulates motility of EHEC O157:H7 directly as well as indirectly by controlling the transcription of qseBC. PMID:24465756

The Agrobacterium tumefaciens rnd Homolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid tra Gene Expression

PubMed Central

Luo, Zhao-Qing; Farrand, Stephen K.

2001-01-01

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-l-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58ΔaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D. PMID:11395455

Inactivation and regrowth of multidrug resistant bacteria in urban wastewater after disinfection by solar-driven and chlorination processes.

PubMed

Fiorentino, Antonino; Ferro, Giovanna; Alferez, María Castro; Polo-López, Maria Inmaculada; Fernández-Ibañez, Pilar; Rizzo, Luigi

2015-07-01

Solar disinfection and solar-driven advanced oxidation processes (AOPs) (namely H2O2/sunlight, TiO2/sunlight, H2O2/TiO2/sunlight, solar photo-Fenton) were evaluated in the inactivation of indigenous antibiotic-resistant bacteria (ARB) in real urban wastewater. A multidrug resistant (MDR) Escherichia coli strain isolated from the effluent of the biological process of an urban wastewater treatment plant was the target ARB. The higher inactivation rates (residual density under detection limit, 2 CFUm L(-1)) were achieved with H2O2/TiO2/sunlight (cumulative energy per unit of volume (QUV) in the range 3-5 kJ L(-1), depending on H2O2/TiO2 ratio) and H2O2/sunlight (QUV of 8 kJ L(-1)) processes. All investigated processes did not affect antibiotic resistance of survived colonies. Moreover, H2O2/sunlight was compared with conventional chlorination process to evaluate bacterial regrowth potential and particularly the proportion of indigenous MDR E. coli with respect to total indigenous E. coli population. Chlorination (1.0 mg Cl2 L(-1)) was more effective than H2O2/sunlight (50 mg H2O2 L(-1)) to achieve total inactivation of MDR E. coli (15 min Vs 90 min) but less effective in controlling their regrowth (24 h Vs 48 h). Interestingly, the percentage of MDR E. coli in H2O2/sunlight treated samples decreased as incubation time increased; the opposite was observed for chlorinated samples. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2012-11-01

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

Truncated Autoinducing Peptide Conjugates Selectively Recognize and Kill Staphylococcus aureus.

PubMed

Tsuchikama, Kyoji; Shimamoto, Yasuhiro; Anami, Yasuaki

2017-06-09

The accessory gene regulator (agr) of Staphylococcus aureus coordinates various pathogenic events and is recognized as a promising therapeutic target for virulence control. S. aureus utilizes autoinducing peptides (AIPs), cyclic-peptide signaling molecules, to mediate the agr system. Despite the high potency of synthetic AIP analogues in agr inhibition, the potential of AIP molecules as a delivery vehicle for antibacterial agents remains unexplored. Herein, we report that truncated AIP scaffolds can be fused with fluorophore and cytotoxic photosensitizer molecules without compromising their high agr inhibitory activity, binding affinity to the receptor AgrC, or cell specificity. Strikingly, a photosensitizer-AIP conjugate exhibited 16-fold greater efficacy in a S. aureus cell-killing assay than a nontargeting analogue. These findings highlight the potential of truncated AIP conjugates as useful chemical tools for in-depth biological studies and as effective anti-S. aureus agents.

The Quorum-Sensing Regulon of Vibriofischeri: Novel Components of the Autoinducer/LuxR Regulatory Circuit

DTIC Science & Technology

1999-06-01

cpdP, from the marine symbiotic bacterium Vibrio fische ri 160 Table of abbreviations 30C6-HSL AI-1 AI-2 C8-HSL CHAPS CNP EDTA FMN GFP HPLC ...using a Zorbax C18 1.0 mm by 150 mm reverse-phase column on a Hewlett-Packard 1090 HPLC /1040 diode array detector at the Harvard Microchemistry...separated by reversed-phase HPLC , and sequenced (Table 2; 10-PK12, 10-PK39, and 10-PK51). From two of the three peptide sequences (Materials and

Heterologous Expression of sahH Reveals That Biofilm Formation Is Autoinducer-2-independent in Streptococcus sanguinis but Is Associated with an Intact Activated Methionine Cycle*

PubMed Central

Redanz, Sylvio; Standar, Kerstin; Podbielski, Andreas; Kreikemeyer, Bernd

2012-01-01

Numerous studies have claimed deleterious effects of LuxS mutation on many bacterial phenotypes, including bacterial biofilm formation. Genetic complementation mostly restored the observed mutant phenotypes to WT levels, leading to the postulation that quorum sensing via a family of molecules generically termed autoinducer-2 (AI-2) is essential for many phenotypes. Because LuxS mutation has dual effects, this hypothesis needs to be investigated into the details for each bacterial species. In this study we used S. sanguinis SK36 as a model biofilm bacterium and employed physiological characterization and transcriptome approaches on WT and luxS-deficient strains, in combination with chemical, luxS, and sahH complementation experiments. SahH enables a direct conversion of SAH to homocysteine and thereby restores the activated methionine cycle in a luxS-negative background without formation of the AI-2 precursor 4,5-dihydroxy-2,3-pentanedione. With this strategy we were able to dissect the individual contribution of LuxS and AI-2 activity in detail. Our data revealed that S. sanguinis biofilm formation is independent from AI-2 substance pools and is rather supported by an intact activated methyl cycle. Of 216 differentially transcribed genes in the luxS mutant, 209 were restored by complementation with a gene encoding the S-adenosylhomocysteine hydrolase. Only nine genes, mainly involved in natural competence, were directly affected by the AI-2 quorum-sensing substance pool. Cumulatively, this suggested that biofilm formation in S. sanguinis is not under control of AI-2. Our study suggests that previously evaluated LuxS mutants in other species need to be revisited to resolve the precise contribution of AI-2 substance pools and the methionine pathways. PMID:22942290

Cloning, expression and characterization of two S-ribosylhomocysteine lyases from Lactobacillus plantarum YM-4-3: Implication of conserved and divergent roles in quorum sensing.

PubMed

Song, Xiao-Dong; Liu, Chen-Jian; Huang, Shi-Hao; Li, Xiao-Ran; Yang, En; Luo, Yi-Yong

2018-05-01

Quorum sensing (QS) is a means of cell-to-cell communication that regulates, via small signalling molecules, expression of a series of genes and controls multicellular behaviour in many bacterial species. The enzyme S-ribosylhomocysteine lyase (LuxS) transforms S-ribosylhomocysteine (SRH) into 4, 5-dihydroxy-2, 3-pentanedione (DPD), the precursor of the interspecies QS signalling molecule autoinducer-2 (AI-2). In this study, two LuxS-coding genes, luxS1 and luxS2, with 70% sequence identity were isolated from Lactobacillus plantarum YM-4-3, and overexpressed in Escherichia coli BL21 (DE3), and the protein products were purified successfully. After incubation of LuxS1 or LuxS2 with SRH, the reaction products were able to induce Vibrio harveyi BB170 bioluminescence, clearly demonstrating that both LuxS1 and LuxS2 synthesize AI-2 from SRH in vitro. Ellman's assay results revealed optimal temperatures for LuxS1 and LuxS2 of 45 and 37 °C, respectively, and their activities were stimulated or inhibited by several metal ions and chemical reagents. In addition, enzyme kinetics data showed that K m , V max and K cat value of LuxS1 for the substrate (SRH) were higher than that of LuxS2. These results suggest that LuxS1 and LuxS2 mediate QS in a temperature-dependent manner and may play conserved roles in AI-2 synthesis but exhibit different activities in response to external environmental stress. To our knowledge, this paper is the first report of two luxS genes present in one bacterial genome and the subsequent comparative elucidation of their functions in AI-2 production. Collectively, our study provides a solid basis for future research concerning the AI-2/LuxS QS system in L. plantarum YM-4-3. Copyright © 2018 Elsevier Inc. All rights reserved.

Role and regulation of the orphan AphA protein of quorum sensing in pathogenic Vibrios.

PubMed

Lu, Renfei; Osei-Adjei, George; Huang, Xinxiang; Zhang, Yiquan

2018-03-01

Quorum sensing (QS), a cell-to-cell communication process, is widely distributed in the bacterial kingdom. Bacteria use QS to control gene expression in response to cell density by detecting the signal molecules called autoinducers. AphA protein is the master QS regulator of vibrios operating at low cell density. It regulates the expression of a variety of genes, especially those encoding virulence factors, flagella/motility and biofilm formation. The role and regulation of AphA in vibrios, especially in human pathogenic vibrios, are summarized in this review. Clarification of the roles of AphA will help us to understand the pathogenesis of vibrios.

Pathogen espionage: multiple bacterial adrenergic sensors eavesdrop on host communication systems.

PubMed

Karavolos, Michail H; Winzer, Klaus; Williams, Paul; Khan, C M Anjam

2013-02-01

The interactions between bacterial pathogens and their eukaryotic hosts are vital in determining the outcome of infections. Bacterial pathogens employ molecular sensors to detect and facilitate adaptation to changes in their niche. The sensing of these extracellular signals enables the pathogen to navigate within mammalian hosts. Intercellular bacterial communication is facilitated by the production and sensing of autoinducer (AI) molecules via quorum sensing. More recently, AI-3 and the host neuroendocrine (NE) hormones adrenaline and noradrenaline were reported to display cross-talk for the activation of the same signalling pathways. Remarkably, there is increasing evidence to suggest that enteric bacteria sense and respond to the host NE stress hormones adrenaline and noradrenaline to modulate virulence. These responses can be inhibited by α and β-adrenergic receptor antagonists implying a bacterial receptor-based sensing and signalling cascade. In Escherichia coli O157:H7 and Salmonella, QseC has been proposed as the adrenergic receptor. Strikingly, there is an increasing body of evidence that not all the bacterial adrenergic responses require signalling through QseC. Here we provide additional hypotheses to reconcile these observations implicating the existence of alternative adrenergic receptors including BasS, QseE and CpxA and their associated signalling cascades with major roles in interkingdom communication. © 2012 Blackwell Publishing Ltd.

Efficacy of supercritical carbon dioxide for nonthermal inactivation of Escherichia coli K12 in apple cider

USDA-ARS?s Scientific Manuscript database

This study evaluated the efficacy of a supercritical carbon dioxide (SCCO2) system with a gas-liquid porous metal contactor for eliminating Escherichia coli K12 in apple cider. Pasteurized, preservative-free apple cider was inoculated with E. coli K12 and processed using the SCCO2 system at CO2 conc...

[Acute toxicity analysis performance of CellSense biosensor with E. coli].

PubMed

Wang, Xue-Jiang; Wang, Hong; Zhao, Jian-Fu; Xia, Si-Qing; Zhao, Hong-Ning

2009-04-15

E. coli microbial electrodes for CellSense biosensor were prepared by polycarbonate membrane immobilization process, and their performance for heavy metals and toxic organic compounds acute toxicity determination were studied. The results showed that when E. coli was in logarithmic and stationary phase, the CellSense biosensor with E. coli showed good performance in heavy metal ions and organic pollutants acute toxicity analysis, when E. coli was in its decline phase, the stability and sensitivity of the CellSense biosensor was poor. The EC50 values of Hg2+, Cu2+, Zn2+, o-chlorophenol (2-CP) and p-nitrophenol (4-NP) detected by CellSense biosensor with E. coli were 0.6, 3.1, 5.8, 180 and 94 microg/mL, respectively. The immobilized E. coli electrodes could still suit for acute toxicity assessment after 2 months storage at 4 degrees C.

Dietary boron: possible roles in human and animal physiology

USDA-ARS?s Scientific Manuscript database

Boron is a bioactive element of low molecular weight. Since discovery of the first boron biomolecule, boromycin, in 1967, several other similar biomolecules are now well-characterized. Most recently described was a bacterial cell-to-cell communication signal that requires boron, autoinducer-II. Boro...

Culturable heterotrophic bacteria from the marine sponge Dendrilla nigra: isolation and phylogenetic diversity of actinobacteria

NASA Astrophysics Data System (ADS)

Selvin, Joseph; Gandhimathi, R.; Kiran, G. Seghal; Priya, S. Shanmugha; Ravji, T. Rajeetha; Hema, T. A.

2009-09-01

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “ Micromonospora-Saccharomonospora-Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.

Nonthermal inactivation of Escherichia coli K12 in buffered peptone water using a pilot-plant scale supercritical carbon dioxide system with gas-liquid porous metal contractor

USDA-ARS?s Scientific Manuscript database

This study evaluated the effectiveness of a supercritical carbon dioxide (SCCO2) system, with a gas-liquid CO2 contactor, for reducing Escherichia coli K12 in diluted buffered peptone water. 0.1% (w/v) buffered peptone water inoculated with E. coli K12 was processed using the SCCO2 system at CO2 con...

Reduction of verotoxigenic Escherichia coli in production of fermented sausages.

PubMed

Holck, Askild L; Axelsson, Lars; Rode, Tone Mari; Høy, Martin; Måge, Ingrid; Alvseike, Ole; L'abée-Lund, Trine M; Omer, Mohamed K; Granum, Per Einar; Heir, Even

2011-11-01

After a number of foodborne outbreaks of verotoxigenic Escherichia coli involving fermented sausages, some countries have imposed regulations on sausage production. For example, the US Food Safety and Inspection Service requires a 5 log(10) reduction of E. coli in fermented products. Such regulations have led to a number of studies on the inactivation of E. coli in fermented sausages by changing processing and post-processing conditions. Several factors influence the survival of E. coli such as pre-treatment of the meat, amount of NaCl, nitrite and lactic acid, water activity, pH, choice of starter cultures and addition of antimicrobial compounds. Also process variables like fermentation temperature and storage time play important roles. Though a large variety of different production processes of sausages exist, generally the reduction of E. coli caused by production is in the range 1-2 log(10). In many cases this may not be enough to ensure microbial food safety. By optimising ingredients and process parameters it is possible to increase E. coli reduction to some extent, but in some cases still other post process treatments may be required. Such treatments may be storage at ambient temperatures, specific heat treatments, high pressure processing or irradiation. HACCP analyses have identified the quality of the raw materials, low temperature in the batter when preparing the sausages and a rapid pH drop during fermentation as critical control points in sausage production. This review summarises the literature on the reduction verotoxigenic E. coli in production of fermented sausages. Copyright © 2011 Elsevier Ltd. All rights reserved.

The effectiveness of sewage treatment processes to remove faecal pathogens and antibiotic residues

PubMed Central

Hendricks, Rahzia; Pool, Edmund John

2012-01-01

Pathogens and antibiotics enter the aquatic environment via sewage effluents and may pose a health risk to wild life and humans. The aim of this study was to determine the levels of faecal bacteria, and selected antibiotic residues in raw wastewater and treated sewage effluents from three different sewage treatment plants in the Western Cape, South Africa. Sewage treatment plant 1 and 2 use older technologies, while sewage treatment plant 3 has been upgraded and membrane technologies were incorporated in the treatment processes. Coliforms and Escherichia coli (E. coli) were used as bioindicators for faecal bacteria. A chromogenic test was used to screen for coliforms and E. coli. Fluoroquinolones and sulfamethoxazole are commonly used antibiotics and were selected to monitor the efficiency of sewage treatment processes for antibiotic removal. Enzyme Linked Immunosorbent Assays (ELISAs) were used to quantitate antibiotic residues in raw and treated sewage. Raw intake water at all treatment plants contained total coliforms and E. coli. High removal of E. coli by treatment processes was evident for treatment plant 2 and 3 only. Fluoroquinolones and sulfamethoxazole were detected in raw wastewater from all sewage treatment plants. Treatment processes at plant 1 did not reduce the fluoroquinolone concentration in treated sewage effluents. Treatment processes at plant 2 and 3 reduced the fluoroquinolone concentration by 21% and 31%, respectively. Treatment processes at plant 1 did not reduce the sulfamethoxazole concentration in treated sewage effluents. Treatment processes at plant 2 and 3 reduced sulfamethoxazole by 34% and 56%, respectively. This study showed that bacteria and antibiotic residues are still discharged into the environment. Further research needs to be undertaken to improve sewage treatment technologies, thereby producing a better quality treated sewage effluent. PMID:22242882

Lethality Prediction for Escherichia Coli O157:H7 and Uropathogenic E. coli in Ground Chicken Treated with High Pressure Processing and Trans-Cinnamaldehyde.

PubMed

Sheen, Shiowshuh; Huang, Chi-Yun; Ramos, Rommel; Chien, Shih-Yung; Scullen, O Joseph; Sommers, Christopher

2018-03-01

Pathogenic Escherichia coli, intestinal (O157:H7) as well as extraintestinal types (for example, Uropathogenic E. coli [UPEC]) are commonly found in many foods including raw chicken meat. The resistance of E. coli O157:H7 to UPEC in chicken meat under the stresses of high hydrostatic Pressure (HHP, also known as HPP-high pressure processing) and trans-cinnamaldehyde (an essential oil) was investigated and compared. UPEC was found slightly less resistant than O157:H7 in our test parameter ranges. With the addition of trans-cinnamaldehyde as an antimicrobial to meat, HPP lethality enhanced both O157:H7 and UPEC inactivation. To facilitate the predictive model development, a central composite design (CCD) was used to assess the 3-parameter effects, that is, pressure (300 to 400 MPa), trans-cinnamaldehyde dose (0.2 to 0.5%, w/w), and pressure-holding time (15 to 25 min), on the inactivation of E. coli O157:H7 and UPEC in ground chicken. Linear models were developed to estimate the lethality of E. coli O157:H7 (R 2 = 0.86) and UPEC (R 2 = 0.85), as well as dimensionless nonlinear models. All models were validated with data obtained from separated CCD combinations. Because linear models of O157:H7 and UPEC had similar R 2 and the significant lethality difference of CCD points was only 9 in 20; all data were combined to generate models to include both O157:H7 and UPEC. The results provide useful information/tool to predict how pathogenic E. coli may survive HPP in the presence of trans-cinnamaldehyde and to achieve a great than 5 log CFU/g reduction in chicken meat. The models may be used for process optimization, product development and to assist the microbial risk assessment. The study provided an effective means to reduce the high hydrostatic pressure level with incorporation of antimicrobial compound to achieve a 5-log reduction of pathogenic E. coli without damaging the raw meat quality. The developed models may be used to predict the high pressure processing lethality (and process optimization), product development (ingredient selection), and to assist the microbial risk assessment. © 2018 Institute of Food Technologists®.

Fate of Escherichia coli O157 Cells Inoculated into Lightly Pickled Chinese Cabbage during Processing, Storage and Incubation in Artificial Gastric Juice.

PubMed

Inatsu, Yasuhiro; Ohata, Yukiko; Ananchaipattana, Chiraporn; Latiful Bari, Md; Hosotani, Yukie; Kawasaki, Susumu

2016-01-01

Fate of Escherichia coli O157 cells was evaluated when inoculated into each step after production of lightly pickled Chinese cabbage. The efficacy of surface sterilization by 100 mg/L of chlorine water for 10 min on raw leaves (6.0 log CFU/g) was 2.2 log CFU/g reduction. No meaningful change of the population of E. coli O157 (3.5 log CFU/g to 1.5 log MPN/g) contaminated into 19 kinds of products was observed. These results indicated the difficulty of estimating the viable count of the cells between contaminated on farms and further processing and storage steps. The population of E. coli O157 (3 log CFU/g to 1 log MPN/g) inoculated into the Chinese cabbage products was reduced less than 0.6 log CFU/g after 2 h-incubation at 37℃ in artificial gastric juice. Prevention from initial contamination of E. coli O157 on the ingredients of Chinese cabbage products is important to reduce the risk of food poisoning because the reduction of the bacterial counts after processing and consumption are limited.

[Characteristics of Bacillus cereus dissociants].

PubMed

Doroshenko, E V; Loĭko, N G; Il'inskaia, O N; Kolpakov, A I; Gornova, I B; Klimanova, E V; El'-Registan, G I

2001-01-01

The autoregulation of the phenotypic (populational) variability of the Bacillus cereus strain 504 was studied. The isolated colonial morphotypes of this bacterium were found to differ in their growth characteristics and the synthesis of extracellular proteases. The phenotypic variabilities of vegetative proliferating cells and those germinated from endospores and cystlike refractory cells were different. Bacterial variants also differed in the production of the d1 and d2 factors (the autoinducers of dormancy and autolysis, respectively) and sensitivity to them. The possible role of these factors in the dissociation of microorganisms is discussed.

The Janthinobacterium sp. HH01 Genome Encodes a Homologue of the V. cholerae CqsA and L. pneumophila LqsA Autoinducer Synthases

PubMed Central

Hornung, Claudia; Poehlein, Anja; Haack, Frederike S.; Schmidt, Martina; Dierking, Katja; Pohlen, Andrea; Schulenburg, Hinrich; Blokesch, Melanie; Plener, Laure; Jung, Kirsten; Bonge, Andreas; Krohn-Molt, Ines; Utpatel, Christian; Timmermann, Gabriele; Spieck, Eva; Pommerening-Röser, Andreas; Bode, Edna; Bode, Helge B.; Daniel, Rolf; Schmeisser, Christel; Streit, Wolfgang R.

2013-01-01

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes. PMID:23405110

Microbial interactions in building of communities

PubMed Central

Wright, Christopher J.; Burns, Logan H.; Jack, Alison A.; Back, Catherine R.; Dutton, Lindsay C.; Nobbs, Angela H.; Lamont, Richard J.; Jenkinson, Howard F.

2012-01-01

SUMMARY Establishment of a community is considered to be essential for microbial growth and survival in the human oral cavity. Biofilm communities have increased resilience to physical forces, antimicrobial agents, and nutritional variations. Specific cell-to-cell adherence processes, mediated by adhesin-receptor pairings on respective microbial surfaces, are able to direct community development. These interactions co-localize species in mutually beneficial relationships, such as streptococci, veillonellae, Porphyromonas gingivalis and Candida albicans. In transition from the planktonic mode of growth to a biofilm community, microorganisms undergo major transcriptional and proteomic changes. These occur in response to sensing of diffusible signals, such as autoinducer molecules, and to contact with host tissues or other microbial cells. Underpinning many of these processes are intracellular phosphorylation events that regulate a large number of microbial interactions relevant to community formation and development. PMID:23253299

Characterization of Diarrheagenic Escherichia coli Isolated in Organic Waste Products (Cattle Fecal Matter, Manure and, Slurry) from Cattle's Markets in Ouagadougou, Burkina Faso.

PubMed

Bako, Evariste; Kagambèga, Assèta; Traore, Kuan Abdoulaye; Bagre, Touwendsida Serge; Ibrahim, Hadiza Bawa; Bouda, Soutongnooma Caroline; Bonkoungou, Isidore Juste Ouindgueta; Kaboré, Saidou; Zongo, Cheikna; Traore, Alfred Sababenejo; Barro, Nicolas

2017-09-22

Cattle farming can promote diarrheal disease transmission through waste, effluents or cattle fecal matter. The study aims to characterize the diarrheagenic Escherichia coli (DEC) isolated from cattle feces, manure in the composting process and slurry, collected from four cattle markets in Ouagadougou. A total of 585 samples (340 cattle feces, 200 slurries and 45 manures in the composting process) were collected from the four cattle markets between May 2015 and May 2016. A multiplex Polymerase Chain Reaction (PCR), namely 16-plex PCR, was used to screen simultaneously the virulence genes specific for shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC). DEC was detected in 10.76% of samples. ETEC was the most prevalent (9.91%). STEC and EAEC have been observed with the same rate (0.51%). ETEC were detected in 12.64% of cattle feces, in 6.66% of manure in the composting process and in 5% of slurry. STEC were detected in 0.58% of cattle feces and in 2.22% of manure in the composting process. EAEC was detected only in 1% of slurry and in 2.22% of manure in the composting process. ETEC strains were identified based on estIa gene and/or estIb gene and/or elt gene amplification. Of the 58 ETEC, 10.34% contained astA , 17.24% contained elt , 3.44% contained estIa and 79.31% contained estIb . The two positive EAEC strains contained only the aggR gene, and the third was positive only for the pic gene. The results show that effluent from cattle markets could contribute to the spreading of DEC in the environment in Burkina Faso.

Development of molecularly imprinted polymers to block quorum sensing and inhibit bacterial biofilm formation.

PubMed

Ma, Luyao; Feng, Shaolong; de la Fuente-Nunez, Cesar; Hancock, Robert E W; Lu, Xiaonan

2018-05-16

Bacterial biofilms are responsible for most clinical infections and show increased antimicrobial resistance. In this study, molecularly imprinted polymers (MIPs) were developed to specifically capture prototypical quorum sensing autoinducers [i.e., N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12AHL)], interrupt quorum sensing, and subsequently inhibit biofilm formation of Pseudomonas aeruginosa, an important human nosocomial pathogen. The synthesis of MIPs was optimized by considering the amount and type of the functional monomers itaconic acid (IA) and 2-hydroxyethyl methacrylate (HEMA). IA-based MIPs showed high adsorption affinity towards 3-oxo-C12AHL with an imprinting factor of 1.68. Compared to IA-based MIPs, the adsorption capacity of HEMA-based MIPs was improved 5-fold. HEMA-based MIPs significantly reduced biofilm formation (by ~65%), while biofilm suppression by IA-based MIPs was neutralized due to increased bacterial attachment. The developed MIPs represent promising alternative biofilm intervention agents that can be applied to surfaces relevant to clinical settings and food processing equipment.

Optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 by heterologous expression in Escherichia coli.

PubMed

Jasniewski, Jordane; Cailliez-Grimal, Catherine; Gelhaye, Eric; Revol-Junelles, Anne-Marie

2008-04-01

An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.

Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes.

PubMed

Boettcher, K J; Ruby, E G

1990-07-01

Bioluminescent marine bacteria of the species Vibrio fischeri are the specific light organ symbionts of the sepiolid squid Euprymna scolopes. Although they share morphological and physiological characteristics with other strains of V. fischeri, when cultured away from the light organ association the E. scolopes symbionts depress their maximal luminescence over 1,000-fold. The primary cause of this reduced luminescence is the underproduction by these bacteria of luciferase autoinducer, a molecule involved in the positive transcriptional regulation of the V. fischeri lux operon. Such an absence of visible light production outside of the symbiotic association has not been previously reported among light organ symbionts of this or any other species of luminous bacteria. Levels of luminescence approaching those of the E. scolopes bacteria in the intact association can be restored by the addition of exogenous autoinducer to bacteria in laboratory culture and are affected by the presence of cyclic AMP. We conclude that some condition(s) specific to the internal environment of the light organ is necessary for maximal autoinduction of luminescence in the symbionts of this squid-bacterial association.

Association of Escherichia coli O157:H7 with Houseflies on a Cattle Farm†

PubMed Central

Alam, Muhammad J.; Zurek, Ludek

2004-01-01

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 × 101 to 1.5 × 105 CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment. PMID:15574966

Association of Escherichia coli O157:H7 with houseflies on a cattle farm.

PubMed

Alam, Muhammad J; Zurek, Ludek

2004-12-01

The ecology of Escherichia coli O157:H7 is not well understood. The aims of this study were to determine the prevalence of and characterize E. coli O157:H7 associated with houseflies (HF). Musca domestica L. HF (n = 3,440) were collected from two sites on a cattle farm over a 4-month period and processed individually for E. coli O157:H7 isolation and quantification. The prevalence of E. coli O157:H7 was 2.9 and 1.4% in HF collected from feed bunks and a cattle feed storage shed, respectively. E. coli O157:H7 counts ranged from 3.0 x 10(1) to 1.5 x 10(5) CFU among the positive HF. PCR analysis of the E. coli O157:H7 isolates revealed that 90.4, 99.2, 99.2, and 100% of them (n = 125) possessed the stx1, stx2, eaeA, and fliC genes, respectively. Large populations of HF on cattle farms may play a role in the dissemination of E. coli O157:H7 among animals and to the surrounding environment.

Expression of tropodithietic acid biosynthesis is controlled by a novel autoinducer.

PubMed

Geng, Haifeng; Belas, Robert

2010-09-01

The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaABCDEF (tdaA-F), as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4-fold, respectively) when cells were grown in standing liquid broth compared to their levels with shaking liquid culturing. No transcription of tdaC was detected when a tdaCp::lacZ transcriptional fusion was placed in 11 of the 12 Tda(-) mutant backgrounds, with cysI being the sole exception. The expression of tdaC could be restored to 9 of the remaining 11 Tda(-) mutants-tdaA and tdaH failed to respond-by placing wild-type (Tda(+)) strains in close proximity or by supplying exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal.

Explaining and modeling the concentration and loading of Escherichia coli in a stream-A case study.

PubMed

Wang, Chaozi; Schneider, Rebecca L; Parlange, Jean-Yves; Dahlke, Helen E; Walter, M Todd

2018-09-01

Escherichia coli (E. coli) level in streams is a public health indicator. Therefore, being able to explain why E. coli levels are sometimes high and sometimes low is important. Using citizen science data from Fall Creek in central NY we found that complementarily using principal component analysis (PCA) and partial least squares (PLS) regression provided insights into the drivers of E. coli and a mechanism for predicting E. coli levels, respectively. We found that stormwater, temperature/season and shallow subsurface flow are the three dominant processes driving the fate and transport of E. coli. PLS regression modeling provided very good predictions under stormwater conditions (R 2  = 0.85 for log (E. coli concentration) and R 2  = 0.90 for log (E. coli loading)); predictions under baseflow conditions were less robust. But, in our case, both E. coli concentration and E. coli loading were significantly higher under stormwater condition, so it is probably more important to predict high-flow E. coli hazards than low-flow conditions. Besides previously reported good indicators of in-stream E. coli level, nitrate-/nitrite-nitrogen and soluble reactive phosphorus were also found to be good indicators of in-stream E. coli levels. These findings suggest management practices to reduce E. coli concentrations and loads in-streams and, eventually, reduce the risk of waterborne disease outbreak. Copyright © 2018. Published by Elsevier B.V.

Immobilization and Stabilization of Acylase on Carboxylated Polyaniline Nanofibers for Highly Effective Antifouling Application via Quorum Quenching.

PubMed

Lee, Jeongjoon; Lee, Inseon; Nam, Jahyun; Hwang, Dong Soo; Yeon, Kyung-Min; Kim, Jungbae

2017-05-10

Acylase (AC) was immobilized and stabilized on carboxylated polyaniline nanofibers (cPANFs) for the development of antifouling nanobiocatalysts with high enzyme loading and stability. AC was immobilized via three different approaches: covalent attachment (CA), enzyme coating (EC), and magnetically separable enzyme precipitate coating (Mag-EPC). The enzyme activity per unit weight of cPANFs with Mag-EPC was 75 and 300 times higher than that of those with CA and EC, respectively, representing improved enzyme loading in the form of Mag-EPC. After incubation under shaking at 200 rpm for 20 days, Mag-EPC maintained 55% of its initial activity, whereas CA and EC showed 3 and 16% of their initial activities, respectively. The antifouling of highly loaded and stable Mag-EPC against the biofouling/biofilm formation of Pseudomonas aeruginosa was tested under static- and continuous-flow conditions. Biofilm formation in the presence of 40 μg/mL Mag-EPC under static condition was 5 times lower than that under control condition with no addition of Mag-EPC. Under continuous membrane filtration, Mag-EPC delayed the increase of transmembrane pressure (TMP) more effectively as the concentration of added Mag-EPC increased. When separating Mag-EPC and membranes in two different vessels under internal circulation of the culture solution, Mag-EPC maintained a higher permeability than the control with no Mag-EPC addition. It was also confirmed that the addition of Mag-EPC reduced the generation of N-acyl homoserine lactone (AHL) autoinducers. This result reveals that the inhibition of biofilm formation and biofouling in the presence of Mag-EPC is due to the hydrolysis of AHL autoinducers, catalyzed by the immobilized and stabilized AC in the form of Mag-EPC. Mag-EPC of AC with high enzyme loadings and improved stability has demonstrated its great potential as an antifouling agent by reducing biofilm formation and membrane biofouling based on "enzymatic quorum quenching" of autoinducers.

Validation of time and temperature values as critical limits for the control of Escherichia coli O157:H7 during the production of fresh ground beef.

PubMed

Mann, J E; Brashears, M M

2006-08-01

In order to provide beef processors with valuable data to validate critical limits set for temperature during grinding, a study was conducted to determine Escherichia coli o157:H7 growth at various temperatures in raw ground beef. Fresh ground beef samples were inoculated with a cocktail mixture of streptomycin-resistant E. coli O157:H7 to facilitate recovery in the presence of background flora. Samples were held at 4.4, 7.2, and 10 degrees C, and at room temperature (22.2 to 23.3 degrees C) to mimic typical processing and holding temperatures observed in meat processing environments. E. coli O157:H7 counts were determined by direct plating onto tryptic soy agar with streptomycin (1,000 microg/ml), at 2-h intervals over 12 h for samples held at room temperature. Samples held under refrigeration temperatures were sampled at 4, 8, 12, 24, 48, and 72 h. Less than one log of E. coli O157:H7 growth was observed at 48 h for samples held at 10 degrees C. Samples held at 4.4 and 7.2 degrees C showed less than one log of E. coli O157:H7 growth at 72 h. Samples held at room temperature showed no significant increase in E. coli O157:H7 counts for the first 6 h, but increased significantly afterwards. These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits in their hazard analysis critical control point plans to minimize E. coli O157:H7 growth during the production and storage of ground beef.

Detection and inhibition of bacterial cell-cell communication.

PubMed

Rice, Scott A; McDougald, Diane; Givskov, Michael; Kjelleberg, Staffan

2008-01-01

Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling purposes, including fatty acids, peptides, N-acylated homoserine lactones, and the signals collectively called autoinducer 2 (AI-2). This chapter will focus on the latter two signaling systems as they are present in a range of medically relevant bacteria, and here we describe assays for determining whether an organism produces a particular signal and assays that can be used to identify inhibitors of the signaling cascade. Lastly, the signal detection and inhibition assays will be directly linked to the expression of virulence factors of specific pathogens.

Decreasing biotoxicity of fume particles produced in welding process.

PubMed

Yu, Kuei-Min; Topham, Nathan; Wang, Jun; Kalivoda, Mark; Tseng, Yiider; Wu, Chang-Yu; Lee, Wen-Jhy; Cho, Kuk

2011-01-30

Welding fumes contain heavy metals, such as chromium, manganese, and nickel, which cause respiratory diseases and cancer. In this study, a SiO(2) precursor was evaluated as an additive to the shielding gas in an arc welding process to reduce the biotoxicity caused by welding fume particles. Transmission electron micrographic images show that SiO(2) coats on the surface of welding fume particles and promotes particle agglomeration. Energy dispersive X-ray spectroscopy further shows that the relative amount of silicon in these SiO(2)-coated agglomerates is higher than in baseline agglomerates. In addition, Escherichia coli (E. coli) exposed to different concentrations of pure SiO(2) particles generated from the arc welding process exhibits similar responses, suggesting that SiO(2) does not contribute to welding fume particle toxicity. The trend of E. coli growth in different concentrations of baseline welding fume particle shows the most significant inhibition occurs in higher exposure concentrations. The 50% lethal logarithmic concentrations for E. coli in arc welding particles of baseline, 2%, and 4.2% SiO(2) precursor additives were 823, 1605, and 1800 mg/L, respectively. Taken together, these results suggest that using SiO(2) precursors as an additive to arc welding shielding gas can effectively reduce the biotoxicity of welding fume. Copyright © 2010 Elsevier B.V. All rights reserved.

Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan

PubMed Central

2005-01-01

Abstract A feedlot trial was conducted to assess the efficacy of an Escherichia coli O157:H7 vaccine in reducing fecal shedding of E. coli O157:H7 in 218 pens of feedlot cattle in 9 feedlots in Alberta and Saskatchewan. Pens of cattle were vaccinated once at arrival processing and again at reimplanting with either the E. coli O157:H7 vaccine or a placebo. The E. coli O157:H7 vaccine included 50 μg of type III secreted proteins. Fecal samples were collected from 30 fresh manure patties within each feedlot pen at arrival processing, revaccination at reimplanting, and within 2 wk of slaughter. The mean pen prevalence of E. coli O157:H7 in feces was 5.0%; ranging in pens from 0% to 90%, and varying signif icantly (P < 0.001) among feedlots. There was no signif icant association (P > 0.20) between vaccination and pen prevalence of fecal E. coli O157:H7 following initial vaccination, at reimplanting, or prior to slaughter. PMID:16187717

Characterization of Diarrheagenic Escherichia coli Isolated in Organic Waste Products (Cattle Fecal Matter, Manure and, Slurry) from Cattle’s Markets in Ouagadougou, Burkina Faso

PubMed Central

Bako, Evariste; Kagambèga, Assèta; Traore, Kuan Abdoulaye; Bagre, Touwendsida Serge; Ibrahim, Hadiza Bawa; Bouda, Soutongnooma Caroline; Bonkoungou, Isidore Juste Ouindgueta; Kaboré, Saidou; Zongo, Cheikna; Traore, Alfred Sababenejo; Barro, Nicolas

2017-01-01

Cattle farming can promote diarrheal disease transmission through waste, effluents or cattle fecal matter. The study aims to characterize the diarrheagenic Escherichia coli (DEC) isolated from cattle feces, manure in the composting process and slurry, collected from four cattle markets in Ouagadougou. A total of 585 samples (340 cattle feces, 200 slurries and 45 manures in the composting process) were collected from the four cattle markets between May 2015 and May 2016. A multiplex Polymerase Chain Reaction (PCR), namely 16-plex PCR, was used to screen simultaneously the virulence genes specific for shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and enteroaggregative E. coli (EAEC). DEC was detected in 10.76% of samples. ETEC was the most prevalent (9.91%). STEC and EAEC have been observed with the same rate (0.51%). ETEC were detected in 12.64% of cattle feces, in 6.66% of manure in the composting process and in 5% of slurry. STEC were detected in 0.58% of cattle feces and in 2.22% of manure in the composting process. EAEC was detected only in 1% of slurry and in 2.22% of manure in the composting process. ETEC strains were identified based on estIa gene and/or estIb gene and/or elt gene amplification. Of the 58 ETEC, 10.34% contained astA, 17.24% contained elt, 3.44% contained estIa and 79.31% contained estIb. The two positive EAEC strains contained only the aggR gene, and the third was positive only for the pic gene. The results show that effluent from cattle markets could contribute to the spreading of DEC in the environment in Burkina Faso. PMID:28937656

Quantitative transfer of Escherichia coli O157:H7 to equipment during small-scale production of fresh-cut leafy greens.

PubMed

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

2012-07-01

Postharvest contamination and subsequent spread of Escherichia coli O157:H7 can occur during shredding, conveying, fluming, and dewatering of fresh-cut leafy greens. This study quantified E. coli O157:H7 transfer from leafy greens to equipment surfaces during simulated small-scale commercial processing. Three to five batches (22.7 kg) of baby spinach, iceberg lettuce, and romaine lettuce were dip inoculated with a four-strain cocktail of avirulent, green fluorescent protein-labeled, ampicillinresistant E. coli O157:H7 to contain ∼10(6), 10(4), and 10(2) CFU/g, and then were processed after 1 h of draining at ∼23°C or 24 h of storage at 4°C. Lettuce was shredded using an Urschel TransSlicer at two different blade and belt speeds to obtain normal (5 by 5 cm) and more finely shredded (0.5 by 5 cm) lettuce. Thereafter, the lettuce was step conveyed to a flume tank and was washed and then dried using a shaker table and centrifugal dryer. Product (25-g) and water (40-ml) samples were collected at various points during processing. After processing, product contact surfaces (100 cm(2)) on the shredder (n = 14), conveyer (n = 8), flume tank (n = 11), shaker table (n = 9), and centrifugal dryer (n = 8) were sampled using one-ply composite tissues. Sample homogenates diluted in phosphate or neutralizing buffer were plated, with or without prior 0.45- m m membrane filtration, on Trypticase soy agar containing 0.6% yeast extract supplemented with 100 ppm of ampicillin to quantify green fluorescent protein-labeled E. coli O157:H7 under UV light. During leafy green processing, ∼90% of the E. coli O157:H7 inoculum transferred to the wash water. After processing, E. coli O157:H7 populations were highest on the conveyor and shredder (P<0.05), followed by the centrifugal dryer, flume tank, and shaker table, with ∼29% of the remaining product inoculum lost during centrifugal drying. Overall, less (P<0.05) of the inoculum remained on the product after centrifugally drying iceberg lettuce that was held for 1 h (8.13%) as opposed to 24 h (42.18%) before processing, with shred size not affecting the rate of E. coli O157:H7 transfer.

Differential growth of wrinkled biofilms

NASA Astrophysics Data System (ADS)

Espeso, D. R.; Carpio, A.; Einarsson, B.

2015-02-01

Biofilms are antibiotic-resistant bacterial aggregates that grow on moist surfaces and can trigger hospital-acquired infections. They provide a classical example in biology where the dynamics of cellular communities may be observed and studied. Gene expression regulates cell division and differentiation, which affect the biofilm architecture. Mechanical and chemical processes shape the resulting structure. We gain insight into the interplay between cellular and mechanical processes during biofilm development on air-agar interfaces by means of a hybrid model. Cellular behavior is governed by stochastic rules informed by a cascade of concentration fields for nutrients, waste, and autoinducers. Cellular differentiation and death alter the structure and the mechanical properties of the biofilm, which is deformed according to Föppl-Von Kármán equations informed by cellular processes and the interaction with the substratum. Stiffness gradients due to growth and swelling produce wrinkle branching. We are able to reproduce wrinkled structures often formed by biofilms on air-agar interfaces, as well as spatial distributions of differentiated cells commonly observed with B. subtilis.

Prevalence and relatedness of Escherichia coli O157:H7 strains in the feces and on the hides and carcasses of U.S. meat goats at slaughter.

PubMed

Jacob, M E; Foster, D M; Rogers, A T; Balcomb, C C; Sanderson, M W

2013-07-01

We determined the prevalences of Escherichia coli O157:H7 in feces, hide, and carcasses of meat goats at a U.S. processing plant. Prevalences were 11.1%, 2.7%, and 2.7%, respectively. Sixteen pulsed-field gel electrophoresis (PFGE) subtypes were identified among 49 E. coli O157:H7 isolates, some of which were present on multiple sample types or collection days.

Prevalence and Relatedness of Escherichia coli O157:H7 Strains in the Feces and on the Hides and Carcasses of U.S. Meat Goats at Slaughter

PubMed Central

Foster, D. M.; Rogers, A. T.; Balcomb, C. C.; Sanderson, M. W.

2013-01-01

We determined the prevalences of Escherichia coli O157:H7 in feces, hide, and carcasses of meat goats at a U.S. processing plant. Prevalences were 11.1%, 2.7%, and 2.7%, respectively. Sixteen pulsed-field gel electrophoresis (PFGE) subtypes were identified among 49 E. coli O157:H7 isolates, some of which were present on multiple sample types or collection days. PMID:23584770

[Nitrogen oxide is involved in the regulation of the Fe-S cluster assembly in proteins and the formation of biofilms by Escherichia coli cells].

PubMed

Vasil'eva, S V; Streltsova, D A; Starostina, I A; Sanina, N A

2013-01-01

The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants deltaiscA and deltasufA, and the double paralog mutant deltaiscA/sufA with deletions in the alternative pathways of Fe2+ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe2+ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CE Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.

Studies on occurrence, characterisation and decontamination of emerging pathogenic Escherichia coli (STEC, ETEC and EIEC) in table eggs.

PubMed

Vinayananda, C O; Fairoze, Nadeem; Madhavaprasad, C B; Byregowda, S M; Nagaraj, C S; Bagalkot, Prashanth; Karabasanavar, Nagappa

2017-12-01

1. Escherichia coli is one of the most common facultative anaerobic species present in the gastrointestinal tract of animals and human beings. Usually they occur as commensals, but some serotypes can cause significant illnesses in humans as well as mammals and birds. 2. The occurrence of E. coli in different categories of table eggs collected from markets was evaluated. Isolates were analysed for the presence of virulence genes, antibiotic susceptibility pattern and efficacy of peracetic acid and chlorine for the purpose of decontaminating table eggs. 3. Significant differences were observed in the occurrence of E. coli between different groups viz. processed (cleaned, washed, sanitised and packed eggs), unprocessed (un-cleaned, un-sanitised and loose eggs) and free range (eggs obtained from backyard poultry) table eggs. Overall, E. coli occurred in table eggs at 28.6% with 22.9, 29.2 and 50.0% occurrence in processed, unprocessed and free-range table eggs, respectively. 4. A total of 24 isolates of E. coli were obtained and screened for virulence genes viz. STH, SLT1/2 and INVE genes. Of the 24 isolates recovered, 10 typeable isolates belonged to O141, O119, O9, O120 and O101 serotypes, while the remaining 14 were untypeable. Antibiograms of the isolates showed multiple antimicrobial resistance (MAR) index in the range of 0.13-0.40. 5. Peracetic acid (PAA) and chlorine (CL) were studied for their sanitisation efficacy; concentrations of 100 mg/kg of PAA and 200 mg/kg of CL completely inactivated E. coli over the egg surface and also resulted in 2.58 and 2.38 log reduction in total viable counts (TVC), respectively. 6. The presence of virulence-associated shiga-like toxin (SLT1/2) and invasion E (INVE) genes and antimicrobial resistance among the emerging serotypes of pathogenic E. coli isolated from table eggs has public health implications. It underscores the need to implement better management practices across the production systems and marketing channels to produce E. coli-free wholesome eggs for consumers.

Inhibition of biofilm development and spoilage potential of Shewanella baltica by quorum sensing signal in cell-free supernatant from Pseudomonas fluorescens.

PubMed

Zhao, Aifei; Zhu, Junli; Ye, Xiaofeng; Ge, Yangyang; Li, Jianrong

2016-08-02

The objective of this study was to in vitro evaluate the effect of a cell-free supernatant (CFS) containing quorum sensing (QS) signal of Pseudomonas fluorescens on the growth, biofilm development and spoilage potential of Shewanella baltica, and preliminarily assess the interactive influences of various chemically synthesized autoinducers on spoilage phenotypes of S. baltica. PF01 strain isolated from spoiled Pseudosciaen crocea was identified P. fluorescens. The addition of 25% and 50% CFS to S. baltica culture had no effect on the growth rate during the lag and exponential phase, however, caused cell decline during the stationary phase. The presence of CFS from P. fluorescens significantly inhibited biofilm development, and greatly decreased the production of trimethylamine (TMA) and biogenic amino in S. baltica. Various signal molecules of QS in the CFS of P. fluorescens culture were detected, including seven N-acyl-l-homoserine lactones (AHLs), autoinducer-2 (AI-2) and two diketopiperazines (DKPs). Exogenous supplement of synthesized seven AHLs containing in the CFS decreased biofilm formation and TMA production in S. baltica, while exposure to exogenous cyclo-(l-Pro-l-Leu) was showed to promote spoilage potential, which revealed that S. baltica also sense the two QS molecules. Furthermore, the stimulating effect of cyclo-(l-Pro-l-Leu) was affected when AHL was simultaneously added, suggesting that the inhibitory activity of spoilage phenotypes in S. baltica might be attributed to a competitive effect of these QS compounds in the CFS of P. fluorescens. The present studies provide a good basis for future research on the role of QS in the regulation of spoilage microbial flora. Copyright © 2016 Elsevier B.V. All rights reserved.

Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production.

PubMed

Yuan, Yongbo; Bi, Changhao; Nicolaou, Sergios A; Zingaro, Kyle A; Ralston, Matthew; Papoutsakis, Eleftherios T

2014-10-01

A major challenge in producing chemicals and biofuels is to increase the tolerance of the host organism to toxic products or byproducts. An Escherichia coli strain with superior ethanol and more generally alcohol tolerance was identified by screening a library constructed by randomly integrating Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome via Cre-lox recombination. Sequencing identified the inserted DNA fragment as the murA2 gene and its upstream intergenic 973-bp sequence, both coded on the negative genomic DNA strand. Overexpression of this murA2 gene and its upstream 973-bp sequence significantly enhanced ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 strains by 4.1-fold and 2.0-fold compared to control strains, respectively. Tolerance to n-butanol and i-butanol in E. coli MG1655 was increased by 1.85-fold and 1.91-fold, respectively. We show that the intergenic 973-bp sequence contains a native promoter for the murA2 gene along with a long 5' UTR (286 nt) on the negative strand, while a noncoding, small RNA, named MurA2S, is expressed off the positive strand. MurA2S is expressed in E. coli and may interact with murA2, but it does not affect murA2's ability to enhance alcohol tolerance in E. coli. Overexpression of murA2 with its upstream region in the ethanologenic E. coli KO11 strain significantly improved ethanol production in cultures that simulate the industrial Melle-Boinot fermentation process.

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. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

Salmonella, Escherichia coli and Enterobacteriaceae in the peanut supply chain: From farm to table.

PubMed

Nascimento, M S; Carminati, J A; Silva, I C R N; Silva, D L; Bernardi, A O; Copetti, M V

2018-03-01

Due to recent foodborne outbreaks, peanuts have been considered a potential risk for Salmonella transmission. For this reason, the aim of this study was to determine the prevalence and contamination load of Salmonella, Escherichia coli and Enterobacteriaceae throughout the peanut supply chain in Brazil. Samples of peanuts and peanut-containing processed products from post-harvest (n=129), secondary processing (n=185) and retail market (n=100) were analyzed. The results showed high Enterobacteriaceae counts in the post-harvest samples. At the end of the secondary processing, 16% of the samples remained contaminated by this group of microorganisms. Six peanut samples from primary production and one sample of peanut butter were tested positive for E. coli while Salmonella was detected in nine samples (2.2%): six from post-harvest, two from the initial stage of the secondary processing and one from retail. The Salmonella counts ranged between 0.004 and 0.092MPN/g and five serotypes were identified (Muenster, Miami, Javiana, Oranienburg, Glostrup). The results demonstrated a high prevalence of Enterobacteriaceae and low prevalence of E. coli throughout the peanut supply chain. Furthermore, it was verified that peanuts may become contaminated by Salmonella during different stages of the supply chain, especially at post-harvest. Copyright © 2017 Elsevier Ltd. All rights reserved.

In Helicobacter pylori auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration, regulates motility through modulation of flagellar gene transcription

PubMed Central

2010-01-01

Background LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only. Results We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway) does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the ΔluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2). In contrast, exogenously added cysteine could not restore motility to the ΔluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the ΔluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the ΔluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the ΔluxSHp mutant restored flagellar gene transcription, and the number and length of flagella. Conclusions Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2. PMID:20691071

Verification of the hygienic adequacy of beef carcass cooling processes by microbiological culture and the temperature-function integration technique.

PubMed

Jericho, K W; O'Laney, G; Kozub, G C

1998-10-01

To enhance food safety and keeping quality, beef carcasses are cooled immediately after leaving the slaughter floor. Within hazard analysis and critical control point (HACCP) systems, this cooling process needs to be monitored by the industry and verified by regulatory agencies. This study assessed the usefulness of the temperature-function integration technique (TFIT) for the verification of the hygienic adequacy of two cooling processes for beef carcasses at one abattoir. The cooling process passes carcasses through a spray cooler for at least 17 h and a holding cooler for at least 7 h. The TFIT is faster and cheaper than culture methods. For spray cooler 1, the Escherichia coli generations predicted by TFIT for carcass surfaces (pelvic and shank sites) were compared to estimated E. coli counts from 120 surface excision samples (rump, brisket, and sacrum; 5 by 5 by 0.2 cm) before and after cooling. Counts of aerobic bacteria, coliforms, and E. coli were decreased after spray cooler 1 (P < or = 0.001). The number of E. coli generations (with lag) at the pelvic site calculated by TFIT averaged 0.85 +/- 0.19 and 0.15 +/- 0.04 after emerging from spray coolers 1 and 2, respectively. The TFIT (with lag) was considered convenient and appropriate for the inspection service to verify HACCP systems for carcass cooling processes.

Seasonal prevalence of Shiga toxin-producing Escherichia coli, including O157:H7 and non-O157 serotypes, and Salmonella in commercial beef processing plants.

PubMed

Barkocy-Gallagher, Genevieve A; Arthur, Terrance M; Rivera-Betancourt, Mildred; Nou, Xiangwu; Shackelford, Steven D; Wheeler, Tommy L; Koohmaraie, Mohammad

2003-11-01

The seasonal prevalence of Escherichia coli O157:H7, Salmonella, non-O157 E. coli (STEC), and stx-harboring cells was monitored at three Midwestern fed-beef processing plants. Overall, E. coli O157:H7 was recovered from 5.9% of fecal samples, 60.6% of hide samples, and 26.7% of carcasses sampled before the preevisceration wash. This pathogen also was recovered from 1.2% (15 of 1,232) of carcasses sampled at chilling (postintervention) at approximate levels of <3.0 cells per 100 cm2. In one case, the E. coli O157:H7 concentration dropped from ca. 1,100 cells per 320 cm2 at the preevisceration stage to a level that was undetectable on ca. 2,500 cm2 at the postintervention stage. The prevalence of E. coli O157:H7 in feces peaked in the summer, whereas its prevalence on hide was high from the spring through the fall. Overall, Salmonella was recovered from 4.4, 71.0, and 12.7% of fecal, hide, and preevisceration carcass samples, respectively. Salmonella was recovered from one postintervention carcass (of 1,016 sampled). Salmonella prevalence peaked in feces in the summer and was highest on hide and preevisceration carcasses in the summer and the fall. Non-O157 STEC prevalence also appeared to vary by season, but the efficiency in the recovery of isolates from stx-positive samples ranged from 37.5 to 83.8% and could have influenced these results. Cells harboring stx genes were detected by PCR in 34.3, 92.0, 96.6, and 16.2% of fecal, hide, preevisceration carcass, and postintervention carcass samples, respectively. The approximate level of non-O157 STEC and stx-harboring cells on postintervention carcasses was > or = 3.0 cells per 100 cm2 for only 8 of 199 carcasses (4.0%). Overall, the prevalence of E. coli O157:H7, Salmonella, and non-O157 STEC varied by season, was higher on hides than in feces, and decreased dramatically, along with pathogen levels, during processing and during the application of antimicrobial interventions. These results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.

Periplasmic expression of human interferon-alpha 2c in Escherichia coli results in a correctly folded molecule.

PubMed Central

Voss, T; Falkner, E; Ahorn, H; Krystek, E; Maurer-Fogy, I; Bodo, G; Hauptmann, R

1994-01-01

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine. Images Figure 3 Figure 4 Figure 6 PMID:8141788

Reprint of Design of synthetic microbial communities for biotechnological production processes.

PubMed

Jagmann, Nina; Philipp, Bodo

2014-12-20

In their natural habitats microorganisms live in multi-species communities, in which the community members exhibit complex metabolic interactions. In contrast, biotechnological production processes catalyzed by microorganisms are usually carried out with single strains in pure cultures. A number of production processes, however, may be more efficiently catalyzed by the concerted action of microbial communities. This review will give an overview of organismic interactions between microbial cells and of biotechnological applications of microbial communities. It focuses on synthetic microbial communities that consist of microorganisms that have been genetically engineered. Design principles for such synthetic communities will be exemplified based on plausible scenarios for biotechnological production processes. These design principles comprise interspecific metabolic interactions via cross-feeding, regulation by interspecific signaling processes via metabolites and autoinducing signal molecules, and spatial structuring of synthetic microbial communities. In particular, the implementation of metabolic interdependencies, of positive feedback regulation and of inducible cell aggregation and biofilm formation will be outlined. Synthetic microbial communities constitute a viable extension of the biotechnological application of metabolically engineered single strains and enlarge the scope of microbial production processes. Copyright © 2014 Elsevier B.V. All rights reserved.

The impact of fecal sample processing on prevalence estimates for antibiotic-resistant Escherichia coli.

PubMed

Omulo, Sylvia; Lofgren, Eric T; Mugoh, Maina; Alando, Moshe; Obiya, Joshua; Kipyegon, Korir; Kikwai, Gilbert; Gumbi, Wilson; Kariuki, Samuel; Call, Douglas R

2017-05-01

Investigators often rely on studies of Escherichia coli to characterize the burden of antibiotic resistance in a clinical or community setting. To determine if prevalence estimates for antibiotic resistance are sensitive to sample handling and interpretive criteria, we collected presumptive E. coli isolates (24 or 95 per stool sample) from a community in an urban informal settlement in Kenya. Isolates were tested for susceptibility to nine antibiotics using agar breakpoint assays and results were analyzed using generalized linear mixed models. We observed a <3-fold difference between prevalence estimates based on freshly isolated bacteria when compared to isolates collected from unprocessed fecal samples or fecal slurries that had been stored at 4°C for up to 7days. No time-dependence was evident (P>0.1). Prevalence estimates did not differ for five distinct E. coli colony morphologies on MacConkey agar plates (P>0.2). Successive re-plating of samples for up to five consecutive days had little to no impact on prevalence estimates. Finally, culturing E. coli under different conditions (with 5% CO 2 or micro-aerobic) did not affect estimates of prevalence. For the conditions tested in these experiments, minor modifications in sample processing protocols are unlikely to bias estimates of the prevalence of antibiotic-resistance for fecal E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

The absence of the N-acyl-homoserine-lactone autoinducer synthase genes tral and ngrl increases the copy number of the symbiotic plasmid in Sinorhizobium fredii NGR234

USDA-ARS?s Scientific Manuscript database

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be...

Modeling the inactivatin of Escherichia coli 0157:H7 and uropathogenic E. coli in ground beef by high pressure processing and citral

USDA-ARS?s Scientific Manuscript database

Disease causing Escherichia coli commonly found in meat and poultry include intestinal pathogenic E. coli (iPEC) as well as extraintestinal types such as the Uropathogenic E. coli (UPEC). In this study we compared the resistance of iPEC (O157:H7) to UPEC in ground beef using High Pressure Processing...

Modeling the inactivation of Escherichia coli 0157:H7 and uropathogenic E.coli in ground chicken by high pressure processing and thymol

USDA-ARS?s Scientific Manuscript database

Disease causing Escherichia coli commonly found in meat and poultry include intestinal pathogenic E. coli (iPEC) as well as extraintestinal types such as the Uropathogenic E. coli (UPEC). In this study we compare the resistance of iPEC (O157:H7) to UPEC in chicken meat using High Pressure Processing...

Efficacy of chlorine dioxide on Escherichia coli inactivation during pilot-scale fresh-cut lettuce processing.

PubMed

Banach, J L; van Overbeek, L S; Nierop Groot, M N; van der Zouwen, P S; van der Fels-Klerx, H J

2018-03-23

Controlling water quality is critical in preventing cross-contamination during fresh produce washing. Process wash water (PWW) quality can be controlled by implementing chemical disinfection strategies. The aim of this study was to evaluate the pilot-scale efficacy of chlorine dioxide (ClO 2 ) during processing on the reduction of Escherichia coli in the PWW and on processed fresh-cut 'Lollo Rossa' lettuce. The objective was to have a residual target concentration of either 5 or 3 mg/L ClO 2 in the washing tank (3.5 m 3 ) before and during 800 kg of lettuce processing (90 min). After 90 min., a nonpathogenic, non-Extended Spectrum Beta-Lactamase (ESBL) E. coli inoculum from an overnight culture broth (37 °C) was added to the tank resulting in an approximate final level of 10 6  CFU/mL. PWW and lettuce samples for microbiological and chemical analyses were taken before and after the input and supply halted. ClO 2 concentrations quickly decreased after ClO 2 input halted, yet a residual concentration of ≥2.5 mg/L and ≥2.1 mg/L ClO 2 , respectively for 5 and 3 mg/L pilots, was present 12 min after the supply halted. No detectable levels of E. coli (limit of detection 5 log) were determined in the water within 1 min after E. coli was added to the ClO 2 containing wash water. Results demonstrated that ClO 2 use at the semi-commercial pilot scale was able to reduce the E. coli peak contamination in the PWW. After storage (5 days, 4 °C), background microbial communities (i.e., fluorescent Pseudomonads and total heterotrophic bacteria) grew out on lettuce. Overall, ClO 2 decreased the potential for cross-contamination between batches compared to when no sanitizer was used. Chlorate levels of the lettuce sampled before entering the wash water ranged from 7.3-11.6 μg/kg. The chlorate levels of the lettuce sampled after being washed in the ClO 2 containing wash water, as well as after rinsing and centrifugation, ranged from 22.8-60.4 μg/kg; chlorite levels ranged from 1.3-1.6 mg/kg, while perchlorate levels were below the limit of quantification (LOQ, <5 ng/g). In this study, we report the semi-commercial pilot-scale evaluation of ClO 2 , for its ability to maintain the PWW quality and to prevent cross-contamination in the washing tank during fresh-cut lettuce processing. Furthermore, we provide quantitative values of ClO 2 disinfection by-products chlorate and chlorite as well as of perchlorate from PWW and/or lettuce samples. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Pseudomonas aeruginosa Promotes Escherichia coli Biofilm Formation in Nutrient-Limited Medium

PubMed Central

Culotti, Alessandro; Packman, Aaron I.

2014-01-01

Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions. PMID:25198725

Autoinduced catalysis and inverse equilibrium isotope effect in the frustrated Lewis pair catalyzed hydrogenation of imines.

PubMed

Tussing, Sebastian; Greb, Lutz; Tamke, Sergej; Schirmer, Birgitta; Muhle-Goll, Claudia; Luy, Burkhard; Paradies, Jan

2015-05-26

The frustrated Lewis pair (FLP)-catalyzed hydrogenation and deuteration of N-benzylidene-tert-butylamine (2) was kinetically investigated by using the three boranes B(C6F5)3 (1), B(2,4,6-F3-C6H2)3 (4), and B(2,6-F2-C6H3)3 (5) and the free activation energies for the H2 activation by FLP were determined. Reactions catalyzed by the weaker Lewis acids 4 and 5 displayed autoinductive catalysis arising from a higher free activation energy (2 kcal mol(-1)) for the H2 activation by the imine compared to the amine. Surprisingly, the imine reduction using D2 proceeded with higher rates. This phenomenon is unprecedented for FLP and resulted from a primary inverse equilibrium isotope effect. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

PubMed

Anselmetti, Dario; Bartels, Frank Wilco; Becker, Anke; Decker, Björn; Eckel, Rainer; McIntosh, Matthew; Mattay, Jochen; Plattner, Patrik; Ros, Robert; Schäfer, Christian; Sewald, Norbert

2008-02-19

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

An improved green synthesis method and Escherichia coli antibacterial activity of silver nanoparticles.

PubMed

Van Viet, Pham; Sang, Truong Tan; Bich, Nguyen Ho Ngoc; Thi, Cao Minh

2018-05-01

Silver nanoparticles (Ag NPs) were synthesized by an improved green synthesis method via a photo-reduction process using low-power UV light in the presence of poly (vinyl pyrrolidone) (PVP) as the surface stabilizer. The effective synthesis process was achieved by optimized synthesis parameters such as C 2 H 5 OH: H 2 O ratio, AgNO 3 : PVP ratio, pH value, and reducing time. The formation of Ag NPs was identified by Ultraviolet-visible (UV-vis) absorption spectra, X-ray diffraction pattern (XRD) and Fourier transform infrared spectroscopy (FTIR) spectra. Ag NPs were crystallized according to (111), (200), and (220) planes of the face-centered cubic. The transmission electron microscopy (TEM) image showed that the morphology of Ag NPs was uniform spherical with the average particle size of 16 ± 2 nm. The results of XRD pattern, TEM image, and dynamic light scattering (DLS) analysis proved that Ag crystals with uniform size were formed after the reduction process. The mechanism of the formation of Ag NPs was proposed and confirmed by FTIR spectra. The antibacterial activity of Ag NPs against Escherichia coli (E. coli) was tested and approximately 100% of E. coli was eliminated by Ag NPs 35 ppm. In the future, this study can become a new process for the application of Ag NPs as an antibiotic in the industrial scale. Copyright © 2018 Elsevier B.V. All rights reserved.

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

PubMed Central

Agga, Getahun E.; Bosilevac, Joseph M.; Brichta-Harhay, Dayna M.; Shackelford, Steven D.; Wang, Rong; Wheeler, Tommy L.; Arthur, Terrance M.

2014-01-01

Specific concerns have been raised that third-generation cephalosporin-resistant (3GCr) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr) E. coli, 3GCr Salmonella enterica, and nalidixic acid-resistant (NALr) S. enterica may be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n = 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCr Salmonella was detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALr S. enterica was detected on only one hide. 3GCr E. coli and COTr E. coli were detected on 100.0% of hides during processing. Concentrations of 3GCr E. coli and COTr E. coli on hides were correlated with pre-evisceration carcass contamination. 3GCr E. coli and COTr E. coli were each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenic E. coli (ExPEC) virulence-associated markers. Only two COTr E. coli isolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria. PMID:25398858

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum.

PubMed

Schmidt, John W; Agga, Getahun E; Bosilevac, Joseph M; Brichta-Harhay, Dayna M; Shackelford, Steven D; Wang, Rong; Wheeler, Tommy L; Arthur, Terrance M

2015-01-01

Specific concerns have been raised that third-generation cephalosporin-resistant (3GC(r)) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COT(r)) E. coli, 3GC(r) Salmonella enterica, and nalidixic acid-resistant (NAL(r)) S. enterica may be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n = 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GC(r) Salmonella was detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NAL(r) S. enterica was detected on only one hide. 3GC(r) E. coli and COT(r) E. coli were detected on 100.0% of hides during processing. Concentrations of 3GC(r) E. coli and COT(r) E. coli on hides were correlated with pre-evisceration carcass contamination. 3GC(r) E. coli and COT(r) E. coli were each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenic E. coli (ExPEC) virulence-associated markers. Only two COT(r) E. coli isolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria.

PubMed

Scarascia, Giantommaso; Wang, Tiannyu; Hong, Pei-Ying

2016-12-15

Sulfate-reducing bacteria (SRB) are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS) in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach.

Disinfection of Multidrug Resistant Escherichia coli by Solar-Photocatalysis using Fe-doped ZnO Nanoparticles.

PubMed

Das, Sourav; Sinha, Sayantan; Das, Bhaskar; Jayabalan, R; Suar, Mrutyunjay; Mishra, Amrita; Tamhankar, Ashok J; Stålsby Lundborg, Cecilia; Tripathy, Suraj K

2017-03-07

Spread of antibiotic resistant bacteria through water, is a threat to global public health. Here, we report Fe-doped ZnO nanoparticles (Fe/ZnO NPs) based solar-photocatalytic disinfection (PCD) of multidrug resistant Escherichia coli (MDR E. coli). Fe/ZnO NPs were synthesized by chemical precipitation technique, and when used as photocatalyst for disinfection, proved to be more effective (time for complete disinfection = 90 min) than ZnO (150 min) and TiO 2 (180 min). Lipid peroxidation and potassium (K + ) ion leakage studies indicated compromisation of bacterial cell membrane and electron microscopy and live-dead staining confirmed the detrimental effects on membrane integrity. Investigations indicated that H 2 O 2 was the key species involved in solar-PCD of MDR E. coli by Fe/ZnO NPs. X-ray diffraction and atomic absorption spectroscopy studies showed that the Fe/ZnO NPs system remained stable during the photocatalytic process. The Fe/ZnO NPs based solar-PCD process proved successful in the disinfection of MDR E. coli in real water samples collected from river, pond and municipal tap. The Fe/ZnO NPs catalyst made from low cost materials and with high efficacy under solar light may have potential for real world applications, to help reduce the spread of resistant bacteria.

Survival and distribution of Escherichia coli on diverse fresh-cut baby leafy greens under preharvest through postharvest conditions.

PubMed

Tomás-Callejas, Alejandro; López-Velasco, Gabriela; Camacho, Alex B; Artés, Francisco; Artés-Hernández, Francisco; Suslow, Trevor V

2011-12-02

Escherichia coli O157:H7 has been associated in multiple outbreaks linked to the consumption of whole produce and fresh-cut leafy vegetables. However, plant-based foods had not been traditionally recognized as a host for enteric pathogens until the elevated incidence of produce-related outbreaks became apparent. The survival dynamics of two cocktails of generic E. coli (environmental water, plant and soil isolates) and E. coli O157:H7 within the phyllosphere of Mizuna, Red Chard and Tatsoi during their production, harvest, minimal processing, packaging and storage over two greenhouse production cycles were studied. Genotyping of applied generic E. coli strains to evaluate their comparative survival and relative abundance in the phyllosphere by REP-PCR is also reported. The Mizuna, Red Chard and Tatsoi shoots were grown under standard greenhouse conditions and fertility management. Both E. coli cocktails were spray-inoculated separately and determined to result in an initial mean population density of log 4.2 CFU/cm². Leaves were harvested as mini-greens approximating commercial maturity, minimally processed in a model washing system treated with 3 mg/L of ClO₂ and stored for 7 days at 5 °C. Rapid decline of generic E. coli and E. coli O157:H7 populations was observed for all plant types regardless of the leaf age at the time of inoculation and the irrigation type across both seasonal growth cycle trials. The decline rate of the surviving populations for the fall season was slower than for the summer season. The minimal processing with 3 mg/L of ClO₂ was not sufficient to fully disinfect the inoculated leaves prior to packaging and refrigerated storage. Viable populations of E. coli and E. coli O157:H7 were confirmed throughout storage, including the final time point at the end of acceptable visual leaf quality. In this study, the ability of low populations of E. coli to survive during production and postharvest operations in selected mini-greens has been demonstrated. However, further field-based trials are needed to expand understanding of the post-contamination fate of enteric bacterial pathogens on leafy vegetables. In summary, this research work provides baseline data upon which to develop food safety preventive control guidance during the production and minimal processing of these crops. Copyright © 2011 Elsevier B.V. All rights reserved.

Bacterial biofilms and quorum sensing: fidelity in bioremediation technology.

PubMed

Mangwani, Neelam; Kumari, Supriya; Das, Surajit

Increased contamination of the environment with toxic pollutants has paved the way for efficient strategies which can be implemented for environmental restoration. The major problem with conventional methods used for cleaning of pollutants is inefficiency and high economic costs. Bioremediation is a growing technology having advanced potential of cleaning pollutants. Biofilm formed by various micro-organisms potentially provide a suitable microenvironment for efficient bioremediation processes. High cell density and stress resistance properties of the biofilm environment provide opportunities for efficient metabolism of number of hydrophobic and toxic compounds. Bacterial biofilm formation is often regulated by quorum sensing (QS) which is a population density-based cell-cell communication process via signaling molecules. Numerous signaling molecules such as acyl homoserine lactones, peptides, autoinducer-2, diffusion signaling factors, and α-hydroxyketones have been studied in bacteria. Genetic alteration of QS machinery can be useful to modulate vital characters valuable for environmental applications such as biofilm formation, biosurfactant production, exopolysaccharide synthesis, horizontal gene transfer, catabolic gene expression, motility, and chemotaxis. These qualities are imperative for bacteria during degradation or detoxification of any pollutant. QS signals can be used for the fabrication of engineered biofilms with enhanced degradation kinetics. This review discusses the connection between QS and biofilm formation by bacteria in relation to bioremediation technology.

Persistence of Escherichia coli O157:H7 during pilot-scale processing of iceberg lettuce using flume water containing peroxyacetic acid-based sanitizers and various organic loads.

PubMed

Davidson, Gordon R; Kaminski-Davidson, Chelsea N; Ryser, Elliot T

2017-05-02

In order to minimize cross-contamination during leafy green processing, chemical sanitizers are routinely added to the wash water. This study assessed the efficacy of peroxyacetic acid and mixed peracid against E. coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line using flume water containing various organic loads. Iceberg lettuce (5.4kg) inoculated to contain 10 6 CFU/g of a 4-strain cocktail of non-toxigenic, GFP-labeled, ampicillin-resistant E. coli O157:H7, was shredded using a commercial shredder, step-conveyed to a flume tank, washed for 90s using water alone or two different sanitizing treatments (50ppm peroxyacetic acid or mixed peracid) in water containing organic loads of 0, 2.5, 5 or 10% (w/v) blended iceberg lettuce, and then dried using a shaker table and centrifugal dryer. Thereafter, three 5.4-kg batches of uninoculated iceberg lettuce were identically processed. Various product (25g) and water (50ml) samples collected during processing along with equipment surface samples (100cm 2 ) from the flume tank, shaker table and centrifugal dryer were then assessed for numbers of E. coli O157:H7. Organic load rarely impacted (P>0.05) the efficacy of either peroxyacetic acid or mixed peracid, with typical reductions of >5logCFU/ml in wash water throughout processing for all organic loads. Increases in organic load in the wash water corresponded to changes in total solids, chemical oxygen demand, turbidity, maximum filterable volume, and oxidation/reduction potential. After 90s of exposure to flume water, E. coli O157:H7 reductions on inoculated lettuce ranged from 0.97 to 1.74logCFU/g using peroxyacetic acid, with an average reduction of 1.35logCFU/g for mixed peracid. E. coli O157:H7 persisted on all previously uninoculated lettuce following the inoculated batch, emphasizing the need for improved intervention strategies that can better ensure end-product safety. Copyright © 2017 Elsevier B.V. All rights reserved.

Outbreak of fluoroquinolone-resistant Escherichia coli infections after transrectal ultrasound-guided biopsy of the prostate.

PubMed

Dumford, Donald; Suwantarat, Nuntra; Bhasker, Vineet; Kundrapu, Sirisha; Zabarsky, Trina F; Drawz, Paul; Zhu, Hui; Donskey, Curtis J

2013-03-01

We conducted an investigation after identifying a cluster of 4 serious infections following transrectal ultrasound-guided biopsy of the prostate (TRUBP) during a 2-month period. Veterans Affairs medical center. Patients with urinary tract infection (UTI) after TRUBP and time-matched controls with no evidence of infection. The incidence of UTI within 30 days after TRUBP was calculated from 2002 through 2010. We evaluated the correlation between infection with fluoroquinolone-resistant gram-negative bacilli (GNB) and fluoroquinolone resistance in outpatient Escherichia coli urinary isolates and performed a case-control study to determine risk factors for infection with fluoroquinolone-resistant GNB. Processes for TRUBP prophylaxis, procedures, and equipment sterilization were reviewed. An outbreak of UTI due to fluoroquinolone-resistant E. coli after TRUBP began 2 years before the cluster was identified and was correlated with increasing fluoroquinolone resistance in outpatient E. coli. No deficiencies were identified in equipment processing or biopsy procedures. Fluoroquinolone-resistant E. coli UTI after TRUBP was independently associated with prior infection with fluoroquinolone-resistant GNB (adjusted odds ratio, 20.8; P=.005). A prediction rule including prior UTI, hospitalization in the past year, and previous infection with fluoroquinolone-resistant GNB identified only 17 (49%) of 35 cases. The outbreak of fluoroquinolone-resistant E. coli infections after TRUBP closely paralleled rising rates of fluoroquinolone resistance among outpatient E. coli isolates. The delayed detection of the outbreak and the absence of sensitive predictors of infection suggest that active surveillance for infection after TRUBP is necessary in the context of increasing fluoroquinolone resistance in the United States.

Engineering Escherichia coli K12 MG1655 to use starch

PubMed Central

2014-01-01

Background To attain a sustainable bioeconomy, fuel, or valuable product, production must use biomass as substrate. Starch is one of the most abundant biomass resources and is present as waste or as a food and agroindustry by-product. Unfortunately, Escherichia coli, one of the most widely used microorganisms in biotechnological processes, cannot use starch as a carbon source. Results We engineered an E. coli strain capable of using starch as a substrate. The genetic design employed the native capability of the bacterium to use maltodextrins as a carbon source plus expression and secretion of its endogenous α-amylase, AmyA, in an adapted background. Biomass production improved using 35% dissolved oxygen and pH 7.2 in a controlled bioreactor. Conclusion The engineered E. coli strain can use starch from the milieu and open the possibility of optimize the process to use agroindustrial wastes to produce biofuels and other valuable chemicals. PMID:24886307

Candida krusei isolated from fruit juices ultrafiltration membranes promotes colonization of Escherichia coli O157:H7 and Salmonella enterica on stainless steel surfaces.

PubMed

Tarifa, María Clara; Lozano, Jorge Enrique; Brugnoni, Lorena Inés

2017-02-01

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm 2 , respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm 2 and 0.55 log CFU cm 2 when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm 2 , respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.

Effect of the luxS gene on biofilm formation and antibiotic resistance by Salmonella serovar Dublin.

PubMed

Ju, Xiangyu; Li, Junjie; Zhu, Mengjiao; Lu, Zhaoxin; Lv, Fengxia; Zhu, Xiaoyu; Bie, Xiaomei

2018-05-01

Biofilms are communities of bacterial cells that serve to protect them from external adverse influences and enhance bacterial resistance to antibiotics and sanitizers. Here, we studied the regulatory effects of glucose and sodium chloride on biofilm formation in Salmonella serovar Dublin (S. Dublin). To analyze expression levels of the quorum sensing gene luxS, we created a luxS knockout mutant. Also, antimicrobial resistance, hydrophobicity and autoinducer-2 (AI-2) activity of both the wild-type (WT) and the mutant strain were investigated. Our results revealed that glucose was not essential for S. Dublin biofilm formation but had an inhibitory effect on biofilm formation when the concentration was over 0.1%. NaCl was found to be indispensable in forming biofilm, and it also exerted an inhibitory effect at high concentrations (>1.0%). Both the WT and the mutant strains displayed significant MIC growth after biofilm formation. An increase of up to 32,768 times in the resistance of S. Dublin in biofilm phonotype against antibiotic (ampicillin) compared to its planktonic phonotype was observed. However, S. Dublin luxS knockout mutant only showed slight differences compared to the WT strain in the antimicrobial tests although it displayed better biofilm-forming capacity than the WT strain. The mutant strain also exhibited higher hydrophobicity than the WT strain, which was a feature related to biofilm formation. The production of the quorum sensing autoinducer-2 (AI-2) was significantly lower in the mutant strain than in the WT strain since the LuxS enzyme, encoded by the luxS gene, plays an essential role in AI-2 synthesis. However, the limited biofilm-forming ability in the WT strain indicated AI-2 was not directly related to S. Dublin biofilm formation. Furthermore, gene expression analysis of the WT and mutant strains revealed upregulation of genes related to biofilm stress response and enhanced resistance in the luxS mutant strain, which may provide evidence for the regulatory role of the luxS gene in biofilm formation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Economic feasibility and environmental impact of synthetic spider silk production from escherichia coli.

PubMed

Edlund, Alan M; Jones, Justin; Lewis, Randolph; Quinn, Jason C

2018-05-25

Major ampullate spider silk represents a promising protein-based biomaterial with diverse commercial potential ranging from textiles to medical devices due to its excellent physical and thermal properties. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli). This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative downstream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants of $761 kg -1 and $23 kg -1 with greenhouse gas emissions of 572 kg CO 2-eq. kg -1 and 55 kg CO 2-eq. kg -1 , respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plant includes improved protein yield, process optimization, and an N th plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and the impact of alternative technologies on the system. Copyright © 2018 Elsevier B.V. All rights reserved.

Prevalence and concentration of Escherichia coli O157 in different seasons and cattle types processed in North America: A systematic review and meta-analysis of published research.

PubMed

Ekong, Pius S; Sanderson, Michael W; Cernicchiaro, Natalia

2015-09-01

Systematic review (SR) and meta-analyses (MA) methodologies were used to identify, critically evaluate and synthesize prevalence and concentration estimates for Escherichia coli O157 contamination along the beef production chain, and to illustrate differences based on cattle types and seasonality in North America from the scientific peer-reviewed literature. Four electronic databases were searched to identify relevant articles. Two independent reviewers performed all SR steps. Random effects MA models were used to estimate the pooled prevalence and concentration of E. coli O157 in feces, hides and carcasses of cattle processed in North America, including their seasonal estimates. The potential sources of between studies heterogeneity were identified using meta-regression and sub-group analysis. Results indicated differences in the fecal prevalence of E. coli O157 among cattle types: 10.68% (95% CI: 9.17-12.28%) in fed beef, 4.65% (95% CI: 3.37-6.10%) in adult beef, and 1.79% (95% CI: 1.20-2.48%) in adult dairy. Fed beef fecal prevalence was 10.65% (95% CI: 8.93-12.49%) during summer and 9.17% (95% CI: 5.24-13.98%) during the winter months. For adult beef, the fecal prevalence was 7.86% (95% CI: 5.43-10.66%) during summer, and 4.21% (95% CI: 1.95-7.13%) during winter. Among adult dairy, the fecal prevalence was 2.27% (95% CI: 1.5-3.18%) during summer, and 0.36% (95% CI: 0.09-0.74%) during winter. There was a significantly higher percentage of hides with E. coli O157 concentration ≥ 40 CFU/100 cm(2) on hides of fed beef sampled at the processing plant (23.81%; 95% CI: 14.79-34.15%) compared to those sampled at the feedlot (1.74%; 95% CI: 0.53-3.44%). Prevalence of E. coli O157 on carcass surfaces differed by season only at the post-evisceration stage, but decreased considerably through the subsequent processing stages. Country, study setting, detection method, hide swab area, and study design were identified as significant sources of heterogeneity among studies reporting prevalence of E. coli O157 along the beef production chain. The pooled prevalence and concentration estimates from this study provide a sound and reliable microbiological basis for risk assessment modeling of E. coli O157 and other pathogens in the food chain. Copyright © 2015 Elsevier B.V. All rights reserved.

High-frequency underwater plasma discharge application in antibacterial activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahmed, M. W.; Choi, S.; Lyakhov, K.

Plasma discharge is a novel disinfection and effectual inactivation approach to treat microorganisms in aqueous systems. Inactivation of Gram-negative Escherichia coli (E. coli) by generating high-frequency, high-voltage, oxygen (O{sub 2}) injected and hydrogen peroxide (H{sub 2}O{sub 2}) added discharge in water was achieved. The effect of H{sub 2}O{sub 2} dose and oxygen injection rate on electrical characteristics of discharge and E. coli disinfection has been reported. Microbial log reduction dependent on H{sub 2}O{sub 2} addition with O{sub 2} injection was observed. The time variation of the inactivation efficiency quantified by the log reduction of the initial E. coli population onmore » the basis of optical density measurement was reported. The analysis of emission spectrum recorded after discharge occurrence illustrated the formation of oxidant species (OH{sup •}, H, and O). Interestingly, the results demonstrated that O{sub 2} injected and H{sub 2}O{sub 2} added, underwater plasma discharge had fabulous impact on the E. coli sterilization. The oxygen injection notably reduced the voltage needed for generating breakdown in flowing water and escalated the power of discharge pulses. No impact of hydrogen peroxide addition on breakdown voltage was observed. A significant role of oxidant species in bacterial inactivation also has been identified. Furthermore the E. coli survivability in plasma treated water with oxygen injection and hydrogen peroxide addition drastically reduced to zero. The time course study also showed that the retardant effect on E. coli colony multiplication in plasma treated water was favorable, observed after long time. High-frequency underwater plasma discharge based biological applications is technically relevant and would act as baseline data for the development of novel antibacterial processing strategies.« less

High-frequency underwater plasma discharge application in antibacterial activity

NASA Astrophysics Data System (ADS)

Ahmed, M. W.; Choi, S.; Lyakhov, K.; Shaislamov, U.; Mongre, R. K.; Jeong, D. K.; Suresh, R.; Lee, H. J.

2017-03-01

Plasma discharge is a novel disinfection and effectual inactivation approach to treat microorganisms in aqueous systems. Inactivation of Gram-negative Escherichia coli ( E. coli) by generating high-frequency, high-voltage, oxygen (O2) injected and hydrogen peroxide (H2O2) added discharge in water was achieved. The effect of H2O2 dose and oxygen injection rate on electrical characteristics of discharge and E. coli disinfection has been reported. Microbial log reduction dependent on H2O2 addition with O2 injection was observed. The time variation of the inactivation efficiency quantified by the log reduction of the initial E. coli population on the basis of optical density measurement was reported. The analysis of emission spectrum recorded after discharge occurrence illustrated the formation of oxidant species (OH•, H, and O). Interestingly, the results demonstrated that O2 injected and H2O2 added, underwater plasma discharge had fabulous impact on the E. coli sterilization. The oxygen injection notably reduced the voltage needed for generating breakdown in flowing water and escalated the power of discharge pulses. No impact of hydrogen peroxide addition on breakdown voltage was observed. A significant role of oxidant species in bacterial inactivation also has been identified. Furthermore the E. coli survivability in plasma treated water with oxygen injection and hydrogen peroxide addition drastically reduced to zero. The time course study also showed that the retardant effect on E. coli colony multiplication in plasma treated water was favorable, observed after long time. High-frequency underwater plasma discharge based biological applications is technically relevant and would act as baseline data for the development of novel antibacterial processing strategies.

Assessment of microbial quality of fish processing industrial effluent in bar-mouth at Bhidia landing site, Veraval, Gujarat, India.

PubMed

Sivaraman, G K; Visnuvinayagam, S; Jha, Ashish Kumar; Renuka, V; Remya, S; Vanik, Deesha

2016-07-01

The present study was carried out to assess the microbial quality of fish processing industries effluent at Bhidia bar-mouth, Veraval, Gujarat during April, 2012 to March 2013. The total viable bacterial count (TVBC), total Enterobacteriaceae count, E. coli count (EC), Staphylococcus aureus and Fecal Streptococcal count in effluent ranged from 3.0 x 10(-1) to 6.8 x 10(6), 9.0 x 10(1) to 2.9 x 10(4), 0 to 0. 5 x 10(4), 0 to 0. 4 x 102 and 0.3 x 10(1) to 0. 1 x 10(4) cfu.(-1)respectively. Significantly higher load of TEC, E. coli, S.aureus, Fecal Streptococci, Total coliforms and Fecal coliforms were higher during summer whereas, TVBC was higher in the month of Sept.-Oct. Furthermore, the total coliform and fecal coliform counts were found to be higher with 1400+ /100 ml MPN value throughout the year of the study, except in the month of August. Overall occurrence of pathogenic strains of E. coli, S. aureus and Fecal streptococci were 41.67%, 25.00% and 66.67% respectively during this period. The antibiogram of the isolated E. coli isolates show that almost 50% were resistant to Cefazidime/Clavulanic acid (CAC), Amoxyclav (AMC), Ciprofloxacin (CIF) and Ampicillin (AMP). The present study indicated that the effluent of fish processing industry was heavily contaminated with E. coli, S. aureus and Fecal Streptococci which confirmed improper treatment of fish processing effluent. Moreover, the precedence of antibiotic resistant E. coli may pose threat to public health safety.

Bacterial bioluminescence onset and quenching: a dynamical model for a quorum sensing-mediated property

PubMed Central

Side, Domenico Delle; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Di Salvo, Marco; Talà, Adelfia; Chechkin, Aleksei; Seno, Flavio

2017-01-01

We present an effective dynamical model for the onset of bacterial bioluminescence, one of the most studied quorum sensing-mediated traits. Our model is built upon simple equations that describe the growth of the bacterial colony, the production and accumulation of autoinducer signal molecules, their sensing within bacterial cells, and the ensuing quorum activation mechanism that triggers bioluminescent emission. The model is directly tested to quantitatively reproduce the experimental distributions of photon emission times, previously measured for bacterial colonies of Vibrio jasicida, a luminescent bacterium belonging to the Harveyi clade, growing in a highly drying environment. A distinctive and novel feature of the proposed model is bioluminescence ‘quenching’ after a given time elapsed from activation. Using an advanced fitting procedure based on the simulated annealing algorithm, we are able to infer from the experimental observations the biochemical parameters used in the model. Such parameters are in good agreement with the literature data. As a further result, we find that, at least in our experimental conditions, light emission in bioluminescent bacteria appears to originate from a subtle balance between colony growth and quorum activation due to autoinducers diffusion, with the two phenomena occurring on the same time scale. This finding is consistent with a negative feedback mechanism previously reported for Vibrio harveyi. PMID:29308273

Bacterial bioluminescence onset and quenching: a dynamical model for a quorum sensing-mediated property.

PubMed

Side, Domenico Delle; Nassisi, Vincenzo; Pennetta, Cecilia; Alifano, Pietro; Di Salvo, Marco; Talà, Adelfia; Chechkin, Aleksei; Seno, Flavio; Trovato, Antonio

2017-12-01

We present an effective dynamical model for the onset of bacterial bioluminescence, one of the most studied quorum sensing-mediated traits. Our model is built upon simple equations that describe the growth of the bacterial colony, the production and accumulation of autoinducer signal molecules, their sensing within bacterial cells, and the ensuing quorum activation mechanism that triggers bioluminescent emission. The model is directly tested to quantitatively reproduce the experimental distributions of photon emission times, previously measured for bacterial colonies of Vibrio jasicida , a luminescent bacterium belonging to the Harveyi clade, growing in a highly drying environment. A distinctive and novel feature of the proposed model is bioluminescence 'quenching' after a given time elapsed from activation. Using an advanced fitting procedure based on the simulated annealing algorithm, we are able to infer from the experimental observations the biochemical parameters used in the model. Such parameters are in good agreement with the literature data. As a further result, we find that, at least in our experimental conditions, light emission in bioluminescent bacteria appears to originate from a subtle balance between colony growth and quorum activation due to autoinducers diffusion, with the two phenomena occurring on the same time scale. This finding is consistent with a negative feedback mechanism previously reported for Vibrio harveyi .

Structure And Specificity of a Quorum-Quenching Lactonase (AiiB) From Agrobacterium Tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, D.; Thomas, P.W.; Momb, J.

2009-06-03

N-Acyl-l-homoserine lactone (AHL) mediated quorum-sensing regulates virulence factor production in a variety of Gram-negative bacteria. Proteins capable of degrading these autoinducers have been called 'quorum-quenching' enzymes, can block many quorum-sensing dependent phenotypes, and represent potentially useful reagents for clinical, agricultural, and industrial applications. The most characterized quorum-quenching enzymes to date are the AHL lactonases, which are metalloproteins that belong to the metallo-beta-lactamase superfamily. Here, we report the cloning, heterologous expression, purification, metal content, substrate specificity, and three-dimensional structure of AiiB, an AHL lactonase from Agrobacterium tumefaciens. Much like a homologous AHL lactonase from Bacillus thuringiensis, AiiB appears to be amore » metal-dependent AHL lactonase with broad specificity. A phosphate dianion is bound to the dinuclear zinc site and the active-site structure suggests specific mechanistic roles for an active site tyrosine and aspartate. To our knowledge, this is the second representative structure of an AHL lactonase and the first of an AHL lactonase from a microorganism that also produces AHL autoinducers. This work should help elucidate the hydrolytic ring-opening mechanism of this family of enzymes and also facilitate the design of more effective quorum-quenching catalysts.« less

Prevalence of Escherichia coli O157:H7 and Salmonella in camels, cattle, goats, and sheep harvested for meat in Riyadh.

PubMed

Bosilevac, Joseph M; Gassem, Mustafa A; Al Sheddy, Ibraheem A; Almaiman, Salah A; Al-Mohizea, Ibrahim S; Alowaimer, Abdullah; Koohmaraie, Mohammad

2015-01-01

Escherichia coli O157:H7 and Salmonella are significant foodborne pathogens that can be found in the feces and on the hides of meat animals. When hides are removed during the harvest process, the carcass and subsequent meat products can become contaminated. Camels, cattle, sheep, and goats are harvested for meat in Riyadh, Saudi Arabia. The prevalence of E. coli O157:H7 and Salmonella are unknown in these animals, and it is assumed that if the animals carry the pathogens in their feces or on their hides, meat products are likely to become contaminated. To this end, a minimum of 206 samples each from hides and feces of camels, cattle, goats, and sheep were collected over the course of 8 months and tested for E. coli O157:H7 and Salmonella. It was found that E. coli O157:H7 was present in feces (10.7, 1.4, 2.4, and 2.4%) and on hides (17.9, 8.2, 2.9, and 9.2%) of cattle, goats, camels, and sheep, respectively. The prevalence of Salmonella was 11.2, 13.5, 23.2, and 18.8% in feces and 80.2, 51.2 67.6, and 60.2% on hides of cattle, goats, camels, and sheep, respectively. The prevalence of E coli O157:H7 was nearly zero in all samples collected in June and July, while Salmonella did not exhibit any seasonal variation. These results constitute the first comprehensive study of E. coli O157:H7 and Salmonella prevalence in Saudi Arabian meat animals at harvest.

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

PubMed

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

2010-03-01

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

The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing.

PubMed

Van Donkersgoed, J; Graham, T; Gannon, V

1999-05-01

Fecal samples collected from cattle at processing during a 1-year period were tested for verotoxins (VT1, VT2), Escherichia coli O157:H7, and Salmonella. Verotoxins were detected in 42.6% (95% CI, 39.8% to 45.4%), E. coli O157:H7 in 7.5% (95% CI, 6.1% to 9.1%), and Salmonella in 0.08% (95% CI, 0.004% to 0.5%) of the fecal samples. In yearling cattle, the median within-lot prevalence (percentage of positive samples within a lot) was 40% (range, 0% to 100%) for verotoxins and 0% for E. coli O157:H7 (range, 0% to 100%) and Salmonella (range, 0% to 17%). One or more fecal samples were positive for verotoxins in 80.4% (95% CI, 72.8% to 86.4%) of the lots of yearling cattle, whereas E. coli O157:H7 were detected in 33.6% (95% CI, 26.0% to 42.0%) of the lots. In cull cows, the median within-lot prevalence was 50% (range, 0% to 100%) for verotoxins and 0% (range, 0% to 100%) for E. coli O157:H7 and Salmonella (range, 0% to 0%). Verotoxins were detected in one or more fecal samples from 78.0% (95% CI, 70.4% to 84.2%) of the lots of cull cows, whereas E. coli O157:H7 were detected in only 6.0% (95% CI, 3.0% to 11.4%) of the lots of cull cows. The prevalence of verotoxins in fecal samples was lower in yearling cattle than in cull cows, whereas the prevalence of E. coli O157:H7 in fecal samples was higher in yearling cattle than in cull cows. The prevalence of E. coli O157:H7 in fecal samples was highest in the summer months. Rumen fill, body condition score, sex, type of cattle (dairy, beef), and distance travelled to the plant were not associated with the fecal prevalence of verotoxins or E. coli O157:H7. The prevalence of verotoxins in fecal samples of cull cows was associated with the source of the cattle. It was highest in cows from the auction market (52%) and farm/ranch (47%) and lowest in cows from the feedlot (31%). In rumen samples, the prevalence of verotoxins was 6.4% (95% CI, 4.2% to 9.4%), and it was 0.8% (95% CI, 0.2% to 2.3%) for E. coli O157:H7, and 0.3% (95% CI, 0.007% to 1.5%) for Salmonella.

Contamination Revealed by Indicator Microorganism Levels during Veal Processing.

PubMed

Bosilevac, Joseph M; Wang, Rong; Luedtke, Brandon E; Wheeler, Tommy L; Koohmaraie, Mohammad

2016-08-01

During site visits of veal processors, the U.S. Department of Agriculture, Food Safety Inspection Service (FSIS) has reported processing deficiencies that likely contribute to increased levels of veal contamination. Here, we report the results of measuring aerobic plate count bacteria (APC), Enterobacteriaceae, coliforms (CF), and Escherichia coli during eight sample collections at five veal processors to assess contamination during the harvest of bob veal and formula-fed veal before (n = 5 plants) and after (n = 3 plants) changes to interventions and processing practices. Hides of veal calves at each plant had mean log CFU/100 cm(2) APC, Enterobacteriaceae, CF, and E. coli of 6.02 to 8.07, 2.95 to 5.24, 3.28 to 5.83, and 3.08 to 5.59, respectively. Preintervention carcasses had mean log CFU/100 cm(2) APC, Enterobacteriaceae, CF, and E. coli of 3.08 to 5.22, 1.16 to 3.47, 0.21 to 3.06, and -0.07 to 3.10, respectively, before and 2.72 to 4.50, 0.99 to 2.76, 0.69 to 2.26, and 0.33 to 2.12, respectively, after changes were made to improve sanitary dressing procedures. Final veal carcasses had mean log CFU/100 cm(2) APC, Enterobacteriaceae, CF, and E. coli of 0.36 to 2.84, -0.21 to 1.59, -0.23 to 1.59, and -0.38 to 1.45 before and 0.44 to 2.64, -0.16 to 1.33, -0.42 to 1.20, and 0.48 to 1.09 after changes were made to improve carcass-directed interventions. Whereas the improved dressing procedures resulted in improved carcass cleanliness, the changes to carcass-directed interventions were less successful, and veal processors are urged to use techniques that ensure uniform and consistent delivery of antimicrobials to carcasses. Analysis of results comparing bob veal to formula-fed veal found bob veal hides, preintervention carcasses, and final carcasses to have increased (P < 0.05) APC, Enterobacteriaceae, CF, and E. coli (with the exception of hide Enterobacteriaceae; P > 0.05) relative to formula fed veal. When both veal categories were harvested at the same plant on the same day, similar results were observed. Since identification by FSIS, the control of contamination during veal processing has started to improve, but challenges still persist.

Evaluation of Bacteriophage Application to Cattle in Lairage at Beef Processing Plants to Reduce Escherichia coli O157:H7 Prevalence on Hides and Carcasses.

PubMed

Arthur, Terrance M; Kalchayanand, Norasak; Agga, Getahun E; Wheeler, Tommy L; Koohmaraie, Mohammad

2017-01-01

Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. Bacteriophages have been proposed as a novel preharvest antimicrobial intervention to reduce the levels of E. coli O157:H7 on cattle hides. The objective of this study was to evaluate a commercialized phage application administered in the lairage area of commercial beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses. Cattle lots either received phage spray treatment (n = 289) or did not (n = 301), as they entered the lairage environments in two separate experiments at two different commercial beef processing plants. Hide and carcass samples were collected and analyzed for E. coli O157:H7 prevalence and concentration. Cattle hides receiving phage treatment had an E. coli O157:H7 prevalence of 51.8%, whereas untreated hides had a prevalence of 57.6%. For carcass samples, the E. coli O157 prevalence in treated and untreated samples was 17.1% and 17.6%, respectively. The results obtained from these experiments demonstrated that the treatment of cattle hides with bacteriophages before processing did not produce a significant reduction of E. coli O157:H7 on cattle hides or beef carcasses during processing.

Surface water disinfection by chlorination and advanced oxidation processes: Inactivation of an antibiotic resistant E. coli strain and cytotoxicity evaluation.

PubMed

Miranda, Andreza Costa; Lepretti, Marilena; Rizzo, Luigi; Caputo, Ivana; Vaiano, Vincenzo; Sacco, Olga; Lopes, Wilton Silva; Sannino, Diana

2016-06-01

The release of antibiotics into the environment can result in antibiotic resistance (AR) spread, which in turn can seriously affect human health. Antibiotic resistant bacteria have been detected in different aquatic environments used as drinking water source. Water disinfection may be a possible solution to minimize AR spread but conventional processes, such as chlorination, result in the formation of dangerous disinfection by-products. In this study advanced oxidation processes (AOPs), namely H2O2/UV, TiO2/UV and N-TiO2/UV, have been compared with chlorination in the inactivation of an AR Escherichia coli (E. coli) strain in surface water. TiO2 P25 and nitrogen doped TiO2 (N-TiO2), prepared by sol-gel method at two different synthesis temperatures (0 and -20°C), were investigated in heterogeneous photocatalysis experiments. Under the investigated conditions, chlorination (1.0 mg L(-1)) was the faster process (2.5 min) to achieve total inactivation (6 Log). Among AOPs, H2O2/UV resulted in the best inactivation rate: total inactivation (6 Log) was achieved in 45 min treatment. Total inactivation was not observed (4.5 Log), also after 120 min treatment, only for N-doped TiO2 synthesized at 0°C. Moreover, H2O2/UV and chlorination processes were evaluated in terms of cytotoxicity potential by means of 3-(4,5-dime-thylthiazol-2-yl)-2,5-diphenylte-trazolium colorimetric test on a human-derived cell line and they similarly affected HepG2 cells viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of an E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass

DOE PAGES

Frederix, Marijke; Mingardon, Florence; Hu, Matthew; ...

2016-04-11

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate (C 2C 1Im][OAc] ) formore » pretreatment of switchgrass biomass. [C 2C 1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C 2C 1Im]-tolerant E. coli strain, where [C 2C 1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C 2C 1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C 2C 1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C 2C 1 Im]-tolerant E. coli strain so as to secrete this [C 2C 1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.« less

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

PubMed

Hartmann, B M; Kaar, W; Yoo, I K; Lua, L H L; Falconer, R J; Middelberg, A P J

2009-12-01

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

Synergistic bactericidal action of phytic acid and sodium chloride against Escherichia coli O157:H7 cells protected by a biofilm.

PubMed

Kim, Nam Hee; Rhee, Min Suk

2016-06-16

The food industry must prevent the build-up of strong Escherichia coli O157:H7 biofilms in food processing environments. The present study examined the bactericidal action of phytic acid (PA), a natural extract from rice bran and the hulls/peels of legumes, against E. coli O157:H7 biofilms. The synergistic bactericidal effects of PA plus sodium chloride (NaCl) were also examined. E. coli O157:H7 biofilms were allowed for form on stainless steel coupons by culture in both rich (tryptic soy broth, TSB) and minimal (M9) medium at 22°C for 6days. Bacterial cells within biofilms grown in M9 medium were significantly more resistant to PA than those grown in TSB (p<0.05); thus M9 medium was selected for further experiments. The anti-biofilm effect of PA was significantly increased by addition of NaCl (2-4%) (p<0.05); indeed, the combination of 0.4% PA plus 3-4% NaCl completely inactivated E. coli O157:H7 biofilms without recovery (a>6.5logCFU/cm(2) reduction). Neither PA nor NaCl alone were this effective (PA, 1.6-2.7logCFU/cm(2) reduction; NaCl, <0.5logCFU/cm(2) reduction). Confocal laser scanning microscopy images of propidium iodide-treated cells showed that PA (0.4%) plus NaCl (2-4%) had marked membrane permeabilizing effects. These results suggest that a sanitizer that combines these two naturally occurring antimicrobial agents may be useful to food safety managers who encounter thick biofilm formation in food processing environments. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevalence and Antibiogram Profiling of Escherichia coli Pathotypes Isolated from the Kat River and the Fort Beaufort Abstraction Water

PubMed Central

Nontongana, Nolonwabo; Sibanda, Timothy; Ngwenya, Elvis; Okoh, Anthony I.

2014-01-01

Escherichia coli is a widespread bacterium encompassing a variety of strains, ranging from highly pathogenic strains, causing worldwide outbreaks of severe diseases to avirulent, well characterized safe laboratory strains. This study evaluated the prevalence and antibiogram profiles of E. coli pathotypes isolated from the Kat River and Fort Beaufort abstraction water. A total of 171 out of 278 confirmed E. coli isolates were positive for at least one pathogenic determinant and these included enteropathogenic E. coli (6%), enterotoxigenic E. coli (47%), uropathogenic E. coli (2%), neonatal meningitis E. coli (5%), diffusely adherent E. coli (1%) and enterohaemorrhagic E. coli (1%). Interestingly, enteroinvasive and enteroaggregative E. coli were not detected. The phenotypic antibiogram profiles of the isolates revealed that all were resistant to penicillin G, while 98% and 38% of the pathotypes were resistant to ampicillin and trimethoprim-sulphamethoxazole, respectively. About 8% of the isolates were resistant to streptomycin. More than half of the isolates exhibited multiple antibiotic resistance with 44% being resistant to three antibiotics and 8% resistant to four antibiotics. We conclude that the Kat River is a reservoir of potentially virulent antibiotic resistant E. coli strains that can cause serious health risks to humans who drink raw water from this river, or in the case that consumption of treated drinking water coincides with failed drinking water processes. PMID:25119699

Mechanisms of Inactivation of Dry Escherichia coli by High-Pressure Carbon Dioxide

PubMed Central

Chen, Yuan Yao; Temelli, Feral

2017-01-01

ABSTRACT High-pressure carbon dioxide processing is a promising technology for nonthermal food preservation. However, few studies have determined the lethality of high-pressure CO2 on dry bacterial cells, and the mechanism of inactivation remains unknown. This study explored the mechanisms of inactivation by using Escherichia coli AW1.7 and mutant strains differing in heat and acid resistance, in membrane composition based on disruption of the locus of heat resistance, and in genes coding for glutamate decarboxylases and cyclopropane fatty acid synthase. The levels of lethality of treatments with liquid, gaseous, and supercritical CO2 were compared. The cell counts of E. coli AW1.7 and mutants with a water activity (aW) of 1.0 were reduced by more than 3 log10 (CFU/ml) after supercritical CO2 treatment at 35°C for 15 min; increasing the pressure generally enhanced inactivation, except for E. coli AW1.7 ΔgadAB. E. coli AW1.7 Δcfa was more susceptible than E. coli AW1.7 after treatment at 10 and 40 MPa; other mutations did not affect survival. Dry cells of E. coli were resistant to treatments with supercritical and liquid CO2 at any temperature. Treatments with gaseous CO2 at 65°C were more bactericidal than those with supercritical CO2 or treatments at 65°C only. Remarkably, E. coli AW1.7 was more susceptible than E. coli AW1.7 Δcfa when subjected to the gaseous CO2 treatment. This study identified CO2-induced membrane fluidization and permeabilization as causes of supercritical mediated microbial inactivation, and diffusivity was a dominant factor for gaseous CO2. IMPORTANCE The safety of dry foods is of increasing concern for public health. Desiccated microorganisms, including pathogens, remain viable over long periods of storage and generally tolerate environmental insults that are lethal to the same organisms at high water activity. This study explored the use of high-pressure carbon dioxide to determine its lethality for dried Escherichia coli and to provide insight into the mechanisms of inactivation. The lethality of high-pressure CO2 and the mechanisms of CO2-mediated inactivation of dry E. coli depended on the physical state of CO2. Liquid and supercritical CO2 were ineffective in reducing the cell counts of dry E. coli isolates, and the effectiveness of gaseous CO2 was related to the diffusivity of CO2. Results provide a novel and alternative method for the food industry to enhance the safety of low aW products. PMID:28283526

Mechanisms of Inactivation of Dry Escherichia coli by High-Pressure Carbon Dioxide.

PubMed

Chen, Yuan Yao; Temelli, Feral; Gänzle, Michael G

2017-05-15

High-pressure carbon dioxide processing is a promising technology for nonthermal food preservation. However, few studies have determined the lethality of high-pressure CO 2 on dry bacterial cells, and the mechanism of inactivation remains unknown. This study explored the mechanisms of inactivation by using Escherichia coli AW1.7 and mutant strains differing in heat and acid resistance, in membrane composition based on disruption of the locus of heat resistance, and in genes coding for glutamate decarboxylases and cyclopropane fatty acid synthase. The levels of lethality of treatments with liquid, gaseous, and supercritical CO 2 were compared. The cell counts of E. coli AW1.7 and mutants with a water activity (a W ) of 1.0 were reduced by more than 3 log 10 (CFU/ml) after supercritical CO 2 treatment at 35°C for 15 min; increasing the pressure generally enhanced inactivation, except for E. coli AW1.7 Δ gadAB E. coli AW1.7 Δ cfa was more susceptible than E. coli AW1.7 after treatment at 10 and 40 MPa; other mutations did not affect survival. Dry cells of E. coli were resistant to treatments with supercritical and liquid CO 2 at any temperature. Treatments with gaseous CO 2 at 65°C were more bactericidal than those with supercritical CO 2 or treatments at 65°C only. Remarkably, E. coli AW1.7 was more susceptible than E. coli AW1.7 Δ cfa when subjected to the gaseous CO 2 treatment. This study identified CO 2 -induced membrane fluidization and permeabilization as causes of supercritical mediated microbial inactivation, and diffusivity was a dominant factor for gaseous CO 2 IMPORTANCE The safety of dry foods is of increasing concern for public health. Desiccated microorganisms, including pathogens, remain viable over long periods of storage and generally tolerate environmental insults that are lethal to the same organisms at high water activity. This study explored the use of high-pressure carbon dioxide to determine its lethality for dried Escherichia coli and to provide insight into the mechanisms of inactivation. The lethality of high-pressure CO 2 and the mechanisms of CO 2 -mediated inactivation of dry E. coli depended on the physical state of CO 2 Liquid and supercritical CO 2 were ineffective in reducing the cell counts of dry E. coli isolates, and the effectiveness of gaseous CO 2 was related to the diffusivity of CO 2 Results provide a novel and alternative method for the food industry to enhance the safety of low a W products. Copyright © 2017 American Society for Microbiology.

Photoinactivation of mcr-1 positive Escherichia coli

NASA Astrophysics Data System (ADS)

Caires, C. S. A.; Leal, C. R. B.; Rodrigues, A. C. S.; Lima, A. R.; Silva, C. M.; Ramos, C. A. N.; Chang, M. R.; Arruda, E. J.; Oliveira, S. L.; Nascimento, V. A.; Caires, A. R. L.

2018-01-01

The emergence of plasmid-mediated colistin resistance in Enterobacteriaceae, mostly in Escherichia coli due to the mcr-1 gene, has revealed the need to develop alternative approaches in treating mcr-1 positive bacterial infections. This is because colistin is a broad-spectrum antibiotic and one of the ‘last-resort’ antibiotics for multidrug resistant bacteria. The present study evaluated for the first time, to the best of our knowledge, the efficacy of photoinactivation processes to kill a known mcr-1 positive E. coli strain. Eosin methylene-blue (EMB) was investigated as a photoantimicrobial agent for inhibiting the growth of a mcr-1 positive E. coli strain obtained from a patient with a diabetic foot infection. The photoantimicrobial activity of EMB was also tested in a non-multidrug resistant E. coli strain. The photoinactivation process was tested using light doses in the 30-45 J cm-2 range provided by a LED device emitting at 625 nm. Our findings demonstrate that a mcr-1 positive E. coli strain is susceptible to photoinactivation. The results show that the EMB was successfully photoactivated, regardless of the bacterial multidrug resistance; inactivating the bacterial growth by oxidizing the cells in accordance with the generation of the oxygen reactive species. Our results suggest that bacterial photoinactivation is an alternative and effective approach to kill mcr-1 positive bacteria.

Evaluation of Escherichia coli biotype 1 as a surrogate for Escherichia coli O157:H7 for cooking, fermentation, freezing, and refrigerated storage in meat processes.

PubMed

Keeling, Carisa; Niebuhr, Steven E; Acuff, Gary R; Dickson, James S

2009-04-01

Five Escherichia coli biotype I isolates were compared with E. coli O157:H7 under four common meat processing conditions. The processes that were evaluated were freezing, refrigerating, fermentation, and thermal inactivation. For each study, at least one surrogate organism was not statistically different when compared with E. coli O157:H7. However, the four studies did not consistently show the same isolate as having this agreement. The three studies that involved temperature as a method of controlling or reducing the E. coli population all had at least one possible surrogate in common. In the fermentation study, only one isolate (BAA-1429) showed no statistical difference when compared with E. coli O157:H7. However, the population reductions that were observed indicated the isolates BAA-1427 and BAA-1431 would overestimate the surviving E. coli O157:H7 population in a fermented summer sausage. When all of the data from all of the surrogates were examined, it was found that isolates BAA-1427, BAA-1429, and BAA-1430 would be good surrogates for all four of the processes that were examined in this study. There was no statistical difference noted between these three isolates and E. coli O157:H7 in the refrigeration study. These isolates resulted in smaller population reductions than did E. coli O157:H7 in the frozen, fermentation, and thermal inactivation studies. This would indicate that these isolates would overpredict the E. coli O157:H7 population in these three instances. This overprediction results in an additional margin of safety when using E. coli biotype 1 as a surrogate.

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

PubMed

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

2018-03-02

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

Hygienization performances of innovative sludge treatment solutions to assure safe land spreading.

PubMed

Levantesi, C; Beimfohr, C; Blanch, A R; Carducci, A; Gianico, A; Lucena, F; Tomei, M C; Mininni, G

2015-05-01

The present research aims at the evaluation of the hygienization performances of innovative sludge treatment processes applied for the separated treatment of secondary sludge. Namely, two digestion pretreatments (sonication and thermal hydrolysis) and two sequential biological processes (mesophilic/thermophilic and anaerobic/aerobic digestion) were compared to the mesophilic (MAD) and thermophilic anaerobic digestion (TAD). Microbial indicators (Escherichia coli, somatic coliphages and Clostridium perfringens spores) and pathogens (Salmonella and enteroviruses), which show different resistances to treatment processes, were monitored in untreated and treated sludge. Overall, microbial load in secondary sludge was shown to be similar or lower than previously reported in literature for mixed sludge. Notably, the anaerobic/aerobic digestion process increased the removal of E. coli and somatic coliphages compared to the simple MAD and always achieved the hygienization requirement (2-log-unit removal of E. coli) proposed by EU Commission in the 3rd Working Document on sludge (April 2000) for the use of treated sludges in agriculture with restriction on their application. The microbial quality limits for the unrestricted use of sludge in agriculture (no Salmonella in 50 g wet weight (WW) and E. coli <500 CFU/g) were always met when thermal digestion or pretreatment was applied; however, the required removal level (6-log-unit removal of E. coli) could not be assessed due to the low level of this microorganism in raw sludge. Observed levels of indicator removal showed a higher resistance of viral particles to thermal treatment compared with bacterial cells and confirmed the suitability of somatic coliphages as indicators in thermal treatment processes.

Impact of Built-up-Litter and Commercial Antimicrobials on Salmonella and Campylobacter Contamination of Broiler Carcasses Processed at a Pilot Mobile Poultry-Processing Unit

PubMed Central

Li, KaWang; Lemonakis, Lacey; Glover, Brian; Moritz, Joseph; Shen, Cangliang

2017-01-01

The small-scale mobile poultry-processing unit (MPPU) produced raw poultry products are of particular food safety concern due to exemption of USDA poultry products inspection act. Limited studies reported the microbial quality and safety of MPPU-processed poultry carcasses. This study evaluated the Salmonella and Campylobacter prevalence in broiler ceca and on MPPU-processed carcasses and efficacy of commercial antimicrobials against Campylobacter jejuni on broilers. In study I, straight-run Hubbard × Cobb broilers (147) were reared for 38 days on clean-shavings (CS, 75) or built-up-litter (BUL, 72) and processed at an MPPU. Aerobic plate counts (APCs), coliforms, Escherichia coli, and yeast/molds (Y/M) of carcasses were analyzed on petrifilms. Ceca and carcass samples underwent microbial analyses for Salmonella and Campylobacter spp. using the modified USDA method and confirmed by API-20e test (Salmonella), latex agglutination immunoassay (Campylobacter), and Gram staining (Campylobacter). Quantitative polymerase chain reaction (CadF gene) identified the prevalence of C. jejuni and Campylobacter coli in ceca and on carcasses. In study II, fresh chilled broiler carcasses were spot inoculated with C. jejuni (4.5 log10 CFU/mL) and then undipped, or dipped into peroxyacetic acid (PAA) (1,000 ppm), lactic acid (5%), lactic and citric acid blend (2.5%), sodium hypochlorite (69 ppm), or a H2O2–PAA mix (SaniDate® 5.0, 0.25%) for 30 s. Surviving C. jejuni was recovered onto Brucella agar. APCs, coliforms, and E. coli populations were similar (P > 0.05) on CS and BUL carcasses. Carcasses of broilers raised on BUL contained a greater (P < 0.05) Y/M population (2.2 log10 CFU/mL) than those reared on CS (1.8 log10 CFU/mL). Salmonella was not detected in any ceca samples, whereas 2.8% of the carcasses from BUL were present with Salmonella. Prevalence of Campylobacter spp., C. jejuni was lower (P < 0.05), and C. coli was similar (P > 0.05) in CS-treated ceca than BUL samples. Prevalence of Campylobacter spp., C. jejuni, and C. coli was not different (P > 0.05) on CS- and BUL-treated carcasses. All antimicrobials reduced C. jejuni by 1.2–2.0 log CFU/mL on carcasses compared with controls. Hence, raising broilers on CS and applying post-chilling antimicrobial treatment can reduce Salmonella and Campylobacter on MPPU-processed broiler carcasses. PMID:28649571

Impact of Built-up-Litter and Commercial Antimicrobials on Salmonella and Campylobacter Contamination of Broiler Carcasses Processed at a Pilot Mobile Poultry-Processing Unit.

PubMed

Li, KaWang; Lemonakis, Lacey; Glover, Brian; Moritz, Joseph; Shen, Cangliang

2017-01-01

The small-scale mobile poultry-processing unit (MPPU) produced raw poultry products are of particular food safety concern due to exemption of USDA poultry products inspection act. Limited studies reported the microbial quality and safety of MPPU-processed poultry carcasses. This study evaluated the Salmonella and Campylobacter prevalence in broiler ceca and on MPPU-processed carcasses and efficacy of commercial antimicrobials against Campylobacter jejuni on broilers. In study I, straight-run Hubbard × Cobb broilers (147) were reared for 38 days on clean-shavings (CS, 75) or built-up-litter (BUL, 72) and processed at an MPPU. Aerobic plate counts (APCs), coliforms, Escherichia coli , and yeast/molds (Y/M) of carcasses were analyzed on petrifilms. Ceca and carcass samples underwent microbial analyses for Salmonella and Campylobacter spp. using the modified USDA method and confirmed by API-20e test ( Salmonella ), latex agglutination immunoassay ( Campylobacter ), and Gram staining ( Campylobacter ). Quantitative polymerase chain reaction (CadF gene) identified the prevalence of C. jejuni and Campylobacter coli in ceca and on carcasses. In study II, fresh chilled broiler carcasses were spot inoculated with C. jejuni (4.5 log 10  CFU/mL) and then undipped, or dipped into peroxyacetic acid (PAA) (1,000 ppm), lactic acid (5%), lactic and citric acid blend (2.5%), sodium hypochlorite (69 ppm), or a H 2 O 2 -PAA mix (SaniDate ® 5.0, 0.25%) for 30 s. Surviving C. jejuni was recovered onto Brucella agar. APCs, coliforms, and E. coli populations were similar ( P  > 0.05) on CS and BUL carcasses. Carcasses of broilers raised on BUL contained a greater ( P  < 0.05) Y/M population (2.2 log 10  CFU/mL) than those reared on CS (1.8 log 10  CFU/mL). Salmonella was not detected in any ceca samples, whereas 2.8% of the carcasses from BUL were present with Salmonella . Prevalence of Campylobacter spp., C. jejuni was lower ( P  < 0.05), and C. coli was similar ( P  > 0.05) in CS-treated ceca than BUL samples. Prevalence of Campylobacter spp., C. jejuni , and C. coli was not different ( P  > 0.05) on CS- and BUL-treated carcasses. All antimicrobials reduced C. jejuni by 1.2-2.0 log CFU/mL on carcasses compared with controls. Hence, raising broilers on CS and applying post-chilling antimicrobial treatment can reduce Salmonella and Campylobacter on MPPU-processed broiler carcasses.

Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

PubMed Central

Scarascia, Giantommaso; Wang, Tiannyu; Hong, Pei-Ying

2016-01-01

Sulfate-reducing bacteria (SRB) are one of the main protagonist groups of biocorrosion in the seawater environment. Given their principal role in biocorrosion, it remains a crucial task to develop strategies to reduce the abundance of SRBs. Conventional approaches include the use of biocides and antibiotics, which can impose health, safety, and environmental concerns. This review examines an alternative approach to this problem. This is achieved by reviewing the role of quorum sensing (QS) in SRB populations and its impact on the biofilm formation process. Genome databases of SRBs are mined to look for putative QS systems and homologous protein sequences representative of autoinducer receptors or synthases. Subsequently, this review puts forward the potential use of quorum quenchers as natural biocides against SRBs and outlines the potential strategies for the implementation of this approach. PMID:27983678

Acyl-homoserine lactone quorum sensing: from evolution to application.

PubMed

Schuster, Martin; Sexton, D Joseph; Diggle, Stephen P; Greenberg, E Peter

2013-01-01

Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

Ligand-Induced Asymmetry in Histidine Sensor Kinase Complex Regulates Quorum Sensing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neiditch,M.; Federle, M.; Pompeani, A.

2006-01-01

Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement inmore » which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.« less

The response of Serratia marcescens JG to environmental changes by quorum sensing system.

PubMed

Sun, Shu-Jing; Liu, Hui-Jun; Weng, Cai-Hong; Lai, Chun-Fen; Ai, Liu-Ying; Liu, Yu-Chen; Zhu, Hu

2016-08-01

Many bacterial cells are known to regulate their cooperative behaviors and physiological processes through a molecular mechanism called quorum sensing. Quorum sensing in Serratia marcescens JG is mediated by the synthesis of autoinducer 2 (AI-2) which is a furanosyl borate diester. In this study, the response of quorum sensing in S. marcescens JG to environment changes such as the initial pH, carbon sources and boracic acid was investigated by a bioreporter and real-time PCR analysis. The results show that glucose can affect AI-2 synthesis to the greatest extent, and 2.0 % glucose can stimulate S. marcescens JG to produce more AI-2, with a 3.5-fold increase in activity compared with control culture. Furthermore, the response of quorum sensing to changes in glucose concentration was performed by changing the amount of luxS RNA transcripts. A maximum of luxS transcription appeared during the exponential growth phase when the glucose concentration was 20.0 g/L. AI-2 production was also slightly impacted by the low initial pH. It is significant for us that the addition of boracic acid at microdosage (0.1-0.2 g/L) can also induce AI-2 synthesis, which probably demonstrated the feasible fact that the 4,5-dihydroxy-2, 3-pentanedione cyclizes by the addition of borate and the loss of water, is hydrated and is converted to the final AI-2 in S. marcescens JG.

Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation

NASA Astrophysics Data System (ADS)

Ouyang, Kai; Dai, Ke; Walker, Sharon L.; Huang, Qiaoyun; Yin, Xixiang; Cai, Peng

2016-05-01

Efficient photocatalytic disinfection of Escherichia coli O157:H7 was achieved by using a C70 modified TiO2 (C70-TiO2) hybrid as a photocatalyst under visible light (λ > 420 nm) irradiation. Disinfection experiments showed that 73% of E. coli O157:H7 died within 2 h with a disinfection rate constant of k = 0.01 min-1, which is three times that measured for TiO2. The mechanism of cell death was investigated by using several scavengers combined with a partition system. The results revealed that diffusing hydroxyl radicals play an important role in the photocatalytically initiated bacterial death, and direct contact between C70-TiO2 hybrid and bacteria is not indispensable in the photocatalytic disinfection process. Extracellular polymeric substances (EPS) of bacteria have little effect on the disinfection efficiency. Analyses of the inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 showed a decrease of the bacterial concentration from 3 × 108 to 38 cfu mL-1 in the solution with C70-TiO2 thin film in the first 2 h of irradiation and a complete inhibition of the growth of E. coli O157:H7 in the later 24 h irradiation.

Inactivation of Escherichia coli inoculated onto fresh-cut chopped cabbage using electron-beam processing.

PubMed

Grasso, Elizabeth M; Uribe-Rendon, Roberto M; Lee, Ken

2011-01-01

During the past decade there were more than 50 reported outbreaks involving leafy green vegetables contaminated with foodborne pathogens. Leafy greens, including cabbage, are fresh foods rarely heated before consumption, which enables foodborne illness. The need for improved safety of fresh food drives the demand for nonthermal food processes to decrease the risk of pathogens while maintaining fresh quality. This study examines the efficacy of electron-beam (e-beam) irradiation in decreasing indigenous microflora on fresh-cut cabbage and determines the optimal dosage to pasteurize fresh-cut cabbage inoculated with Escherichia coli K-12. Fresh-cut cabbage (100 g) was inoculated with ∼8 log E. coli K-12 and e-beam irradiated at doses of 0, 1.0, 2.3, or 4.0 kGy. At 2.3 kGy there was <1.0 log indigenous microflora remaining, indicating greater than a 4.0-log reduction by e-beam. At a 4.0-kGy dose there was >7-log reduction of E. coli K-12 in the fresh-cut cabbage. The D(10)-value for E. coli K-12 in fresh-cut cabbage was 0.564 kGy. E-beam irradiation is thus a viable nonthermal treatment that extends the shelf life and increases the safety of fresh cabbage by reducing or eliminating indigenous microflora and unwanted pathogens.

Efficacy of hypobromous acid as a hide-on carcass antimicrobial intervention.

PubMed

Schmidt, John W; Wang, Rong; Kalchayanand, Norasak; Wheeler, Tommy L; Koohmaraie, Mohammad

2012-05-01

Escherichia coli O157:H7 and Salmonella on cattle hides at slaughter are the main source of beef carcass contamination by these foodborne pathogens during processing. Hypobromous acid (HOBr) has been approved for various applications in meat processing, but the efficacy of HOBr as a hide antimicrobial has not been determined. In this study, the antimicrobial properties of HOBr were determined by spraying cattle hides at either of two concentrations, 220 or 500 ppm. Treatment of hides with 220 ppm of HOBr reduced the prevalence of E. coli O157:H7 on hides from 25.3 to 10.1% (P < 0.05) and reduced the prevalence of Salmonella from 28.3 to 7.1% (P < 0.05). Treatment of hides with 500 ppm of HOBr reduced (P < 0.05) the prevalence of E. coli O157:H7 on hides from 21.2 to 10.1% and the prevalence of Salmonella from 33.3 to 8.1%. The application of 220 ppm of HOBr reduced (P < 0.05) aerobic plate counts, total coliform counts, and E. coli counts on hides by 2.2 log CFU/ 100 cm(2). The use of 500 ppm of HOBr resulted in reductions (P < 0.05) of aerobic plate counts, total coliform counts, and E. coli counts by 3.3, 3.7, and 3.8 log CFU/100 cm(2), respectively, demonstrating that the use of higher concentrations of HOBr on hides resulted in additional antimicrobial activity. These results indicate that the adoption of a HOBr hide wash will reduce hide concentrations of spoilage bacteria and pathogen prevalence, resulting in a lower risk of carcass contamination.

Evaluating the efficacy of three USDA-approved antimicrobial sprays for reducing surrogate Shiga toxin-producing cells of "Escherichia coli on bob veal carcasses

USDA-ARS?s Scientific Manuscript database

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®...

Application of high pressure processing to reduce verotoxigenic E. coli in two types of dry-fermented sausage.

PubMed

Omer, M K; Alvseike, O; Holck, A; Axelsson, L; Prieto, M; Skjerve, E; Heir, E

2010-12-01

The effect of high pressure processing (HPP) on the survival of verotoxigenic Escherichia coli (VTEC) in two types of Norwegian type dry-fermented sausages was studied. Two different types of recipes for each sausage type were produced. The sausage batter was inoculated with 6.8 log(10) CFU/g of VTEC O103:H25. After fermentation, drying and maturation, slices of finished sausages were vacuum packed and subjected to two treatment regimes of HPP. One group was treated at 600 MPa for 10 min and another at three cycles of 600 MPa for 200 s per cycle. A generalized linear model split by recipe type showed that these two HPP treatments on standard recipe sausages reduced E. coli by 2.9 log(10) CFU/g and 3.3 log(10) CFU/g, respectively. In the recipe with higher levels of dextrose, sodium chloride and sodium nitrite E. coli reduction was 2.7 log(10) CFU/g in both treatments. The data show that HPP has a potential to make the sausages safer and also that the effect depends somewhat on recipe. Copyright © 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.

Efficacy of commercial produce sanitizers against nontoxigenic Escherichia coli O157:H7 during processing of iceberg lettuce in a pilot-scale leafy green processing line.

PubMed

Davidson, Gordon R; Buchholz, Annemarie L; Ryser, Elliot T

2013-11-01

Chemical sanitizers are routinely used during commercial flume washing of fresh-cut leafy greens to minimize cross-contamination from the water. This study assessed the efficacy of five commercial sanitizer treatments against Escherichia coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line. Iceberg lettuce (5.4 kg) was inoculated to contain 10(6) CFU/g of a four-strain cocktail of nontoxigenic, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 and processed after 1 h of draining at ~22 °C. Lettuce was shredded using a commercial slicer, step-conveyed to a flume tank, washed for 90 s using six different treatments (water alone, 50 ppm of peroxyacetic acid, 50 ppm of mixed peracid, or 50 ppm of available chlorine either alone or acidified to pH 6.5 with citric acid [CA] or T-128), and then dried using a shaker table and centrifugal dryer. Various product (25-g) and water (50-ml) samples collected during processing along with equipment surface samples (100 cm(2)) from the flume tank, shaker table, and centrifugal dryer were homogenized in neutralizing buffer and plated on tryptic soy agar. During and after iceberg lettuce processing, none of the sanitizers were significantly more effective (P ≤ 0.05) than water alone at reducing E. coli O157:H7 populations on lettuce, with reductions ranging from 0.75 to 1.4 log CFU/g. Regardless of the sanitizer treatment used, the centrifugal dryer surfaces yielded E. coli O157:H7 populations of 3.49 to 4.98 log CFU/100 cm(2). Chlorine, chlorine plus CA, and chlorine plus T-128 were generally more effective (P ≤ 0.05) than the other treatments, with reductions of 3.79, 5.47, and 5.37 log CFU/ml after 90 s of processing, respectively. This indicates that chlorine-based sanitizers will likely prevent wash water containing low organic loads from becoming a vehicle for cross-contamination.

Effective ethanol production from whey powder through immobilized E. coli expressing Vitreoscilla hemoglobin.

PubMed

Sar, Taner; Stark, Benjamin C; Yesilcimen Akbas, Meltem

2017-03-04

Ethanol production from whey powder was investigated by using free as well as alginate immobilized E. coli and E. coli expressing Vitreoscilla hemoglobin (VHb) in both shake flask and fermenter cultures. Media with varying levels of whey (lactose contents of 3%, 5%, 8% or 15%) and yeast extract (0.3% or 0.5%) were evaluated with fermentation times of 48-96 h. Immobilization and VHb expression resulted in higher ethanol production with all media; the increases ranged from 2% to 89% for immobilization and from 2% to 182% for VHb expression. It was determined that growth medium containing 8% lactose with 0.5% yeast extract yielded the highest ethanol production for free or immobilized strains, with or without VHb expression, in both shake flask and fermenter cultures. Immobilization with alginate was found to be a promising process for ethanol production by VHb-expressing ethanologenic E. coli.

Effective ethanol production from whey powder through immobilized E. coli expressing Vitreoscilla hemoglobin

PubMed Central

Sar, Taner; Stark, Benjamin C.; Yesilcimen Akbas, Meltem

2017-01-01

ABSTRACT Ethanol production from whey powder was investigated by using free as well as alginate immobilized E. coli and E. coli expressing Vitreoscilla hemoglobin (VHb) in both shake flask and fermenter cultures. Media with varying levels of whey (lactose contents of 3%, 5%, 8% or 15%) and yeast extract (0.3% or 0.5%) were evaluated with fermentation times of 48–96 h. Immobilization and VHb expression resulted in higher ethanol production with all media; the increases ranged from 2% to 89% for immobilization and from 2% to 182% for VHb expression. It was determined that growth medium containing 8% lactose with 0.5% yeast extract yielded the highest ethanol production for free or immobilized strains, with or without VHb expression, in both shake flask and fermenter cultures. Immobilization with alginate was found to be a promising process for ethanol production by VHb-expressing ethanologenic E. coli. PMID:27579556

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

PubMed

Visvalingam, Jeyachchandran; Liu, Yang; Yang, Xianqin

2017-03-06

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

Bifidobacterium pseudolongum are efficient indicators of animal fecal contamination in raw milk cheese industry

PubMed Central

2011-01-01

Background The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after second maturation - Brie -, after removal from the mold and during ripening) using bifidobacteria as indicators of fecal contamination. Results Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. B. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2.40 log cfu ml-1) of Brie cheeses samples. Mean counts of B. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further characterization of these populations is currently under process. Forty-eight percents (St-Marcellin) and 70% (Brie) of the samples were B. pseudolongum positive/E. coli negative while only 10% (St-Marcellin) and 3% (Brie) were B. pseudolongum negative/E. coli positive. Conclusions The increase of total bifidobacteria during ripening in Marcellin's process does not allow their use as fecal indicator. The presence of B. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. B. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. B. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses. PMID:21816092

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

PubMed

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

2017-12-01

A study was conducted to investigate the efficacy of a sulfuric acid-sodium sulfate blend (SSS) against Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), Salmonella, and nonpathogenic E. coli biotype I on prerigor beef surface tissue. The suitability of using the nonpathogenic E. coli as a surrogate for in-plant validation studies was also determined by comparing the data obtained for the nonpathogenic inoculum with those for the pathogenic inocula. Prerigor beef tissue samples (10 by 10 cm) were inoculated (ca. 6 log CFU/cm 2 ) on the adipose side in a laboratory-scale spray cabinet with multistrain mixtures of E. coli O157:H7 (5 strains), non-O157 STEC (12 strains), Salmonella (6 strains), or E. coli biotype I (5 strains). Treatment parameters evaluated were two SSS pH values (1.5 and 1.0) and two spray application pressures (13 and 22 lb/in 2 ). Untreated inoculated beef tissue samples served as controls for initial bacterial populations. Overall, the SSS treatments lowered inoculated (6.1 to 6.4 log CFU/cm 2 ) bacterial populations by 0.6 to 1.5 log CFU/cm 2 (P < 0.05), depending on inoculum type and recovery medium. There were no main effects (P ≥ 0.05) of solution pH or spray application pressure when SSS was applied to samples inoculated with any of the tested E. coli inocula; however, solution pH did have a significant effect (P < 0.05) when SSS was applied to samples inoculated with Salmonella. Results indicated that the response of the nonpathogenic E. coli inoculum to the SSS treatments was similar (P ≥ 0.05) to that of the pathogenic inocula tested, making the E. coli biotype I strains viable surrogate organisms for in-plant validation of SSS efficacy on beef. The application of SSS at the tested parameters to prerigor beef surface tissue may be an effective intervention for controlling pathogens in a commercial beef harvest process.

Reprogramming of Escherichia coli K-12 Metabolism during the Initial Phase of Transition from an Anaerobic to a Micro-Aerobic Environment

PubMed Central

Trotter, Eleanor W.; Rolfe, Matthew D.; Hounslow, Andrea M.; Craven, C. Jeremy; Williamson, Michael P.; Sanguinetti, Guido; Poole, Robert K.; Green, Jeffrey

2011-01-01

Background Many bacteria undergo transitions between environments with differing O2 availabilities as part of their natural lifestyles and during biotechnological processes. However, the dynamics of adaptation when bacteria experience changes in O2 availability are understudied. The model bacterium and facultative anaerobe Escherichia coli K-12 provides an ideal system for exploring this process. Methods and Findings Time-resolved transcript profiles of E. coli K-12 during the initial phase of transition from anaerobic to micro-aerobic conditions revealed a reprogramming of gene expression consistent with a switch from fermentative to respiratory metabolism. The changes in transcript abundance were matched by changes in the abundances of selected central metabolic proteins. A probabilistic state space model was used to infer the activities of two key regulators, FNR (O2 sensing) and PdhR (pyruvate sensing). The model implied that both regulators were rapidly inactivated during the transition from an anaerobic to a micro-aerobic environment. Analysis of the external metabolome and protein levels suggested that the cultures transit through different physiological states during the process of adaptation, characterized by the rapid inactivation of pyruvate formate-lyase (PFL), a slower induction of pyruvate dehydrogenase complex (PDHC) activity and transient excretion of pyruvate, consistent with the predicted inactivation of PdhR and FNR. Conclusion Perturbation of anaerobic steady-state cultures by introduction of a limited supply of O2 combined with time-resolved transcript, protein and metabolite profiling, and probabilistic modeling has revealed that pyruvate (sensed by PdhR) is a key metabolic signal in coordinating the reprogramming of E. coli K-12 gene expression by working alongside the O2 sensor FNR during transition from anaerobic to micro-aerobic conditions. PMID:21980479

Combination of minimal processing and irradiation to improve the microbiological safety of lettuce ( Lactuca sativa, L.)

NASA Astrophysics Data System (ADS)

Goularte, L.; Martins, C. G.; Morales-Aizpurúa, I. C.; Destro, M. T.; Franco, B. D. G. M.; Vizeu, D. M.; Hutzler, B. W.; Landgraf, M.

2004-09-01

The feasibility of gamma radiation in combination with minimal processing (MP) to reduce the number of Salmonella spp. and Escherichia coli O157:H7 in iceberg lettuce ( Lactuca sativa, L.) (shredded) was studied in order to increase the safety of the product. The reduction of the microbial population during the processing, the D10-values for Salmonella spp. and E. coli O157:H7 inoculated on shredded iceberg lettuce as well as the sensory evaluation of the irradiated product were evaluated. The immersion in chlorine (200 ppm) reduced coliform and aerobic mesophilic microorganisms by 0.9 and 2.7 log, respectively. D-values varied from 0.16 to 0.23 kGy for Salmonella spp. and from 0.11 to 0.12 kGy for E. coli O157:H7. Minimally processed iceberg lettuce exposed to 0.9 kGy does not show any change in sensory attributes. However, the texture of the vegetable was affected during the exposition to 1.1 kGy. The exposition of MP iceberg lettuce to 0.7 kGy reduced the population of Salmonella spp. by 4.0 log and E. coli by 6.8 log without impairing the sensory attributes. The combination of minimal process and gamma radiation to improve the safety of iceberg lettuce is feasible if good hygiene practices begins at farm stage.

Growth of Mycobacterium avium subsp. paratuberculosis, Escherichia coli, and Salmonella Enteritidis during Preparation and Storage of Yogurt

PubMed Central

Cirone, K.; Huberman, Y.; Morsella, C.; Méndez, L.; Jorge, M.; Paolicchi, F.

2013-01-01

The purpose of this study was to determine the viability of Mycobacterium avium subsp. paratuberculosis (MAP), Escherichia coli (E. coli), and Salmonella Enteritidis (S. Enteritidis) during preparation and refrigerated storage of yogurt. Three yogurts were prepared using pasteurized commercial milk. Each yogurt was artificially contaminated with (1) MAP, (2) E. coli + S. Enteritidis, and (3) MAP + E. coli + S. Enteritidis. Samples were taken during and after the fermentation process until day 20 after inoculation. MAP was not detected during their preparation and short-term storage but was recuperated after starting at 180 min after inoculation storage. Live bacterial counts of E. coli, and S. Enteritidis increased during the first 24 hours, followed by a slight decrease towards the end of the study. In this study it was shown how MAP, E. coli, and S. Enteritidis resisted the acidic conditions generated during the preparation of yogurt and low storage temperatures. This work contributes to current knowledge regarding survival of MAP, E. coli, and S. Enteritidis during preparation and refrigerated storage of yogurt and emphasizes the need to improve hygiene measures to ensure the absence of these pathogenic microorganisms in dairy products. PMID:24455399

Supercritical carbon dioxide process for pasteurization of fruit juices

USDA-ARS?s Scientific Manuscript database

Supercritical carbon dioxide (SCCO2) nonthermal processing inactivates microorganisms in juices using non-toxic and non-reactive CO2. However, data is lacking on the inactivation of E. coli K12 and L. plantarum in apple cider using pilot plant scale SCCO2 equipment. For this study, pasteurized pres...

Salmonella and Escherichia coli O157:H7 prevalence in cattle and on carcasses in a vertically integrated feedlot and harvest plant in Mexico.

PubMed

Narvaez-Bravo, C; Miller, M F; Jackson, T; Jackson, S; Rodas-Gonzalez, A; Pond, K; Echeverry, A; Brashears, M M

2013-05-01

To determine the prevalence of Salmonella and Escherichia coli O157:H7 in cattle feedlots and the impact of subsequent contamination on carcasses in a Mexican Federal Inspection Type Standards harvest facility, 250 animals were tagged and sampled in each step of the slaughter process. Samples were taken from hides and fecal grabs, and composite samples were taken from three anatomical carcass sites (hindshank, foreshank, and inside round) during the slaughter process, at preevisceration (PE), prior to entering the hot box (PHB), and after 24 h of dry chilling (DC). Additionally, 250 fecal samples were collected from the feedlot (FL), holding pens (HP), and intestinal feces (IF), and water samples were taken from the HP area. E. coli O157:H7 and Salmonella detection were carried out with the BAX System, immunomagnetic separation, and conventional methods. Overall Salmonella prevalence was 52.5%. The highest prevalence (92.4%) was found on hides, followed by feces from the HP (91.0%), FL (55.56%), PE (49.0%), IF (46.8%), and PHB (24.8%), for all sampling periods combined. The lowest prevalence of 6.0% was found after DC. The overall prevalence of E. coli O157:H7 was as follows: 11.7% for hides, 5.2% for IF, 2.7% for FL, 2.0% for HP, 0.8% for PE, 0.4% for PHB, and 0.4% for the cooler. High prevalence of Salmonella in IF and on hides present a significant risk factor for contamination by Salmonella at the different processing steps. These results serve as a warning as to the risks of contamination in meats for these pathogens and the importance of following good manufacturing practices during beef production processes.

Disinfection of wastewater effluents with the Fenton-like process induced by electromagnetic fields.

PubMed

Rodríguez-Chueca, J; Mediano, A; Ormad, M P; Mosteo, R; Ovelleiro, J L

2014-09-01

This research work is focused on the application and assessment of effectiveness of the Fenton-like processes induced by radiofrequency for the inactivation of faecal bacteria (Escherichia coli and Enterococcus sp.) present in treated urban wastewater effluents. Fenton processes were carried out at near neutral pH (pH 5) with different iron sources, such as iron salts (ferric chloride, 5, 50 and 100 mg/L Fe(3+)), magnetite (1 g/L) and clay (80 g/L), hydrogen peroxide (25 mg/L) and in absence and presence of radiofrequency. Two different electromagnetic field intensities (1.57 and 3.68 kA/m) were used in Fenton processes induced by radiofrequency. Different agents used in the Fenton processes induced by electromagnetic fields (iron source, hydrogen peroxide and RF) were analyzed individually and in combination under the same experimental conditions. First assays of ferromagnetic material/H2O2/radiofrequency processes achieved promising results in terms of bacterial inactivation. For instance, Fe(3+)/H2O2/Radiofrequency achieved a maximum level of E. coli inactivation of 3.55 log after 10 min of treatment. These results are higher than those obtained in absence of radiofrequency. The thermal activation of iron atoms allows the Fenton reaction to intensify, increasing the final yield of the treatment. On the other hand, different behavior was observed in the inactivation of E. coli and Enterococcus sp. due to the structural differences between Gram-negative and Gram-positive bacteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of Escherichia coli O157:H7 growth media for use in test-and-hold procedures for ground beef processing.

PubMed

Guerini, Michael N; Arthur, Terrance M; Shackelford, Steven D; Koohmaraie, Mohammad

2006-05-01

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7--Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX) BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42 degrees C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.

The fate of verocytotoxigenic Escherichia coli C600φ3538(Δvtx2 ::cat) and its vtx2 prophage during grass silage preparation.

PubMed

Nyambe, S; Burgess, C; Whyte, P; O'Kiely, P; Bolton, D

2017-05-01

Silage is grass, preserved by fermentation and used as winter feed for cattle. The impact of a range of current grass silage preparation practices on the survival of Escherichia coli C600φ3538(Δvtx 2 ::cat) and on the induction, release and infectivity of free phage were investigated. Wilted and fresh grass samples, from plots with and without slurry application, were ensiled with or without formic acid. Each treatment combination was inoculated with approximately 6 log 10 CFU per g E. coli C600φ3538(Δvtx 2 ::cat) (donor strain) and E. coli C600::kanamycin R (recipient strain) in test-tube model silos and incubated in the dark at 15°C. The physico-chemical (pH, ammonia, ethanol, lactic acid and volatile fatty acids) and microbiological (total viable counts, TVC, total Enterobacteriaceae counts, TEC, E. coli counts, ECC and lactic acid bacteria, LAB) properties of each fermentation were monitored throughout the experiment as were the concentrations of E. coli C600φ3538(Δvtx 2 ::cat), E. coli C600::kanamycin R , free phage and transductants, using culture and PCR-based methods. Over the course of the experiment the pH of the grass samples typically decreased by 2 pH units. TVC, TEC and ECC decreased by up to 2·3, 6·4 and 6·2 log 10 CFU per g, respectively, while the LAB counts remained relatively stable at 5·2-7·1 log 10 CFU per g. Both donor and recipient strains decreased by approximately 5 log 10 CFU per g. Free phages were detected in all treatments and transductants were detected and confirmed by PCR in the silo containing wilted grass, pretreated with slurry and ensiled without formic acid. Verocytotoxigenic E. coli may survive the ensiling process and the conditions encountered are sufficient to induce vtx 2 bacteriophage leading to low levels of phage-mediated vtx 2 gene transfer. These studies suggest that the ensiling of grass may create an environment which facilitates the emergence of new verocytotoxigenic E. coli. © 2017 The Society for Applied Microbiology.

Survival and growth of Listeria monocytogenes and enterohemorrhagic Escherichia coli O157:H7 in minimally processed artichokes.

PubMed

Sanz, Susana; Giménez, Mercedes; Olarte, Carmen

2003-12-01

The ability of Listeria monocytogenes and Escherichia coli O157:H7 inoculated by immersion (at 4.6 and 5.5 log CFU/ g, respectively) to survive on artichokes during various stages of preparation was determined. Peeling, cutting, and disinfecting operations (immersion in 50 ppm of a free chlorine solution at 4 degrees C for 5 min) reduced populations of L. monocytogenes and E. coli O157:H7 by only 1.6 and 0.8 log units, respectively. An organic acid rinse (0.02% citric acid and 0.2% ascorbic acid) was more effective than a tap water rinse in removing these pathogens. Given the possibility of both pathogens being present on artichokes at the packaging stage, their behavior during the storage of minimally processed artichokes was investigated. For this purpose, batches of artichokes inoculated with L. monocytogenes or E. coli O157:H7 (at 5.5 and 5.2 log CFU/g, respectively) were packaged in P-Plus film bags and stored at 4 degrees C for 16 days. During this period, the equilibrium atmosphere composition and natural background microflora (mesophiles, psychrotrophs, anaerobes, and fecal coliforms) were also analyzed. For the two studied pathogens, the inoculum did not have any effect on the final atmospheric composition (10% O2, 13% CO2) or on the survival of the natural background microflora of the artichokes. L. monocytogenes was able to survive during the entire storage period in the inoculated batches, while the E. coli O157:H7 level increased by 1.5 log units in the inoculated batch during the storage period. The modified atmosphere was unable to control the behavior of either pathogen.

Glyco-Immune Diagnostic Signatures and Therapeutic Targets of Mesothelioma

DTIC Science & Technology

2014-07-01

Huflejt ME. Processing and analysis of serum antibody binding signals from Printed Glycan Arrays for diagnostic and prognostic applications. Int J...rats 542 LeCLex1-6’(LeC1-3’)Lac-sp4 543 Lex1-6’(LeB1-3’)Lac-sp4 641 E.coli oligosaccharide -2 (1208) 642 E.coli oligosaccharide -3 (1210) 254 More

Simultaneous atrazine degradation and E. coli inactivation by simulated solar photo-Fenton-like process using persulfate.

PubMed

Garkusheva, Natalya; Matafonova, Galina; Tsenter, Irina; Beck, Sara; Batoev, Valeriy; Linden, Karl

2017-07-29

This work evaluated the feasibility of a photo-Fenton-like process using persulfate (PS) and ferrous iron (Fe 2+ ) under simulated solar radiation for degrading the herbicide atrazine (ATZ, 6-Chloro-N-ethyl-N'-isopropyl-1,3,5-triazine-2,4-diamine) and inactivating E. coli. Milli Q water, lake water, and diluted wastewater effluents were spiked both simultaneously and separately with ATZ (4 mg/L) and E. coli (10 5 CFU/mL), and exposed to treatment. A method for determining the average irradiance throughout the water media in the UV(A+B) range of the Xe lamp emission was developed for bench-scale experiments. These values were used to calculate the UV(A+B) fluences and the solar UV(A+B) energy doses per unit of volume (Q UV(A+B) , kJ/L). The obtained kinetic data were presented versus energy dose. Treatment of lake water at near-neutral pH was ineffective via the photo-Fenton-like process, attaining only 20% ATZ removal and 1-log reduction of E. coli. In Milli Q water and wastewater, the complete degradation of ATZ in the absence of bacteria was observed at an average energy dose of 1.5 kJ/L (60 min), while in the presence of cells the degradation efficiency was ∼60%. When ATZ was present, E. coli inactivation was also affected in Milli Q water, with 1.4-log reduction (93%) at a dose of 1.6 kJ/L (60 min), whereas in wastewater complete inactivation was achieved at a lower dose of 1.3 kJ/L (45 min). The energy requirements on a Q UV(A+B) basis for simultaneous 90% ATZ removal and 99.99% E. coli inactivation in Milli Q water and wastewater were shown to be less than 10 kJ/L. This suggests the solar/PS/Fe 2+ system is promising for simultaneous treatment and disinfection of wastewater effluents.

Escherichia coli and Cronobacter sakazakii in 'Tommy Atkins' minimally processed mangos: Survival, growth and effect of UV-C and electrolyzed water.

PubMed

Santo, David; Graça, Ana; Nunes, Carla; Quintas, Célia

2018-04-01

These studies were aimed at assessing the growing capacity of Escherichia coli and Cronobacter sakazakii and the effectiveness of Ultraviolet-C (UV-C) radiation, acidic electrolyzed (AEW) and neutral electrolyzed (NEW) waters in the inhibition of these bacteria on minimally processed 'Tommy Atkins' mangoes (MPM). The fruits were contaminated by dip inoculation and kept 10 days at 4, 8, 12 and 20 °C while enumerating bacteria. Contaminated mangoes were disinfected using UV-C (2.5, 5, 7.5 and 10 kJ/m 2 ), AEW, NEW and sodium hypochlorite (SH) and the microorganisms were monitored. None of the enterobacteria grew at 4, 8 and 12 °C regardless of having persisted during the 10-day period. At 20 °C, E. coli and C. sakazakii grew, after adaption phases of 48 h and 24 h, to values of 8.7 and 8.5 log cfu/g at day eight, respectively. E. coli showed the highest reduction counts on the MPM washed with NEW and SH (2.2 log cfu/g). UV-C was more effective in reducing C. sakazakii (2.4-2.6 log cfu/g), when compared to AEW, NEW and SH (1.2-1.8 log cfu/g). The efficacy of decontamination technologies depends on microorganisms, highlighting the importance of preventing contamination at the primary production and of combining different methods to increase the safety of fresh-cut fruits. Copyright © 2017 Elsevier Ltd. All rights reserved.

Escherichia coli counting using lens-free imaging for sepsis diagnosis

NASA Astrophysics Data System (ADS)

Moon, Sangjun; Manzur, Fahim; Manzur, Tariq; Klapperich, Catherine; Demirci, Utkan

2009-09-01

Sepsis causes 9.3% of overall deaths in United States. To diagnose sepsis, cell/bacteria capture and culturing methods have been widely investigated in the medical field. Escherichia Coli (E. Coli) is used as a model organism for sepsis in blood stream since wide variety of antibodies are established and the genetic modification process is well documented for fluorescent tagging. In point-of-care testing applications, the sepsis diagnostics require fast monitoring, inexpensive testing, and reliable results at resource limited settings, i.e. battle field, home care for dialysis. However, the cell/E.coli are hard to directly capture and see at the POCT because of the small size, 2 μm long and 0.5 μm in diameter, and the bacteria are rare in the blood stream in sepsis. Here, we propose a novel POCT platform to image and enumerate cell/E.coli on a microfluidic surface to diagnose sepsis at resource limited conditions. We demonstrate that target cells are captured from 5 μl of whole blood using specific antibodies and E.coli are imaged using a lens-free imaging platform, 2.2 μm pixel CMOS based imaging sensor. This POCT cell/bacteria capture and enumeration approach can further be used for medical diagnostics of sepsis. We also show approaches to rapidly quantify white blood cell counts from blood which can be used to monitor immune response.

Prevention and control of enterohaemorrhagic Escherichia coli (EHEC) infections: memorandum from a WHO meeting. WHO Consultation on Prevention and Control of Enterohaemorrhagic Escherichia coli (EHEC) Infections.

PubMed Central

Reilly, A.

1998-01-01

Escherichia coli is a commonly occurring inhabitant of the intestine of humans and other animals, but there are several pathogenic types of E. coli which cause a variety of human diseases. One of these pathogenic types, E. coli O157:H7, belongs to the group of enterohaemorrhagic E. coli (EHEC) which produce potent toxins and cause a particularly severe form of disease, haemorrhagic colitis (HC). About 10% of patients with HC can go on to develop haemolytic uraemic syndrome (HUS), a life-threatening complication of E. coli O157:H7 infection that is characterized by acute renal failure, haemolytic anaemia, and thrombocytopenia. These sequelae are particularly serious in young children and older people. On average, 2-7% of patients with HUS die, but in some outbreaks among the elderly the mortality rate has been as high as 50%. This Memorandum reviews the growing importance of E. coli O157:H7 as a foodborne pathogen and reports on the issues of surveillance, outbreak investigation, and control strategies with respect to EHEC infections that were discussed at the WHO Consultation on Prevention and Control of EHEC Infections, held in Geneva on 28 April to 1 May 1997. Recommended measures for prevention and control include the following: use of potable water in food production; presentation of clean animals at slaughter; improved hygiene throughout the slaughter process; appropriate use of food processing measures; thorough cooking of foods; and the education of food handlers, abattoir workers, and farm workers on the principles and application of food hygiene. PMID:9744244

Evidence for the benefits of food chain interventions on E. coli 0157:H7/NM prevalence in retail ground beef and human disease incidence: A success story.

PubMed

Pollari, Frank; Christidis, Tanya; Pintar, Katarina D M; Nesbitt, Andrea; Farber, Jeff; Lavoie, Marie-Claude; Gill, Alex; Kirsch, Penelope; Johnson, Roger P

2017-04-20

Human infection with Escherichia coli O157:H7/NM has historically been associated with consumption of undercooked ground beef. The purpose of this paper is to investigate the correlation of the decline in E. coli O157:H7/NM infections in Canada with the introduction of control efforts in ground beef by industry. The human incidence of E. coli O157:H7/NM, prevalence in ground beef and interventions from 1996 to 2014 were analyzed. Pathogen prevalence data were obtained from federal government and industry surveillance and inspection/compliance programs. A survey of the largest ground beef producers in Canada was conducted to identify when interventions were implemented. The incidence of E. coli O157:H7/NM infections in Canada declined from ∼4 cases/100 000 to ∼1 case/100 000 from 2000 to 2010. Verotoxigenic Escherichia coli (VTEC) prevalence in ground beef sold at retail declined from about 30% around the year 2000 to <2% since 2012. Other measures of the prevalence of E. coli, VTEC, and E. coli O157:H7/NM in beef and ground beef also declined. The number and types of interventions implemented in the major beef processing establishments in Canada increased from 1996 to 2016. The observed decline in human illnesses and pathogen levels in relation to retail meats was associated with the introduction of control efforts by industry, federal and provincial/territorial governments, and the general population. Industry-led changes in beef processing along with the introduction of food safety policies, regulations, and public education have led to improved food safety in Canada.

Efficacy of Slightly Acidic Electrolyzed Water and UV-Ozonated Water Combination for Inactivating Escherichia Coli O157:H7 on Romaine and Iceberg Lettuce during Spray Washing Process.

PubMed

Pang, Yu-Hsin; Hung, Yen-Con

2016-07-01

Spray washing is a common sanitizing method for the fresh produce industry. The purpose of this research was to investigate the antimicrobial effect of spraying slightly acidic electrolyzed water (SAEW) and a combination of ozonated water with ultraviolet (UV) in reducing Escherichia coli O157:H7 on romaine and iceberg lettuces. Both romaine and iceberg lettuces were spot inoculated with 100 μL of a 3 strain mixture of E. coli O157:H7 to achieve an inoculum of 6 log CFU/g on lettuce. A strong antimicrobial effect was observed for the UV-ozonated water combination, which reduced the population of E. coli by 5 log CFU/g of E. coli O157:H7 on both lettuces. SAEW achieved about 5 log CFU/g reductions in the bacterial counts on romaine lettuce. However, less than 2.5 log CFU/g in the population of E. coli O157:H7 was reduced on iceberg lettuce. The difference may be due to bacteria aggregation near and within stomata for iceberg lettuce but not for romaine lettuce. The UV light treatment may stimulate the opening of the stomata for the UV-ozonated water treatment and hence achieve better bacterial inactivation than the SAEW treatment for iceberg lettuce. Our results demonstrated that the combined treatment of SAEW and UV-ozonated water in the spray washing process could more effectively reduce E. coli O157:H7 on lettuce, which in turn may help reduce incidences of E. coli O157:H7 outbreaks. © 2016 Institute of Food Technologists®

Evaluation of bacteriophage application to cattle in lairage at beef processing plants to reduce Escherichia coli O157:H7 prevalence on hides and carcasses

USDA-ARS?s Scientific Manuscript database

Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing faci...

Efficient Photocatalytic Disinfection of Escherichia coli O157:H7 using C70-TiO2 Hybrid under Visible Light Irradiation

PubMed Central

Ouyang, Kai; Dai, Ke; Walker, Sharon L.; Huang, Qiaoyun; Yin, Xixiang; Cai, Peng

2016-01-01

Efficient photocatalytic disinfection of Escherichia coli O157:H7 was achieved by using a C70 modified TiO2 (C70-TiO2) hybrid as a photocatalyst under visible light (λ > 420 nm) irradiation. Disinfection experiments showed that 73% of E. coli O157:H7 died within 2 h with a disinfection rate constant of k = 0.01 min−1, which is three times that measured for TiO2. The mechanism of cell death was investigated by using several scavengers combined with a partition system. The results revealed that diffusing hydroxyl radicals play an important role in the photocatalytically initiated bacterial death, and direct contact between C70-TiO2 hybrid and bacteria is not indispensable in the photocatalytic disinfection process. Extracellular polymeric substances (EPS) of bacteria have little effect on the disinfection efficiency. Analyses of the inhibitory effect of C70-TiO2 thin films on E. coli O157:H7 showed a decrease of the bacterial concentration from 3 × 108 to 38 cfu mL−1 in the solution with C70-TiO2 thin film in the first 2 h of irradiation and a complete inhibition of the growth of E. coli O157:H7 in the later 24 h irradiation. PMID:27161821

Microbial Inactivation by Ultrasound Assisted Supercritical Fluids

NASA Astrophysics Data System (ADS)

Benedito, Jose; Ortuño, Carmen; Castillo-Zamudio, Rosa Isela; Mulet, Antonio

A method combining supercritical carbon dioxide (SC-CO2) and high power ultrasound (HPU) has been developed and tested for microbial/enzyme inactivation purposes, at different process conditions for both liquid and solid matrices. In culture media, using only SC-CO2, the inactivation rate of E. coli and S. cerevisiae increased with pressure and temperature; and the total inactivation (7-8 log-cycles) was attained after 25 and 140 min of SC-CO2 (350 bar, 36 °C) treatment, respectively. Using SC-CO2+HPU, the time for the total inactivation of both microorganisms was reduced to only 1-2 min, at any condition selected. The SC-CO2+HPU inactivation of both microorganisms was slower in juices (avg. 4.9 min) than in culture media (avg. 1.5 min). In solid samples (chicken, turkey ham and dry-cured pork cured ham) treated with SC-CO2 and SC-CO2+HPU, the inactivation rate of E. coli increased with temperature. The application of HPU to the SC-CO2 treatments accelerated the inactivation rate of E. coli and that effect was more pronounced in treatments with isotonic solution surrounding the solid food samples. The application of HPU enhanced the SC-CO2 inactivation mechanisms of microorganisms, generating a vigorous agitation that facilitated the CO2 solubilization and the mass transfer process. The cavitation generated by HPU could damage the cell walls accelerating the extraction of vital constituents and the microbial death. Thus, using the combined technique, reasonable industrial processing times and mild process conditions could be used which could result into a cost reduction and lead to the minimization in the food nutritional and organoleptic changes.

Survival of pathogenic bacteria in compost with special reference to Escherichia coli.

PubMed

Gong, Chun-ming; Koichi, Inoue; Shunji, Inanaga; Takashi, Someya

2005-01-01

Application of compost in agricultural practice could potentially cause contamination of foodstuffs with pathogenic bacteria such as Escherichia coli O157:H7 (E. Coli O157). We investigated pathogenic bacteria in compost collected from the compost facilities, and evaluated the survival of E. coli K12 and O157 in laboratory experiments. Out of 19 compost product samples, coliform bacteria and salmonella were detected in 7 and 3 samples respectively. The number of coliform bacteria was 1.8 x 10(2) to 2.5 x 10(6) CFU/g dw and that of salmonella was 4.2 x 10(1) to 6.0 x 10(3) CFU/g dw. Moreover, coliform bacteria, fecal coliform, E. coli and salmonella were detected during composting at 54 degrees C to 67 degrees C. The results indicated that moisture content was a very important factor to the heat sensitivity of pathogenic bacteria in compost, E. coil in compost of high moisture content was more sensitive than that in compost of low moisture content, cells harvested in logarithmic phase was more sensitive than these in stationary phase, and E. coli K12 was more sensitive than E. coli O157. Based on the D values, the lethal time of E. coli K12 and O157 from l0(8) to 10(0) CFU/g dw were 16.3 and 28.8 min, respectively, at 60 degrees C in compost with 40% moisture content. However, some E. coil cells survived in composting process at 54 degrees C to 67 degrees C. Water potential (low moisture content) and physiological aspects of bacteria (stationary phase) could explain only in part of the prolonged survival of E. coil in compost, and there should be some other factors that are conducive to bacterial survival in compost.

Inactivation of Escherichia coli O157:H7 on stainless steel upon exposure to Paenibacillus polymyxa biofilms.

PubMed

Kim, Seonhwa; Bang, Jihyun; Kim, Hoikyung; Beuchat, Larry R; Ryu, Jee-Hoon

2013-11-01

We investigated the potential use of biofilm formed by a competitive-exclusion (CE) microorganism to inactivate Escherichia coli O157:H7 on a stainless steel surface. Five microorganisms showing inhibitory activities against E. coli O157:H7 were isolated from vegetable seeds and sprouts. The microorganism with the greatest antimicrobial activity was identified as Paenibacillus polymyxa (strain T5). In tryptic soy broth (TSB), strain T5 reached a higher population at 25 °C than at 12 or 37 °C without losing inhibitory activity against E. coli O157:H7. When P. polymyxa (6 log CFU/mL) was co-cultured with E. coli O157:H7 (2, 3, 4, or 5 log CFU/mL) in TSB at 25 °C, the number of E. coli O157:H7 decreased significantly within 24h. P. polymyxa formed a biofilm on stainless steel coupons (SSCs) in TSB at 25 °C within 24h, and cells in biofilms, compared to attached cells without biofilm formation, showed significantly increased resistance to a dry environment (43% relative humidity [RH]). With the exception of an inoculum of 4 log CFU/coupon at 100% RH, upon exposure to biofilm formed by P. polymyxa on SSCs, populations of E. coli O157:H7 (2, 4, or 6 log CFU/coupon) were significantly reduced within 48 h. Most notably, when E. coli O157:H7 at 2 log CFU/coupon was applied to SSCs on which P. polymyxa biofilm had formed, it was inactivated within 1h, regardless of RH. These results will be useful when developing strategies using biofilms produced by competitive exclusion microorganisms to inactivate foodborne pathogens in food processing environments. © 2013.

Antimicrobial susceptibility of hazard analysis critical control point Escherichia coli isolates from federally inspected beef processing plants in Alberta, Saskatchewan, and Ontario.

PubMed

Van Donkersgoed, Joyce; Manninen, Ken; Potter, Andy; McEwen, Scott; Bohaychuk, Valerie; Klashinsky, Sandy; Deckert, Anne; Irwin, Rebecca

2003-09-01

A survey to estimate the prevalence of antimicrobial resistance in Escherichia coli was conducted in 7 Canadian federally inspected processing plants during 2001. Escherichia coli isolates were recovered during routine hazard analysis critical control point sampling from beef carcasses and trim and subsequently tested for their antimicrobial susceptibility by using susceptibility panels. Of the 2653 isolates analyzed, 68% were sensitive to all 18 antimicrobials tested. For 14 of the 18 antimicrobials evaluated, the percentage of resistant isolates was < or = 1. Twenty-five percent of the isolates were resistant to tetracycline, 9% to sulfamethoxazole, 7% to streptomycin, and 3% to ampicillin. Multiple resistance was found in 12% of the isolates, with 7% showing resistance to 2 antimicrobials, 2% to 3 antimicrobials, 2% to 4 antimicrobials, and 1% to 5 or more antimicrobials. Forty-five different antimicrobial resistance patterns were observed. The reasons for the development of the antimicrobial resistance were not investigated in this study. This study was useful as a pilot to help to develop a national antimicrobial resistance surveillance program in Canada. This study indicates that laboratory standardization is possible for consistent results across the country and that the indicator organism, E. coli, is fairly easy to obtain for surveillance but Salmonella are not, due to their low prevalence in beef.

Antimicrobial susceptibility of hazard analysis critical control point Escherichia coli isolates from federally inspected beef processing plants in Alberta, Saskatchewan, and Ontario

PubMed Central

Van Donkersgoed, Joyce; Manninen, Ken; Potter, Andy; McEwen, Scott; Bohaychuk, Valerie; Klashinsky, Sandy; Deckert, Anne; Irwin, Rebecca

2003-01-01

A survey to estimate the prevalence of antimicrobial resistance in Escherichia coli was conducted in 7 Canadian federally inspected processing plants during 2001. Escherichia coli isolates were recovered during routine hazard analysis critical control point sampling from beef carcasses and trim and subsequently tested for their antimicrobial susceptibility by using susceptibility panels. Of the 2653 isolates analyzed, 68% were sensitive to all 18 antimicrobials tested. For 14 of the 18 antimicrobials evaluated, the percentage of resistant isolates was ≤ 1. Twenty-five percent of the isolates were resistant to tetracycline, 9% to sulfamethoxazole, 7% to streptomycin, and 3% to ampicillin. Multiple resistance was found in 12% of the isolates, with 7% showing resistance to 2 antimicrobials, 2% to 3 antimicrobials, 2% to 4 antimicrobials, and 1% to 5 or more antimicrobials. Forty-five different antimicrobial resistance patterns were observed. The reasons for the development of the antimicrobial resistance were not investigated in this study. This study was useful as a pilot to help to develop a national antimicrobial resistance surveillance program in Canada. This study indicates that laboratory standardization is possible for consistent results across the country and that the indicator organism, E. coli, is fairly easy to obtain for surveillance but Salmonella are not, due to their low prevalence in beef. PMID:14524625

Virulence profiles of Shiga Toxin-Producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina.

PubMed

Pizarro, M A; Orozco, J H; Degarbo, S M; Calderón, A E; Nardello, A L; Laciar, A; Rüttler, M E

2013-12-01

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

Characterization of bacterial strains isolated from a beef-processing plant following cleaning and disinfection - Influence of isolated strains on biofilm formation by Sakaï and EDL 933 E. coli O157:H7.

PubMed

Marouani-Gadri, Nesrine; Augier, Gladys; Carpentier, Brigitte

2009-07-31

The objective of this study was to investigate the effects on Escherichia coli O157:H7 biofilm formation of bacteria isolated from meat site surfaces following cleaning and disinfection. We first isolated and identified, to the genus level, strains of the latter organisms. Samples were obtained by swabbing the surfaces of equipment or floors over areas ranging from 315 to 3200 cm(2) in a slaughter hall, a meat cutting room and a meat boning room of a meat-processing plant. The number of bacteria recovered from these surfaces ranged from <1 to> 10(5) CFU/cm(2). In the slaughter hall, stainless steel was in one case one of the most contaminated materials and in other cases one of the less contaminated. The same observation was made for conveyor belts made of polyvinyl chloride in the boning room. Dominant genera in the meat plant were Staphylococcus and Bacillus which were both 34% of the isolates from the slaughter hall and 14 and 4% respectively of the isolates from the cutting room. Randomly selected isolates of each of the genera recovered from the slaughter hall were cultured with E. coli O157:H7 in meat exudate at 15 degrees C to form dual-organism biofilms on polyurethane. In all cases but one, the isolates increased the numbers of attached E. coli O157:H7. The effects ranged from 0.37 to 1.11 for EDL 933 strain and from 0.19 to 1.38 log (CFU/cm(2)) for Sakaï strain. This is the first time that a resident microbiota of a meat-processing plant has been shown to have a favourable effect on E. coli O157:H7 colonization of a solid surface, which is of great interest from a food safety standpoint.

Prevalence of antimicrobial-resistant Escherichia coli from raw vegetables in Lebanon.

PubMed

Faour-Klingbeil, Dima; Kuri, Victor; Fadlallah, Sukayna; Matar, Ghassan M

2016-04-28

Fresh produce has been implicated in a number of documented outbreaks of foodborne illness caused by bacteria, viruses, and parasites. Shiga toxin-producing Escherichia coli (STEC) have been detected on vegetables, raising concerns about the prevalence of E. coli contamination in produce, which can take place at various points from farm to fork. This study aimed to detect the presence of STEC and multidrug-resistant (MDR) E. coli on fresh vegetables and water from different sources along the fresh produce supply chain in Lebanon. E. coli isolates (n = 60) were group serotyped using trivalent antisera (trivalent 1 [O111+O55+O26], trivalent 2 [O86+O119+O127], trivalent 3 [O125; O126; O128], and trivalent 4 [O114+O124+O142]) and tested for stx1 and stx2 genes by polymerase chain reaction (PCR) assay. Resistance to antimicrobial agents was determined using the disk diffusion method. The virulence genes stx1 and stx2 were not detected in any of the isolates. However, 60% of the isolates were MDR and predominantly observed in trivalent 2 (32%). It is postulated that the inadequate post-harvest washing contributed to transmission of antimicrobial-resistant E. coli at wholesale and retail levels. Fresh vegetables harbor MDR E. coli and their consumption poses risks of increasing the reservoir of antimicrobial resistance in the intestines of the Lebanese population. Greater emphasis should be placed on vigilant sanitation measures at the consumption level, and effective national risk mitigation strategies are crucial to minimize fecal contamination in the early stages of production, particularly in the post-harvest washing processes.

Designer cells programming quorum-sensing interference with microbes.

PubMed

Sedlmayer, Ferdinand; Hell, Dennis; Müller, Marius; Ausländer, David; Fussenegger, Martin

2018-05-08

Quorum sensing is a promising target for next-generation anti-infectives designed to address evolving bacterial drug resistance. The autoinducer-2 (AI-2) is a key quorum-sensing signal molecule which regulates bacterial group behaviors and is recognized by many Gram-negative and Gram-positive bacteria. Here we report a synthetic mammalian cell-based microbial-control device that detects microbial chemotactic formyl peptides through a formyl peptide sensor (FPS) and responds by releasing AI-2. The microbial-control device was designed by rewiring an artificial receptor-based signaling cascade to a modular biosynthetic AI-2 production platform. Mammalian cells equipped with the microbial-control gene circuit detect formyl peptides secreted from various microbes with high sensitivity and respond with robust AI-2 production, resulting in control of quorum sensing-related behavior of pathogenic Vibrio harveyi and attenuation of biofilm formation by the human pathogen Candida albicans. The ability to manipulate mixed microbial populations through fine-tuning of AI-2 levels may provide opportunities for future anti-infective strategies.

Validation of intervention strategies to control Escherichia coli O157:H7 and Salmonella typhimurium DT 104 in mechanically tenderized and brine-enhanced beef.

PubMed

Echeverry, Alejandro; Brooks, J Chance; Miller, Markus F; Collins, Jesse A; Loneragan, Guy H; Brashears, Mindy M

2009-08-01

After three different outbreaks were linked to the consumption of nonintact meat products contaminated with Escherichia coli O157:H7, the U.S. Department of Agriculture, Food Safety and Inspection Service published notice requiring establishments producing mechanically tenderized and moisture-enhanced beef products to reassess their respective hazard analysis and critical control point system, due to potential risk to the consumers. The purpose of this study was to determine the effectiveness of different intervention strategies (lactic acid, lactic acid bacteria, and acidified sodium chlorite) to control E. coli O157:H7 and Salmonella enterica serotype Typhimurium definitive phage type 104 in mechanically tenderized and brine-enhanced beef strip loins when applied to the steaks prior to packaging and shipment for processing. After the mechanical process, translocation of E. coli O157:H7 and Salmonella Typhimurium DT 104 from the surface into the internal muscles occurred at levels between 2.0 and 4.0 log CFU/g (from an initial inoculation level of 5.0 log) after mechanical tenderization, and at levels of 1.0 to 3.0 log CFU/g after injection, with all the interventions consistently presenting lower microbial counts (P < 0.05) than did the controls. Lactic acid bacteria reduced internal E. coli O157:H7 loads 1.2 to > 2.2 log cycles, while the acidified sodium chlorite and lactic acid reduced them between 0.8 and 3.0 log, respectively. Salmonella Typhimurium DT 104 was also reduced internally after application of all interventions between 0.9 and 2.2 log. The application of antimicrobials to the steaks prior to packaging and shipment on day 0 was effective in reducing internalization of both pathogens in nonintact beef products stored for both 14 and 21 days.

Cloud-enabled microscopy and droplet microfluidic platform for specific detection of Escherichia coli in water.

PubMed

Golberg, Alexander; Linshiz, Gregory; Kravets, Ilia; Stawski, Nina; Hillson, Nathan J; Yarmush, Martin L; Marks, Robert S; Konry, Tania

2014-01-01

We report an all-in-one platform - ScanDrop - for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a "cloud" network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2-4 days for other currently available standard detection methods.

Cloud-Enabled Microscopy and Droplet Microfluidic Platform for Specific Detection of Escherichia coli in Water

PubMed Central

Kravets, Ilia; Stawski, Nina; Hillson, Nathan J.; Yarmush, Martin L.; Marks, Robert S.; Konry, Tania

2014-01-01

We report an all-in-one platform – ScanDrop – for the rapid and specific capture, detection, and identification of bacteria in drinking water. The ScanDrop platform integrates droplet microfluidics, a portable imaging system, and cloud-based control software and data storage. The cloud-based control software and data storage enables robotic image acquisition, remote image processing, and rapid data sharing. These features form a “cloud” network for water quality monitoring. We have demonstrated the capability of ScanDrop to perform water quality monitoring via the detection of an indicator coliform bacterium, Escherichia coli, in drinking water contaminated with feces. Magnetic beads conjugated with antibodies to E. coli antigen were used to selectively capture and isolate specific bacteria from water samples. The bead-captured bacteria were co-encapsulated in pico-liter droplets with fluorescently-labeled anti-E. coli antibodies, and imaged with an automated custom designed fluorescence microscope. The entire water quality diagnostic process required 8 hours from sample collection to online-accessible results compared with 2–4 days for other currently available standard detection methods. PMID:24475107

[Effect of Pseudomonas aeruginosa exometabolites on planktonic and biofilm cultures of Escherichia coli].

PubMed

Kuznetsova, M V; Karpunina, T I; Maslennikova, I L; Nesterova, L Iu; Demakov, V A

2012-01-01

Study the effect of P. aeruginosa exometabolites on planktonic and biofilm cultures of bioluminescent E. coli strain. E. coli K12 TG1 (pF1 lux+ Ap(r)) recombinant bioluminescent strain, P. aeruginosa ATCC 27853 reference strain and 2 nosocomial isolates were used. Pyocyanin and pyoverdin content in supernatant of P. aeruginosa over-night cultures was evaluated according to E. Deziel et al. (2001). Planktonic and biofilm cultures of E. coli were obtained in 96-well plates (LB, statically, 37 degrees C), optical density of plankton, film biomass (OD600, OD580) and bioluminescence in plankton and biofilm were evaluated in microplate reader Infiniti M200 (Tecan, Austria). P. aeruginosa exometabolites increased the duration of lag-phase in E. coli, and short term exposition inhibited luminescence of planktonic cells. These effects are determined by bactericidal action ofpyocyanin and pyoverdin. Supernatants ofover-night cultures of P. aeruginosa inhibit formation of biofilm and disrupt the formed biofilm of E. coli. Effect of pyocyanin and pyoverdin on these processes is not established, other factors may have higher significance. Bioluminescence of E. coli K12 TGI that reflects the energetic status of the cell allows to evaluate and prognose the character of coexistence of P. aeruginosa in combined with E. coli planktonic and biofilm culture.

High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses.

PubMed

Chen, Chih-Ming; Ke, Se-Chin; Li, Chia-Ru; Wu, Ying-Chen; Chen, Ter-Hsin; Lai, Chih-Ho; Wu, Xin-Xia; Wu, Lii-Tzu

2017-10-01

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to β-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum β-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Meat industry wastewater: microbiological quality and antimicrobial susceptibility of E. coli and Salmonella sp. isolates, case study in Vojvodina, Serbia.

PubMed

Stošić, Milena; Čučak, Dragana; Kovačević, Srđan; Perović, Marija; Radonić, Jelena; Turk Sekulić, Maja; Vojinović Miloradov, Mirjana; Radnović, Dragan

2016-01-01

Wastewater from meat processing industries is a fusion of compounds with a high load of organic matter, and pathogen microorganisms like Escherichia coli, and Salmonella sp. The aim of this research was to determine microbiological characteristics of the wastewater discharged from the meat processing industry in order to get a more detailed insight into meat industry wastewater pollution, and to evaluate the resistance of bacterial strains E. coli and Salmonella sp. to antibiotics. The evaluation of the antimicrobial susceptibility was performed on 37 strains of E. coli and eight strains of Salmonella sp. to nine different antibiotics. The number of faecal pollution indicators was very high in all samples. From a total of 37 strains of E. coli, a moderate degree of resistance was shown to tetracycline (37.83%); a low degree of resistance to ampicillin (21.62%), streptomycin (24.32%), trimethoprim-sulfamethoxazol (18.92%) and nalidixic acid (16.22%); and very low to: chloramphenicol (13.51%), ciprofloxacin (2.7%), gentamicin and cefotaxime (0.0%). The results for eight strains of Salmonella sp. show that all eight isolates had some degree of susceptibility to nine tested antimicrobial agents and six strains were fully susceptible to all tested antibiotics.

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.

Impact of persistent and nonpersistent generic Escherichia coli and Salmonella sp. recovered from a beef packing plant on biofilm formation by E. coli O157.

PubMed

Visvalingam, J; Ells, T C; Yang, X

2017-12-01

To examine the influence of meat plant Escherichia coli and Salmonella sp. isolates on E. coli O157 biofilm formation. Biofilm formation was quantified by crystal violet staining (A 570 nm ) and viable cell numbers for up to 6 days at 15°C. All five persistent E. coli genotypes formed strong biofilms when cultured alone or co-cultured with E. coli O157, with A 570 nm values reaching ≥4·8 at day 4, while only two of five nonpersistent genotypes formed such biofilms. For E. coli O157:H7 co-culture biofilms with E. coli genotypes 136 and 533, its numbers were ≥1·5 and ≥1 log CFU per peg lower than those observed for its mono-culture biofilm at days 2 and 4, respectively. The number of E. coli O157:NM in similar co-culture biofilms was 1 log CFU per peg lower than in its mono-culture biofilm at day 4 and 6, respectively. Salmonella sp. lowered the number of E. coli O157:NM by 0·5 log unit, once, at day 6. Generic E. coli may outcompete E. coli O157 strains while establishing biofilms. Findings advance knowledge regarding inter-strain competition for a similar ecological niche and may aid development of biocontrol strategies for E. coli O157 in food processing environments. © 2017 Her Majesty the Queen in Right of Canada. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology Reproduced with the permission of the Minister of the Department of Agriculture and Agri-Food Canada.

Interaction of Escherichia coli with growing salad spinach plants.

PubMed

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

2003-10-01

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

Liquid egg white pasteurization using a centrifugal UV irradiator.

PubMed

Geveke, David J; Torres, Daniel

2013-03-01

Studies are limited on UV nonthermal pasteurization of liquid egg white (LEW). The objective of this study was to inactivate Escherichia coli using a UV irradiator that centrifugally formed a thin film of LEW on the inside of a rotating cylinder. The LEW was inoculated with E. coli K12 to approximately 8 log cfu/ml and was processed at the following conditions: UV intensity 1.5 to 9.0 mW/cm²; cylinder rotational speed 450 to 750 RPM, cylinder inclination angle 15° to 45°, and flow rate 300 to 900 ml/min, and treatment time 1.1 to 3.2s. Appropriate dilutions of the samples were pourplated with tryptic soy agar (TSA). Sublethal injury was determined using TSA+4% NaCl. The regrowth of surviving E. coli during refrigerated storage for 28 days was investigated. The electrical energy of the UV process was also determined. The results demonstrated that UV processing of LEW at a dose of 29 mJ/cm² at 10°C reduced E. coli by 5 log cfu/ml. Inactivation significantly increased with increasing UV dose and decreasing flow rate. The results at cylinder inclination angles of 30° and 45° were similar and were significantly better than those at 15°. The cylinder rotational speed had no significant effect on inactivation. The occurrence of sublethal injury was detected. Storage of UV processed LEW at 4° and 10°C for 21 days further reduced the population of E. coli to approximately 1 log cfu/ml where it remained for an additional 7 days. The UV energy applied to the LEW to obtain a 5 log reduction of E. coli was 3.9 J/ml. These results suggest that LEW may be efficiently pasteurized, albeit at low flow rates, using a nonthermal UV device that centrifugally forms a thin film. Published by Elsevier B.V.

Evaluation of the treatment of both sides of raw chicken breasts with an atmospheric pressure plasma jet for the inactivation of Escherichia coli.

PubMed

Yong, Hae In; Kim, Hyun-Joo; Park, Sanghoo; Choe, Wonho; Oh, Mi Wha; Jo, Cheorun

2014-08-01

Atmospheric pressure plasma (APP) is an emerging nonthermal microbial inactivation technique. In this study, agar and raw chicken breast were inoculated with Escherichia coli and treated with an APP jet based on cold arc plasma. The aim of this study was to investigate the optimum conditions for the plasma treatment of an APP jet in order to maximize the efficiency of E. coli inactivation. The combination of N2+O2 (10 standard cubic centimeters per minute) and a longer treatment time (10 min) resulted in the highest inactivation of E. coli on agar plates with an optimum treatment distance of 20 mm. The samples in dry and wet conditions showed similar reductions in E. coli count when one side of the samples was treated at a given treatment time. Treating both sides-2.5 min on each side-resulted in a higher growth inhibition of E. coli than treatment of a single side only for 5 min. However, there was no significant difference between one-side treated samples (10 min) and both-sides treated samples (5+5 min). When the concentration of E. coli in the chicken breast sample was 10(4) colony-forming units (CFU)/g, the reduction rate of the E. coli was the highest, followed by 10(5), 10(6), and 10(7) CFU/g; however, no difference was found between 10(3) and 10(4) CFU/g. In conclusion, various treatment conditions may affect the inactivation efficiency of E. coli. In the present study, the optimum condition was determined as the treatment distance of 20 mm and longer treatment time (10 min) with the addition of oxygen to the nitrogen gas flow. Furthermore, the cell concentration of sample was an important parameter for the efficacy of the inactivation process.

Effects of lactic acid and commercial chilling processes on survival of Salmonella, Yersinia enterocolitica, and Campylobacter coli in pork variety meats.

PubMed

King, Amanda M; Miller, Rhonda K; Castillo, Alejandro; Griffin, Davey B; Hardin, Margaret D

2012-09-01

Current industry chilling practices with and without the application of 2% L-lactic acid were compared for their effectiveness at reducing levels of Salmonella, Yersinia enterocolitica, and Campylobacter coli on pork variety meats. Pork variety meats (livers, intestines, hearts, and stomachs) were inoculated individually with one of the three pathogens and subjected to five different treatment combinations that included one or more of the following: water wash (25°C), lactic acid spray (2%, 40 to 50°C), chilling (4°C), and freezing (-15°C). Samples were analyzed before treatment, after each treatment step, and after 2, 4, and 6 months of frozen storage. Results showed that when a lactic acid spray was used in combination with water spray, immediate reductions were approximately 0.5 log CFU per sample of Salmonella, 0.8 log CFU per sample of Y. enterocolitica, and 1.1 log CFU per sample of C. coli. Chilling, both alone and in combination with spray treatments, had little effect on pathogens, while freezing resulted in additional 0.5-log CFU per sample reductions in levels of Salmonella and Y. enterocolitica, and an additional 1.0-log CFU per sample reduction in levels of C. coli. While reductions of at least 1 log CFU per sample were observed on variety meats treated with only a water wash and subsequently frozen, samples treated with lactic acid had greater additional reductions than those treated with only a water spray throughout frozen storage. The results of this study suggest that the use of lactic acid as a decontamination intervention, when used in combination with good manufacturing practices during processing, causes significant reductions in levels of Salmonella, Y. enterocolitica, and C. coli on pork variety meats.

2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

PubMed

Sethupathy, Sivasamy; Nithya, Chari; Pandian, Shunmugiah Karutha

2015-01-01

The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

Escherichia coli as a fatty acid and biodiesel factory: current challenges and future directions.

PubMed

Rahman, Ziaur; Rashid, Naim; Nawab, Javed; Ilyas, Muhammad; Sung, Bong Hyun; Kim, Sun Chang

2016-06-01

Biodiesel has received widespread attention as a sustainable, environment-friendly, and alternative source of energy. It can be derived from plant, animal, and microbial organisms in the form of vegetable oil, fats, and lipids, respectively. However, biodiesel production from such sources is not economically feasible due to extensive downstream processes, such as trans-esterification and purification. To obtain cost-effective biodiesel, these bottlenecks need to be overcome. Escherichia coli, a model microorganism, has the potential to produce biodiesel directly from ligno-cellulosic sugars, bypassing trans-esterification. In this process, E. coli is engineered to produce biodiesel using metabolic engineering technology. The entire process of biodiesel production is carried out in a single microbial cell, bypassing the expensive downstream processing steps. This review focuses mainly on production of fatty acid and biodiesel in E. coli using metabolic engineering approaches. In the first part, we describe fatty acid biosynthesis in E. coli. In the second half, we discuss bottlenecks and strategies to enhance the production yield. A complete understanding of current developments in E. coli-based biodiesel production and pathway optimization strategies would reduce production costs for biofuels and plant-derived chemicals.

Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica.

PubMed

Lee, Soo Youn; Lee, Jiho; Lee, Hye Sun; Chang, Jeong Ho

2014-03-15

We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step. © 2013 Elsevier B.V. All rights reserved.

Electron crystallography of PhoE porin, an outer membrane, channel- forming protein from E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walian, P.J.

1989-11-01

One approach to studying the structure of membrane proteins is the use of electron crystallography. Dr. Bing Jap has crystallized PhoE pore-forming protein (porin) from the outer membrane of escherichia coli (E. coli) into monolayer crystals. The findings of this research and those of Jap (1988, 1989) have determined these crystals to be highly ordered, yielding structural information to a resolution of better than 2.8 angstroms. The task of this thesis has been to collect and process the electron diffraction patterns necessary to generate a complete three-dimensional set of high resolution structure factor amplitudes of PhoE porin. Fourier processing ofmore » these amplitudes when combined with the corresponding phase data is expected to yield the three-dimensional structure of PhoE porin at better than 3.5 angstroms resolution. 92 refs., 33 figs., 3 tabs. (CBS)« less

Vascular endothelial growth factor receptor 1 contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells through the phosphatidylinositol 3-kinase/Akt signaling pathway.

PubMed

Zhao, Wei-Dong; Liu, Wei; Fang, Wen-Gang; Kim, Kwang Sik; Chen, Yu-Hua

2010-11-01

Escherichia coli is the most common Gram-negative organism causing neonatal meningitis. Previous studies demonstrated that E. coli K1 invasion of brain microvascular endothelial cells (BMEC) is required for penetration into the central nervous system, but the microbe-host interactions that are involved in this process remain incompletely understood. Here we report the involvement of vascular endothelial growth factor receptor 1 (VEGFR1) expressed on human brain microvascular endothelial cells (HBMEC) in E. coli K1 invasion of HBMEC. Our results showed that treatment of confluent HBMEC with pan-VEGFR inhibitors significantly inhibited E. coli K1 invasion of HBMEC. Immunofluorescence results indicated the colocalization of VEGFR1 with E. coli K1 during bacterial invasion of HBMEC. The E. coli-induced actin cytoskeleton rearrangements in HBMEC were blocked by VEGFR inhibitors but not by VEGFR2-specific inhibitors. The small interfering RNA (siRNA) knockdown of VEGFR1 in HBMEC significantly attenuated E. coli invasion and the concomitant actin filament rearrangement. Furthermore, we found an increased association of VEGFR1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) in HBMEC infected with E. coli K1 and that E. coli K1-triggered Akt activation in HBMEC was blocked by VEGFR1 siRNA and VEGFR inhibitors. Taken together, our results demonstrate that VEGFR1 contributes to E. coli K1 invasion of HBMEC via recruitment of the PI3K/Akt signaling pathway.

Solar photocatalytic disinfection of E. coli and bacteriophages MS2, ΦX174 and PR772 using TiO2, ZnO and ruthenium based complexes in a continuous flow system.

PubMed

Mac Mahon, Joanne; Pillai, Suresh C; Kelly, John M; Gill, Laurence W

2017-05-01

The performance of photocatalytic treatment processes were assessed using different photocatalysts against E. coli and bacteriophages MS2, ΦX174 and PR772, in a recirculating continuous flow compound parabolic collector system under real sunlight conditions. Suspended TiO 2 and ZnO nanoparticle powders and Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate in solution were tested separately, as well as in combination, using E. coli. For a 3-log reduction of E. coli in distilled water, inactivation rates in terms of cumulative dose were in the order Ru(bpy) 3 Cl 2 >(TiO 2 & Ru(bpy) 3 Cl 2 )>(ZnO & Ru(bpy) 3 Cl 2 )>ZnO>TiO 2 >photolysis. Reactivation of E. coli was observed following all trials despite the detection limit being reached, although the reactivated colonies were observed to be under stress and much slower growing when compared to original colonies. Treatment with Ru(bpy) 3 Cl 2 was also compared against standard photolysis of bacteriophages MS2, ΦX174 and PR772 with the order of photolytic inactivation for a 3-log reduction in terms of cumulative UV-A dose being ΦX174>PR772>MS2. However, MS2 was found to be the most susceptible bacteriophage to treatment with Ru(bpy) 3 Cl 2 , with complete removal of the phage observed within the first 15min of exposure. Ru(bpy) 3 Cl 2 also significantly improved inactivation rates for PR772 and ΦX174. Copyright © 2017 Elsevier B.V. All rights reserved.

Lethality prediction for Escherichia coli 0157:H7 and Uropathogenic E. coli in ground chicken treated with high pressure processing and trans-cinnamaldehyde

USDA-ARS?s Scientific Manuscript database

Pathogenic Escherichia coli, intestinal (O157:H7) as well as extraintestinal types (Uropathogenic E. coli (UPEC)) are commonly found in many foods including chicken meat. In this study we compared the resistance of E. coli O157:H7 to UPEC in chicken meat under the stresses of high hydrostatic pressu...

The influence of quorum sensing in compartment II of the MELiSSA loop

NASA Astrophysics Data System (ADS)

Condori, Sandra; Mastroleo, Felice; Wattiez, Ruddy; Leys, Natalie

MELiSSA (Micro-Ecological Life Support System Alternative) has been conceived as a 5 compartments microorganisms and higher plants recycling system for long haul space flights. Rhodospirillum rubrum S1H colonizes compartment II. Previous work reported that continuous culture of the bacterium in a photobioreactor could lead to thick biofilm formation, leading to bioreactor arrest. Our aim is to investigate the unknown quorum sensing (QS) system of R. rubrum S1H, specifically under MELiSSA relevant culture conditions meaning light anaerobic (LAN) and using acetate as carbon source. In that purpose an autoinducer synthase gene (Rru_A3396) knockout mutant was constructed by allelic exchange generating strain M68. In addition phenotypic comparison between wild type (WT) and M68 was performed. Results of thin layer chromatography assay where Agrobacterium tumefaciens NT1 have been used as reporter strain showed that WT produces acyl-homoserine lactones (AHLs) from C4 to C12 acyl carbon chain length; however, in M68 no AHLs were detected confirming that gene Rru_A3396 (named rruI) encodes an autoinducer synthase. Interestingly under a low shear or static environment M68 showed cell aggregation similar as reported in a closely related bacterium Rhodobacter sphaeroides (cerI mutant). In contrast to WT, M68 did not form biofilm and exhibited a decreased motility and pigment content. M68 vs wild type transcriptomics results showed that 326 genes were statistically significant differentially expressed. Downregulation of genes related to photosynthesis e.g., reaction center subunits, light harvesting complex and photosynthetic assembly proteins was observed. Similar results were obtained for preliminary proteomic analysis. Results obtained showed that in R. rubrum S1H the AHL-based QS system regulates almost 8% of the genome which is linked to biofilm formation among other biological processes described above. Since strain M68 could not be used in compartment II due to its less effective photosynthetic apparatus (among other cellular functions) we are investigating other alternatives to avoid biofilm formation.

Minimal processing of iceberg lettuce has no substantial influence on the survival, attachment and internalization of E. coli O157 and Salmonella.

PubMed

Van der Linden, Inge; Avalos Llano, Karina R; Eriksson, Markus; De Vos, Winnok H; Van Damme, Els J M; Uyttendaele, Mieke; Devlieghere, Frank

2016-12-05

The influence of a selection of minimal processing techniques (sanitizing wash prior to packaging, modified atmosphere, storage conditions under light or in the dark) was investigated in relation to the survival of, attachment to and internalization of enteric pathogens in fresh produce. Cut Iceberg lettuce was chosen as a model for fresh produce, Escherichia coli O157:H7 (E. coli O157) and Salmonella enterica were chosen as pathogen models. Care was taken to simulate industrial post-harvest processing. A total of 50±0.1g of fresh-cut Iceberg lettuce was packed in bags under near ambient atmospheric air with approximately 21% O 2 (NAA) conditions or equilibrium modified atmosphere with 3% O 2 (EMAP). Two lettuce pieces inoculated with E. coli O157 BRMSID 188 or Salmonella Typhimurium labeled with green fluorescent protein (GFP) were added to each package. The bags with cut lettuce were stored under either dark or light conditions for 2days at 7°C. The pathogens' capacity to attach to the lettuce surface and cut edge was evaluated 2days after inoculation using conventional plating technique and the internalization of the bacteria was investigated and quantified using confocal microscopy. The effect of a sanitizing wash step (40mg/L NaClO or 40mg/L peracetic acid+1143mg/L lactic acid) of the cut lettuce prior to packaging was evaluated as well. Our results indicate that both pathogens behaved similarly under the investigated conditions. Pathogen growth was not observed, nor was there any substantial influence of the investigated atmospheric conditions or light/dark storage conditions on their attachment/internalization. The pathogens attached to and internalized via cut edges and wounds, from which they were able to penetrate into the parenchyma. Internalization through the stomata into the parenchyma was not observed, although some bacteria were found in the substomatal cavity. Washing the cut edges with sanitizing agents to reduce enteric pathogen numbers was not more effective than a rinse with precooled tap water prior to packaging. Our results confirm that cut surfaces are the main risk for postharvest attachment and internalization of E. coli O157 and Salmonella during minimal processing and that storage and packaging conditions have no important effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

PubMed

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

PubMed Central

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

Tidal fluctuations influence E. coli concentrations in urban estuaries.

PubMed

Jovanovic, Dusan; Coleman, Rhys; Deletic, Ana; McCarthy, David T

2017-06-15

This study investigated the influence of water level and velocity on Escherichia coli levels over multiple tidal cycles in an urban microtidal estuary in Melbourne, Australia. Over 3,500 E. coli samples and high resolution water level and velocity measurements from two locations within the estuary were used for the analysis. E. coli negatively correlated with water level in the upper estuary which was proposed to be linked to increased resuspension of estuarine sediments during low tide. No relationship was found in the lower estuary, likely due to wet weather inputs dwarfing subtler tidal-related processes. Removal of wet weather data enabled significant relationships to emerge in the lower estuary: 1) positive with water level (when a 9-h shift applied corresponding to the phase shift between water levels and velocities) and; 2) positive with velocity (no shift applied). This supports a link between increased E. coli levels and tidal-related resuspension. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of an integrated electrodisinfection/electrocoagulation process with Al bipolar electrodes for urban wastewater reclamation.

PubMed

Cotillas, Salvador; Llanos, Javier; Cañizares, Pablo; Mateo, Sara; Rodrigo, Manuel A

2013-04-01

In this work, a novel integrated electrochemical process for urban wastewater regeneration is described. The electrochemical cell consists in a Boron Doped Diamond (BDD) or a Dimensionally Stable Anode (DSA) as anode, a Stainless Steel (SS) as cathode and a perforated aluminum plate, which behaves as bipolar electrode, between anode and cathode. Thus, in this cell, it is possible to carry out, at the same time, two different electrochemical processes: electrodisinfection (ED) and electrocoagulation (EC). The treatment of urban wastewater with different anodes and different operating conditions is studied. First of all, in order to check the process performance, experiments with synthetic wastewaters were carried out, showing that it is possible to achieve a 100% of turbidity removal by the electrodissolution of the bipolar electrode. Next, the effect of the current density and the anode material are studied during the ED-EC process of actual effluents. Results show that it is possible to remove Escherichia coli and turbidity simultaneously of an actual effluent from a WasteWater Treatment Facility (WWTF). The use of BDD anodes allows to remove the E. coli completely at an applied electric charge of 0.0077 A h dm(-3) when working with a current density of 6.65 A m(-2). On the other hand, with DSA anodes, the current density necessary to achieve the total removal of E. coli is higher (11.12 A m(-2)) than that required with BDD anodes. Finally, the influence of cell flow path and flow rate have been studied. Results show that the performance of the process strongly depends on the characteristics of the initial effluent (E. coli concentration and Cl(-)/NH(4)(+) initial ratio) and that a cell configuration cathode (inlet)-anode (outlet) and a higher flow rate enhance the removal of the turbidity from the treated effluent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Techno-economic analysis of the industrial production of a low-cost enzyme using E. coli: the case of recombinant β-glucosidase.

PubMed

Ferreira, Rafael da Gama; Azzoni, Adriano Rodrigues; Freitas, Sindelia

2018-01-01

The enzymatic conversion of lignocellulosic biomass into fermentable sugars is a promising approach for producing renewable fuels and chemicals. However, the cost and efficiency of the fungal enzyme cocktails that are normally employed in these processes remain a significant bottleneck. A potential route to increase hydrolysis yields and thereby reduce the hydrolysis costs would be to supplement the fungal enzymes with their lacking enzymatic activities, such as β-glucosidase. In this context, it is not clear from the literature whether recombinant E. coli could be a cost-effective platform for the production of some of these low-value enzymes, especially in the case of on-site production. Here, we present a conceptual design and techno-economic evaluation of the production of a low-cost industrial enzyme using recombinant E. coli . In a simulated baseline scenario for β-glucosidase demand in a hypothetical second-generation ethanol (2G) plant in Brazil, we found that the production cost (316 US$/kg) was higher than what is commonly assumed in the literature for fungal enzymes, owing especially to the facility-dependent costs (45%) and to consumables (23%) and raw materials (25%). Sensitivity analyses of process scale, inoculation volume, and volumetric productivity indicated that optimized conditions may promote a dramatic reduction in enzyme cost and also revealed the most relevant factors affecting production costs. Despite the considerable technical and economic uncertainties that surround 2G ethanol and the large-scale production of low-cost recombinant enzymes, this work sheds light on some relevant questions and supports future studies in this field. In particular, we conclude that process optimization, on many fronts, may strongly reduce the costs of E. coli recombinant enzymes, in the context of tailor-made enzymatic cocktails for 2G ethanol production.

Inactivation mechanisms of pathogenic bacteria in several matrixes during the composting process in a composting toilet.

PubMed

Sossou, S K; Hijikata, N; Sou, M; Tezuka, R; Maiga, A H; Funamizu, N

2014-01-01

This study aimed to compare the inactivation rate and the mechanisms of pathogenic bacteria in three matrixes (sawdust, rice husk and charcoal) during the composting process. The inactivation rate was evaluated with Escherichia coli strain and the damaged parts and/or functions were evaluated with three different media. Normalized inactivation rate constant in three media and from three matrixes had no significant difference in each process (pure, 1 month and 2 months). The value in rice husk was relatively increased during 2 months but there was no significant difference. The inactivation rate constants of Tryptic Soy Agar (TSA) and Compact Dry E. coli/Coliform in pure sawdust and rice husk were relatively lower than that of Desoxycholate Agar, but increased in 2 months. This indicated that damaging part was changed from outer membrane to enzymes and metabolisms during the 2-month composting process. In the case of charcoal, only the TSA value in apure matrix was relatively lower than that of others, but it increased in 2 months. This indicated that damaging part was changed from outer membrane and enzyme to metabolisms during the composting process. Composting matrix and composting process did not significantly affect inactivation rate of pathogenic bacteria during the process but affected the damaging part of the bacteria.

Determination of Sources of Escherichia coli on Beef by Multiple-Locus Variable-Number Tandem Repeat Analysis.

PubMed

Yang, Xianqin; Tran, Frances; Youssef, Mohamed K; Gill, Colin O

2015-07-01

The possible origin of Escherichia coli found on cuts and trimmings in the breaking facility of a beef packing plant was examined using multiple-locus variable-number tandem repeat analysis. Coliforms and E. coli were enumerated in samples obtained from 160 carcasses that would enter the breaking facility when work commenced and after each of the three production breaks throughout the day, from the conveyor belt before work and after each break, and from cuts and trimmings when work commenced and after each break. Most samples yielded no E. coli, irrespective of the surface types. E. coli was recovered from 7 (<5%) carcasses, at numbers mostly ≤1.0 log CFU/160,000 cm(2). The log total numbers of E. coli recovered from the conveyor belt, cuts, and trimmings were mostly between 1 and 2 log CFU/80,000 cm(2). A total of 554 E. coli isolates were recovered. Multiple-locus variable-number tandem repeat analysis of 327 selected isolates identified 80 distinct genotypes, with 37 (46%) each containing one isolate. However, 28% of the isolates were of genotypes that were recovered from more than one sampling day. Of the 80 genotypes, 65 and 2% were found in one or all four sampling periods throughout the day. However, they represented 23 and 14% of the isolates, respectively. Of the genotypes identified for each surface type, at least one contained ≥9 isolates. No unique genotypes were associated with carcasses, but 10, 17, and 19 were uniquely associated with cuts, trimmings, and the belt, respectively. Of the isolates recovered from cuts, 49, 3, and 19% were of genotypes that were found among isolates recovered from the belt, carcasses, or both the belt and carcasses, respectively. A similar composition was found for isolates recovered from trimmings. These findings show that the E. coli found on cuts and trimmings at this beef packing plant mainly originated from the conveyor belt and that small number of E. coli strains survived the daily cleaning and sanitation process, thus persisting in the plant.

Escherichia coli hyperepidemic clone ST410-A harboring blaCTX-M-15 isolated from fresh vegetables in a municipal market in Quito-Ecuador.

PubMed

Ortega-Paredes, David; Barba, Pedro; Mena-López, Santiago; Espinel, Nathaly; Zurita, Jeannete

2018-04-30

Dissemination of Extended spectrum β-lactamases (ESBL) Enterobacteriaceae is a major medical threat. Vegetables and fruits, which are usually consumed raw, are a very suitable pathway for the spread of these bacteria from farm-to-fork. However, limited information exists regarding resistant bacteria and epidemic clones that are disseminated in vegetables and tap water in South America. We processed a total of 90 samples in triplicate of nine typically consumed raw vegetables from a central municipal market, and tap water samples were processed from twenty-one locations in Quito, Ecuador. The samples were analyzed for total coliforms and ESBL Enterobacteriaceae contamination using the dilution filtration method. ESBL Escherichia coli isolates were phenotypically and genotypically characterized. The water was free of Enterobacteriaceae, but all the vegetables and fruits (except for blackberries) presented total coliform counts. Watercress had the highest load of total coliforms (3.3 × 10E4). ESBL E. coli was detected in alfalfa, leaf lettuce and parsley/cilantro samples. Alfalfa had the highest load of ESBL E. coli/total coliforms (1/3.3 × 10E2). We identified E. coli ST44-A and ST410-A harboring bla CTX-M-15 downstream of ISEcp1. Alfalfa and parsley/cilantro were contaminated with hyperepidemic E. coli ST410-A, which was resistant to quinolones and harbored bla CTX-M-15 . For the first time, we report ESBL E. coli ST410-A from vegetables and express an alert regarding the health risk this could represent. Copyright © 2018 Elsevier B.V. All rights reserved.

Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains.

PubMed

Yoshida, Akihito; Nishimura, Taku; Kawaguchi, Hideo; Inui, Masayuki; Yukawa, Hideaki

2006-11-01

We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (DeltaldhA, DeltafrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h(-1) g(-1) dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h(-1) l(-1) (20.2 l h(-1) l(-1) at 37 degrees C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications.

Fluorometric In Situ Monitoring of an Escherichia coli Cell Factory with Cytosolic Expression of Human Glycosyltransferase GalNAcT2: Prospects and Limitations

PubMed Central

Schwab, Karen; Lauber, Jennifer; Hesse, Friedemann

2016-01-01

The glycosyltransferase HisDapGalNAcT2 is the key protein of the Escherichia coli (E. coli) SHuffle® T7 cell factory which was genetically engineered to allow glycosylation of a protein substrate in vivo. The specific activity of the glycosyltransferase requires time-intensive analytics, but is a critical process parameter. Therefore, it has to be monitored closely. This study evaluates fluorometric in situ monitoring as option to access this critical process parameter during complex E. coli fermentations. Partial least square regression (PLS) models were built based on the fluorometric data recorded during the EnPresso® B fermentations. Capable models for the prediction of glucose and acetate concentrations were built for these fermentations with rout mean squared errors for prediction (RMSEP) of 0.19 g·L−1 and 0.08 g·L−1, as well as for the prediction of the optical density (RMSEP 0.24). In situ monitoring of soluble enzyme to cell dry weight ratios (RMSEP 5.5 × 10−4 µg w/w) and specific activity of the glycosyltransferase (RMSEP 33.5 pmol·min−1·µg−1) proved to be challenging, since HisDapGalNAcT2 had to be extracted from the cells and purified. However, fluorescence spectroscopy, in combination with PLS modeling, proved to be feasible for in situ monitoring of complex expression systems. PMID:28952595

Use of total of Escherichia coli counts to assess the hygienic characteristics of a beef carcass dressing process.

PubMed

Gill, C O; McGinnis, J C; Badoni, M

1996-08-01

Swab samples were obtained from 3 sites on the surfaces of beef carcasses passing through a high speed dressing process, with 24 samples from each site being obtained at each of 4 points in the process. The aerobic microflora recovered from each swab after incubation at 25 degrees C was enumerated and characterized, and numbers of coliforms and Escherichia coli were determined. The data on aerobic flora indicated that skinning results in similar contamination of all 3 sites, that further deposition of bacteria at the brisket site occurs after skinning, and that trimming and washing achieve modest decontamination of the neck and brisket site, and extensive decontamination of the rump site. Changes in flora compositions during processing were too limited to much affect the assessment based on the aerobic flora total counts alone. The E. coli data indicated that during skinning the rump site was more heavily contaminated with faecal organisms than the other sites, that contamination of the brisket site is little altered between skinning and carcass splitting, although there is an extensive redistribution of E. coli at the neck site and sporadic, limited decontamination of the rump site, and that trimming and washing do not decontaminate the neck or rump sites, but that the rump site is extensively decontaminated by trimming. There was good correlation between E. coli and coliform counts, but weak correlation between E. coli and aerobic, 25 degrees C, counts. The findings suggest that assessments of beef carcass dressing processes for Hazard Analysis: Critical Control Point (HACCP) purposes should be based on enumerations of E. coli, or perhaps coliforms, rather than of the aerobic flora, to avoid important misunderstandings of the hygienic effects of the various operations in a process.

Explanatory Variables Associated with Campylobacter and Escherichia coli Concentrations on Broiler Chicken Carcasses during Processing in Two Slaughterhouses.

PubMed

Pacholewicz, Ewa; Swart, Arno; Wagenaar, Jaap A; Lipman, Len J A; Havelaar, Arie H

2016-12-01

This study aimed at identifying explanatory variables that were associated with Campylobacter and Escherichia coli concentrations throughout processing in two commercial broiler slaughterhouses. Quantative data on Campylobacter and E. coli along the processing line were collected. Moreover, information on batch characteristics, slaughterhouse practices, process performance, and environmental variables was collected through questionnaires, observations, and measurements, resulting in data on 19 potential explanatory variables. Analysis was conducted separately in each slaughterhouse to identify which variables were related to changes in concentrations of Campylobacter and E. coli during the processing steps: scalding, defeathering, evisceration, and chilling. Associations with explanatory variables were different in the slaughterhouses studied. In the first slaughterhouse, there was only one significant association: poorer uniformity of the weight of carcasses within a batch with less decrease in E. coli concentrations after defeathering. In the second slaughterhouse, significant statistical associations were found with variables, including age, uniformity, average weight of carcasses, Campylobacter concentrations in excreta and ceca, and E. coli concentrations in excreta. Bacterial concentrations in excreta and ceca were found to be the most prominent variables, because they were associated with concentration on carcasses at various processing points. Although the slaughterhouses produced specific products and had different batch characteristics and processing parameters, the effect of the significant variables was not always the same for each slaughterhouse. Therefore, each slaughterhouse needs to determine its particular relevant measures for hygiene control and process management. This identification could be supported by monitoring changes in bacterial concentrations during processing in individual slaughterhouses. In addition, the possibility that management and food handling practices in slaughterhouses contribute to the differences in bacterial contamination between slaughterhouses needs further investigation.

Differences in inactivation of Escherichia coli O157:H7 strains in ground beef following repeated high pressure processing treatments and cold storage.

PubMed

Zhou, Yijing; Karwe, Mukund V; Matthews, Karl R

2016-09-01

High pressure processing (HPP) is a safe non-thermal processing method to effectively improve food safety. In this study, HPP treatment followed by cold storage was investigated to reduce Escherichia coli O157:H7 in ground beef. Experiments were conducted using ground beef contaminated with six E. coli O157:H7 strains one at a time or as a cocktail. Control and inoculated ground beef samples were HPP at 25 °C, 35 °C, and 45 °C, at 400 MPa and pre-determined number of pressure cycles totaling a holding time of 15 min. Optimum HPP parameters were 25 °C, 400 MPa at five pressure cycles of 3 min each which achieved a 5-log reduction of E. coli O157:H7 in ground beef. Storing HPP processed ground beef at 4 °C or -20 °C further decreased (P < 0.05) the E. coli O157:H7 population. An effective HPP treatment (5-log reduction) was developed that could be used post-processing to reduce the risk associated with E. coli O157:H7 contamination in ground beef. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds

PubMed Central

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa

2015-01-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. PMID:25809972

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds.

PubMed

Hamamoto, Kouta; Ueda, Shuhei; Yamamoto, Yoshimasa; Hirai, Itaru

2015-06-01

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Conventional and advanced oxidation processes used in disinfection of treated urban wastewater.

PubMed

Rodríguez-Chueca, J; Ormad, M P; Mosteo, R; Sarasa, J; Ovelleiro, J L

2015-03-01

The purpose of the current study is to compare the inactivation of Escherichia coli in wastewater effluents using conventional treatments (chlorination) and advanced oxidation processes (AOPs) such as UV irradiation, hydrogen peroxide (H2O2)/solar irradiation, and photo-Fenton processes. In addition, an analysis of the operational costs of each treatment is carried out taking into account the optimal dosages of chemicals used. Total inactivation of bacteria (7.5 log) was achieved by means of chlorination and UV irradiation. However, bacterial regrowth was observed 6 hours after the completion of UV treatment, obtaining a disinfection value around 3 to 4 log. On the other hand, the combination H2O2/solar irradiation achieved a maximum inactivation of E. coli of 3.30 ± 0.35 log. The photo-Fenton reaction achieved a level of inactivation of 4.87 ± 0.10 log. The order of disinfection, taking into account the reagent/cost ratio of each treatment, is as follows: chlorination > UV irradiation > photo-Fenton > H2O2/sunlight irradiation.

Microbiological testing of raw, boxed beef in the context of hazard analysis critical control point at a high-line-speed abattoir.

PubMed

Jericho, K W; Kozub, G C; Gannon, V P; Taylor, C M

2000-12-01

The efficacy of cold storage of raw, bagged, boxed beef was assessed microbiologically at a high-line-speed abattoir (270 carcasses per h). At the time of this study, plant management was in the process of creating a hazard analysis critical control point plan for all processes. Aerobic bacteria, coliforms, and type 1 Escherichia coli were enumerated (5 by 5-cm excision samples, hydrophobic grid membrane filter technology) before and after cold storage of this final product produced at six fabrication tables. In addition, the temperature-function integration technique (TFIT) was used to calculate the potential number of generations of E. coli during the first 24 or 48 h of storage of the boxed beef. Based on the temperature histories (total of 60 boxes, resulting from 12 product cuts, five boxes from each of two fabrication tables on each of 6 sampling days, and six types of fabrication tables), TFIT did not predict any growth of E. coli (with or without lag) for the test period. This was verified by E. coli mean log10 values of 0.65 to 0.42 cm2 (P > 0.05) determined by culture before and after the cooling process, respectively. Counts of aerobic bacteria and coliforms were significantly reduced (P < 0.001 and P < 0.05, respectively) during the initial period of the cooling process. There were significant microbiological differences (P < 0.05) between table-cut units.

Anti-aggregatory effect of cyclodextrins in the refolding process of recombinant growth hormones from Escherichia coli inclusion bodies.

PubMed

Bajorunaite, Egle; Cirkovas, Andrejus; Radzevicius, Kostas; Larsen, Kim Lambertsen; Sereikaite, Jolanta; Bumelis, Vladas-Algirdas

2009-06-01

Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-beta-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.

Interference in Bacterial Quorum Sensing: A Biopharmaceutical Perspective

PubMed Central

Rémy, Benjamin; Mion, Sonia; Plener, Laure; Elias, Mikael; Chabrière, Eric; Daudé, David

2018-01-01

Numerous bacteria utilize molecular communication systems referred to as quorum sensing (QS) to synchronize the expression of certain genes regulating, among other aspects, the expression of virulence factors and the synthesis of biofilm. To achieve this process, bacteria use signaling molecules, known as autoinducers (AIs), as chemical messengers to share information. Naturally occurring strategies that interfere with bacterial signaling have been extensively studied in recent years, examining their potential to control bacteria. To interfere with QS, bacteria use quorum sensing inhibitors (QSIs) to block the action of AIs and quorum quenching (QQ) enzymes to degrade signaling molecules. Recent studies have shown that these strategies are promising routes to decrease bacterial pathogenicity and decrease biofilms, potentially enhancing bacterial susceptibility to antimicrobial agents including antibiotics and bacteriophages. The efficacy of QSIs and QQ enzymes has been demonstrated in various animal models and are now considered in the development of new medical devices against bacterial infections, including dressings, and catheters for enlarging the therapeutic arsenal against bacteria. PMID:29563876

The presence of Enterococcus, coliforms and E. coli in a commercial yeast manufacturing process.

PubMed

O'Brien, S S; Lindsay, D; von Holy, A

2004-07-01

This study evaluated a typical commercial yeast manufacturing process for bacterial contamination. Product line samples of a commercial yeast manufacturing process and the corresponding seed yeast manufacturing process were obtained upstream from the final compressed and dry yeast products. All samples were analysed before (non-PI) and after preliminary incubation (PI) at 37 degrees C for 24 h. The PI procedure was incorporated for amplification of bacterial counts below the lower detection limit. Enterococcus, coliform and Escherichia coli counts were quantified by standard pour-plate techniques using selective media. Presence at all stages and progressive increases in counts of Enterococcus, coliforms and E. coli during processing in the commercial manufacturing operation suggested that the primary source of contamination of both compressed and dry yeast with these bacteria was the seed yeast manufacturing process and that contamination was amplified throughout the commercial yeast manufacturing process. This was confirmed by surveys of the seed yeast manufacturing process which indicated that contamination of the seed yeast with Enterococcus, coliforms and E. coli occurred during scale up of seed yeast biomass destined as inoculum for the commercial fermentation.

Occurrence of Coliform and Escherichia coli Contamination and Absence of Escherichia coli O157:H7 on Romaine Lettuce from Retail Stores in the Upper Midwest.

PubMed

Greve, Josephine D; Zietlow, Mark S; Miller, Kevin M; Ellingson, Jay L E

2015-09-01

A total of 720 whole, romaine lettuce heads were purchased from retail locations in the Upper Midwest and assessed for coliform and Escherichia coli contamination and for the presence of E. coli O157:H7. During a 16-month period (August 2010 through December 2011), coliform and E. coli counts were enumerated on Petrifilm, and the presence of E. coli O157:H7 and the virulence gene eae was evaluated by real-time PCR (qPCR). Over half (400 of 720) of the lettuce samples were processed with an immunomagnetic separation step before the qPCR assay. All retail lettuce samples were negative for E. coli O157:H7 when tested with the R.A.P.I.D. LT qPCR targeting a region of the O-antigen, and only two (0.28%) were positive for the eae gene when tested with LightCycler qPCR. On Petrifilm, coliform counts of most lettuce samples (96.4%) were between <10(1) and 10(3) CFU/g, and E. coli counts for nearly all lettuce samples (98.2%) were <10(1) CFU/g. No seasonal trend in coliform and E. coli counts was observed throughout the examination period nor was a difference in coliform counts observed between packaged and nonpackaged lettuce heads. These results contribute to the limited recorded data and understanding of microbial contamination of whole romaine lettuce heads purchased from retail locations, specifically revealing the absence of E. coli O157:H7 and low levels of contamination with coliforms and other E. coli strains.

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

PubMed

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

2017-01-01

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

Effects of in-plant interventions on reduction of enterohemorrhagic Escherichia coli and background indicator microorganisms on veal calf hides.

PubMed

Wang, Rong; Koohmaraie, Mohammad; Luedtke, Brandon E; Wheeler, Tommy L; Bosilevac, Joseph M

2014-05-01

Enterohemorrhagic Escherichia coli (EHEC) serotypes in veal have recently been recognized as a problem. Because hides are considered to be the principal source of EHEC and hide interventions have been shown to be very efficacious in the control of EHEC in beef processing plants, various hide-directed intervention strategies have been implemented in several veal processing plants to mitigate contamination. We evaluated the effectiveness of three different hide interventions used at veal processing plants: A, a water rinse followed by a manual curry comb of the hide; B, application of 200 ppm of chlorine followed by a hot water rinse; and C, a 5-min treatment with chlorine foam followed by a rinse with 180 to 200 ppm of acidified sodium chlorite. The levels of total aerobic bacteria, Enterobacteriaceae, coliforms, and E. coli, as well as the prevalence of Salmonella, E. coli O157:H7, and non-O157 EHEC, were determined on hides pre- and postintervention. Interventions A, B, and C reduced indicator organisms (P < 0.05) by 0.8 to 3.5 log CFU, 2.1 to 2.7 log CFU, and 1.0 to 1.5 log CFU, respectively. No Salmonella was detected on hides prior to intervention. E. coli O157:H7 prevalence was observed at only one plant, so comparison was not possible. Other non-O157 EHECs (O26, O103, and O111) were observed for all interventions studied. Interventions A and B reduced culture-confirmed non-O157 EHEC by 29 and 21 % , respectively, whereas intervention C did not reduce non-O157 EHEC. Our results show that the most effective veal hide intervention for reducing indicator organisms and EHECs was the application of 200 ppm of chlorine followed by hot water rinse. These data provide options that veal processors can consider in their EHEC control program.

Microbiological Quality Assessment of Game Meats at Retail in Japan.

PubMed

Asakura, Hiroshi; Kawase, Jun; Ikeda, Tetsuya; Honda, Mioko; Sasaki, Yoshimasa; Uema, Masashi; Kabeya, Hidenori; Sugiyama, Hiromu; Igimi, Shizunobu; Takai, Shinji

2017-12-01

In this study, we examined the prevalence of Shiga toxin-producing Escherichia coli and Salmonella spp. and the distribution of indicator bacteria in 248 samples of game meats (120 venison and 128 wild boar) retailed between November 2015 and March 2016 in Japan. No Salmonella spp. were detected in any of the samples, whereas Shiga toxin-producing Escherichia coli serotype OUT:H25 (stx 2d + , eae - ) was isolated from one deer meat sample, suggesting a possible source for human infection. Plate count assays indicated greater prevalence of coliforms and E. coli in wild boar meat than in venison, whereas their prevalence in processing facilities showed greater variation than in animal species. The 16S rRNA ion semiconductor sequencing analysis of 24 representative samples revealed that the abundances of Acinetobacter and Arthrobacter spp. significantly correlated with the prevalence of E. coli, and quantitative PCR analyses in combination with selective plate count assay verified these correlations. To our knowledge, this is the first report to characterize the diversity of microorganisms of game meats at retail in Japan, together with identification of dominant microbiota. Our data suggest the necessity of bottom-up hygienic assessment in areas of slaughtering and processing facilities to improve microbiological safety.

Feasibility study and sensory test of turmeric tamarind traditional drink with various processing

NASA Astrophysics Data System (ADS)

Rudyatmi, E.; Bintari, S. H.; Iswari, R. S.

2018-03-01

Turmeric tamarind traditional drink as anti-oxidant, good for health, and has a potency to developed. The objectives of this study are to determine the feasibility and the favorite level of consumer towards two turmerics tamarind traditional drink which processed differently. Feasibility depended on sensory test and microbe's contamination according to a quality standard of BPOM and complemented with an anti-bacterial test. The anti-bacterial test is determined by a diameter of inhibition zone towards E coli and B subtilist. Consumer’s favorites level is determined by organoleptic test to 40 testers. Sensory test results to form, odor, taste, and color are normal. TPC first traditional drink 6,9 x 102 col/gr and TKK ≤1,0x10 col/gr, TPC second traditional drink 2,0 x 101 col/gr and TKK ≤1,0x10 col/gr; all tests toward E coli, Salmonella SP, Staphylococcus aureus, Pseudomonas aeruginosa, shigella sp negative/gr; inhibiting capabilities towards B.subtilis and E coli of first traditional drink > second traditional drink. Inhibiting capabilities towards B.subtilis > E coli. All parameters comply with BPOM standard. Most of the tester love the first method turmeric tamarind traditional drink.

Characterization of recombinant E. coli expressing arsR from Rhodopseudomonas palustris CGA009 that displays highly selective arsenic adsorption.

PubMed

Ke, Changdong; Zhao, Chungui; Rensing, Christopher; Yang, Suping; Zhang, Yi

2018-05-22

Innovative methods to lower arsenic (As) exposure are sought. The As regulatory protein (ArsR) is reported of having high affinity and specificity to arsenite [As(III)]. Rhodopseudomonas palustris CGA009 is a good model organism for studying As detoxification due to at least three ars operons and four diverse arsR RP1-4 on the genome. In this study, four Escherichia coli harboring arsR RP1-4 derived from CGA009 were engineered and tested regarding their As resistance. The results showed that E. coli (arsR RP2 ) displayed robust As(III) resistance, and its growth inhibition rate was only 2.9% when exposed to 3.0 mmol/L As(III). At pH 7.0, E. coli (arsR RP2 ) showed an enhanced As adsorption capacity. As(III) (2.32 mg/g (dry weight, dw)) and 1.47 mg/g arsenate [As(V)] was adsorbed representing a 4.2-fold and 1.3-fold increase respectively compared to the control strain. The adsorption process was well fitted to Langmuir isothermal mode. E. coli (arsR RP2 ) (1.0~12.0 g/L) could remove 30.3~82.2% of As (III) when exposed to 10 μg/L As(III). No increase in absorption to copper(II), zinc(II), chromium(III), and lead(II) could be detected. Our studies revealed that arsR RP1-4 from CGA009 could confer As(III) resistance; E. coli (arsR RP2 ) displayed the highest As resistance, selectivity, and adsorption capacity within a wider pH (5.0~9.0) and salinity (0~15.0 g/L NaCl) range, especially important as it could remove As(III) from low concentration As-containing water.

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

PubMed Central

Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

2013-01-01

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

The binary protein-protein interaction landscape of Escherichia coli

PubMed Central

Rajagopala, Seesandra V.; Vlasblom, James; Arnold, Roland; Franca-Koh, Jonathan; Pakala, Suman B.; Phanse, Sadhna; Ceol, Arnaud; Häuser, Roman; Siszler, Gabriella; Wuchty, Stefan; Emili, Andrew; Babu, Mohan; Aloy, Patrick; Pieper, Rembert; Uetz, Peter

2014-01-01

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (~70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, approximately doubling the number of known binary PPIs in E. coli. Integration of binary PPIs and genetic interactions revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that could be mapped within multi-protein complexes were informative regarding internal topology and indicated that interactions within complexes are significantly more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily significant model microbe. PMID:24561554

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

PubMed

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

2018-04-12

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

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

PubMed Central

Kim, Jin Sun; Park, Su Jung; Kwak, Kyung Jin; Kim, Yeon Ok; Kim, Joo Yeol; Song, Jinkyung; Jang, Boseung; Jung, Che-Hun; Kang, Hunseung

2007-01-01

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process. PMID:17169986

Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens.

PubMed

Clifford, Jennifer C; Buchanan, Alex; Vining, Oliver; Kidarsa, Teresa A; Chang, Jeff H; McPhail, Kerry L; Loper, Joyce E

2016-10-01

Bacteria can be both highly communicative and highly competitive in natural habitats and antibiotics are thought to play a role in both of these processes. The soil bacterium Pseudomonas protegens Pf-5 produces a spectrum of antibiotics, two of which, pyoluteorin and 2,4-diacetylphloroglucinol (DAPG), function in intracellular and intercellular communication, both as autoinducers of their own production. Here, we demonstrate that phloroglucinol, an intermediate in DAPG biosynthesis, can serve as an intercellular signal influencing the expression of pyoluteorin biosynthesis genes, the production of pyoluteorin, and inhibition of Pythium ultimum, a phytopathogenic oomycete sensitive to pyoluteorin. Through analysis of RNAseq data sets, we show that phloroglucinol had broad effects on the transcriptome of Pf-5, significantly altering the transcription of more than two hundred genes. The effects of nanomolar versus micromolar concentrations of phloroglucinol differed both quantitatively and qualitatively, influencing the expression of distinct sets of genes or having opposite effects on transcript abundance of certain genes. Therefore, our results support the concept of hormesis, a phenomenon associated with signalling molecules that elicit distinct responses at different concentrations. Phloroglucinol is the first example of an intermediate of antibiotic biosynthesis that functions as a chemical messenger influencing gene expression in P. protegens. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Survival of Escherichia coli O157:H7 during the manufacture and ripening of Cacioricotta goat cheese.

PubMed

Ioanna, F; Quaglia, N C; Storelli, M M; Castiglia, D; Goffredo, E; Storelli, A; De Rosa, M; Normanno, G; Jambrenghi, A Caputi; Dambrosio, A

2018-04-01

The aim of this study was to assess the growth and survival of Escherichia coli O157:H7 during the manufacturing and ripening of Cacioricotta goat cheese. Goat milk was artificially contaminated with E. coli O157:H7 and the bacterial load was monitored from production up to 90 days of ripening. Goat milk was inoculated with 10 2  cfu ml -1 of E. coli O157:H7 and the bacterial count of the curd at time zero was 2.31 log 10  cfu g -1 . During the first day of ripening, the bacterial load has increased to 5.73 log 10  cfu g -1 to more than 6.20 log 10  cfu g -1 during the first week. The bacterial load remained constant up to 28 days and then slightly decreased until the end of ripening, with values of a w and pH of 0.88 and 5.41 respectively. The results of this study highlighted that E. coli O157:H7 is able to survive the manufacturing process and they suggest that the 90-day period of ripening alone is insufficient to remove E. coli O157:H7 in contaminated Cacioricotta goat cheese. Moreover, these results support the assumption that the presence of a low contamination of milk with E. coli O157:H7 could represent a potential source of infection and a threat to consumers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Virulence and antimicrobial resistance of Salmonella spp. and Escherichia coli in the beef jerky production line.

PubMed

Fernandes, Fernanda Pereira; Voloski, Flávia Liége Schütz; Ramires, Tassiana; Haubert, Louise; Reta, Giulia Giugliani; Mondadori, Rafael Gianella; Silva, Wladimir Padilha da; Conceição, Rita de Cássia Dos Santos da; Duval, Eduarda Hallal

2017-05-01

Intense manipulation during beef jerky production increases the possibility of contamination with pathogenic microorganisms. This study evaluated the contamination by thermotolerant coliforms, Escherichia coli and Salmonella spp., on processing surfaces and raw materials during beef jerky production, as well as in the final product. Thermotolerant coliforms were found on all surfaces tested and in the raw material. Escherichia coli was identified in 6.7% of the surface samples, while Salmonella spp. was found in 3.3% of the surface samples and 8.6% of raw material samples. Virulence genes were detected in Salmonella spp. isolates. One Salmonella spp. isolate was resistant to sulfonamide, while one E. coli isolate was multiresistant, including the presence of resistance genes sul2, strA, strB, tetA and tetB. The presence of coliforms demonstrates failings in hygienic-sanitary procedures. The presence of pathogenic microorganisms causing foodborne diseases in the production line indicates persistent contamination in the production plant. Although the drying process applied to beef jerky should guarantee the safety of the final product, the presence of multiresistant pathogenic microorganisms, presenting virulence genes, should be a matter of concern. Because beef jerky is a ready-to-eat product, a failure in the production process may cause such microorganisms to pose a public health risk. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The detrimental influence of bacteria (E. coli, Shigella and Salmonella) on the degradation of organic compounds (and vice versa) in TiO2 photocatalysis and near-neutral photo-Fenton processes under simulated solar light.

PubMed

Moncayo-Lasso, Alejandro; Mora-Arismendi, Luis Enrique; Rengifo-Herrera, Julián Andrés; Sanabria, Janeth; Benítez, Norberto; Pulgarin, César

2012-05-01

TiO2 photocatalytic and near-neutral photo-Fenton processes were tested under simulated solar light to degrade two models of natural organic matter - resorcinol (R) (which should interact strongly with TiO2 surfaces) and hydroquinone (H) - separately or in the presence of bacteria. Under similar oxidative conditions, inactivation of Escherichia coli, Shigella sonnei and Salmonella typhimurium was carried out in the absence and in the presence of 10 mg L(-1) of R and H. The 100% abatement of R and H by using a TiO2 photocatalytic process in the absence of bacteria was observed in 90 min for R and in 120 min for H, while in the presence of microorganisms abatement was only of 55% and 35% for R and H, respectively. Photo-Fenton reagent at pH 5.0 completely removed R and H in 40 min, whereas in the presence of microorganisms their degradation was of 60% to 80%. On the other hand, 2 h of TiO2 photocatalytic process inactivated S. typhimurium and E. coli cells in three and six orders of magnitude, respectively, while S. sonnei was completely inactivated in 10 min. In the presence of R or H, the bacterial inactivation via TiO2 photocatalysis was significantly decreased. With photo-Fenton reagent at pH 5 all the microorganisms tested were completely inactivated in 40 min of simulated solar light irradiation in the absence of organics. When R and H were present, bacterial photo-Fenton inactivation was less affected. The obtained results suggest that in both TiO2 and iron photo-assisted processes, there is competition between organic substances and bacteria simultaneously present for generated reactive oxygen species (ROS). This competition is most important in heterogeneous systems, mainly when there are strong organic-TiO2 surface interactions, as in the resorcinol case, suggesting that bacteria-TiO2 interactions could play a key role in photocatalytic cell inactivation processes.

Modeling the Inactivation of Intestinal Pathogenic Escherichia coli O157:H7 and Uropathogenic E. coli in Ground Chicken by High Pressure Processing and Thymol

PubMed Central

Chien, Shih-Yung; Sheen, Shiowshuh; Sommers, Christopher H.; Sheen, Lee-Yan

2016-01-01

Disease causing Escherichia coli commonly found in meat and poultry include intestinal pathogenic E. coli (iPEC) as well as extraintestinal types such as the Uropathogenic E. coli (UPEC). In this study we compared the resistance of iPEC (O157:H7) to UPEC in chicken meat using High Pressure Processing (HPP) in with (the hurdle concept) and without thymol essential oil as a sensitizer. UPEC was found slightly more resistant than E. coli O157:H7 (iPEC O157:H7) at 450 and 500 MPa. A central composite experimental design was used to evaluate the effect of pressure (300–400 MPa), thymol concentration (100–200 ppm), and pressure-holding time (10–20 min) on the inactivation of iPEC O157:H7 and UPEC in ground chicken. The hurdle approach reduced the high pressure levels and thymol doses imposed on the food matrices and potentially decreased food quality damaged after treatment. The quadratic equations were developed to predict the impact (lethality) on iPEC O157:H7 (R2 = 0.94) and UPEC (R2 = 0.98), as well as dimensionless non-linear models [Pr > F (<0.0001)]. Both linear and non-linear models were validated with data obtained from separated experiment points. All models may predict the inactivation/lethality within the same order of accuracy. However, the dimensionless non-linear models showed potential applications with parameters outside the central composite design ranges. The results provide useful information of both iPEC O157:H7 and UPEC in regard to how they may survive HPP in the presence or absence of thymol. The models may further assist regulatory agencies and food industry to assess the potential risk of iPEC O157:H7 and UPEC in ground chicken. PMID:27379050

Bioconversion of distillers' grains hydrolysates to advanced biofuels by an Escherichia coli co-culture.

PubMed

Liu, Fang; Wu, Weihua; Tran-Gyamfi, Mary B; Jaryenneh, James D; Zhuang, Xun; Davis, Ryan W

2017-11-09

First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers' grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate 'one-pot' bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains. The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn't compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer. The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.

Microbiological Food Safety Status of Commercially Produced Tomatoes from Production to Marketing.

PubMed

van Dyk, Brigitte N; de Bruin, Willeke; du Plessis, Erika M; Korsten, Lise

2016-03-01

Tomatoes have been implicated in various microbial disease outbreaks and are considered a potential vehicle for foodborne pathogens. Traceback studies mostly implicate contamination during production and/or processing. The microbiological quality of commercially produced tomatoes was thus investigated from the farm to market, focusing on the impact of contaminated irrigation and washing water, facility sanitation, and personal hygiene. A total of 905 samples were collected from three largescale commercial farms from 2012 through 2014. The farms differed in water sources used (surface versus well) and production methods (open field versus tunnel). Levels of total coliforms and Escherichia coli and prevalence of E. coli O157:H7 and Salmonella Typhimurium were determined. Dominant coliforms were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No pathogens or E. coli were detected on any of the tomatoes tested throughout the study despite the high levels of coliforms (4.2 to 6.2 log CFU/g) present on the tomatoes at the market. The dominant species associated with tomatoes belonged to the genera Enterobacter, Klebsiella, and Citrobacter. Water used on the farm for irrigation considered not fit for purpose according to national agricultural irrigation standards, with high E. coli levels resulting from either a highly contaminated source water (river water at 3.19 log most probable number [MPN]/100 ml) or improper storage of source water (stored well water at 1.72 log MPN/100 ml). Salmonella Typhimurium was detected on two occasions on a contact surface in the processing facility of the first farm in 2012. Contact surface coliform counts were 2.9 to 4.8 log CFU/cm(2). Risk areas identified in this study were water used for irrigation and poor sanitation practices in the processing facility. Implementation of effective food safety management systems in the fresh produce industry is of the utmost importance to ensure product safety for consumers.

Fate of naturally occurring Escherichia coli O157:H7 and other zoonotic pathogens during minimally managed bovine feedlot manure composting processes.

PubMed

Berry, Elaine D; Millner, Patricia D; Wells, James E; Kalchayanand, Norasak; Guerini, Michael N

2013-08-01

Reducing Escherichia coli O157:H7 in livestock manures before application to cropland is critical for reducing the risk of foodborne illness associated with produce. Our objective was to determine the fate of naturally occurring E. coli O157:H7 and other pathogens during minimally managed on-farm bovine manure composting processes. Feedlot pen samples were screened to identify E. coli O157:H7-positive manure. Using this manure, four piles of each of three different composting formats were constructed in each of two replicate trials. Composting formats were (i) turned piles of manure plus hay and straw, (ii) static stockpiles of manure, and (iii) static piles of covered manure plus hay and straw. Temperatures in the tops, toes, and centers of the conical piles (ca. 6.0 m(3) each) were monitored. Compost piles that were turned every 2 weeks achieved higher temperatures for longer periods in the tops and centers than did piles that were left static. E. coli O157:H7 was not recovered from top samples of turned piles of manure plus hay and straw at day 28 and beyond, but top samples from static piles were positive for the pathogen up to day 42 (static manure stockpiles) and day 56 (static covered piles of manure plus hay and straw). Salmonella, Campylobacter spp., and Listeria monocytogenes were not found in top or toe samples at the end of the composting period, but E. coli O157:H7 and Listeria spp. were recovered from toe samples at day 84. Our findings indicate that some minimally managed composting processes can reduce E. coli O157:H7 and other pathogens in bovine manure but may be affected by season and/or initial levels of indigenous thermophilic bacteria. Our results also highlight the importance of adequate C:N formulation of initial mixtures for the production of high temperatures and rapid composting, and the need for periodic turning of the piles to increase the likelihood that all parts of the mass are subjected to high temperatures.

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

PubMed

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

2018-05-01

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

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in biofilms by pulsed ultraviolet light.

PubMed

Montgomery, Nedra L; Banerjee, Pratik

2015-06-10

The inactivation of biofilms formed by pathogenic bacteria on ready-to-eat and minimally processed fruits and vegetables by nonthermal processing methods is critical to ensure food safety. Pulsed ultraviolet (PUV) light has shown promise in the surface decontamination of liquid, powdered, and solid foods. In this study, the antimicrobial efficacy of PUV light treatment on nascent biofilms formed by Escherichia coli O157:H7 and Listeria monocytogenes on the surfaces of food packaging materials, such as low-density polyethylene (LDPE), and fresh produce, such as lettuce (Lactuca sativa) leaves, was investigated. The formation of biofilms on Romaine lettuce leaves and LDPE films was confirmed by crystal violet and Alcian blue staining methods. Inactivation of cells in the biofilm was determined by standard plating procedures, and by a luminescence-based bacterial cell viability assay. Upon PUV treatment of 10 s at two different light source to sample distances (4.5 and 8.8 cm), viable cell counts of L. monocytogenes and E. coli O157:H7 in biofilms on the lettuce surface were reduced by 0.6-2.2 log CFU mL(-1) and 1.1-3.8 log CFU mL(-1), respectively. On the LDPE surface, the efficiency of inactivation of biofilm-encased cells was slightly higher. The maximum values for microbial reduction on LDPE were 2.7 log CFU mL(-1) and 3.9 log CFU mL(-1) for L. monocytogenes and E. coli O157:H7, respectively. Increasing the duration of PUV light exposure resulted in a significant (P < 0.05) reduction in biofilm formation by both organisms. The results also revealed that PUV treatment was more effective at reducing E. coli biofilms compared with Listeria biofilms. A moderate increase in temperature (~7-15°C) was observed for both test materials. PUV is an effective nonthermal intervention method for surface decontamination of E. coli O157:H7 and L. monocytogenes on fresh produce and packaging materials.

Lethality of home-style dehydrator processes against Escherichia coli O157:H7 and salmonella serovars in the manufacture of ground-and-formed beef jerky and the potential for using a pathogen surrogate in process validation.

PubMed

Borowski, A G; Ingham, S C; Ingham, B H

2009-10-01

Ground-and-formed beef jerky can be made easily at home with ground beef and kits that include spice, cure, and jerky-forming equipment. Ground beef poses inherent risks of illness due to Escherichia coli O157:H7 and Salmonella contamination, making adequate pathogen lethality important in jerky manufacturing. We evaluated the effectiveness of drying regimes at eliminating E. coli O157:H7 and Salmonella in seasoned ground-and-formed beef jerky manufactured with three home-style dehydrators and one small commercial unit. Inoculated jerky strips were dried for up to 12 or 24 h in a home-style or the commercial unit, respectively, with target drying temperatures ranging from 51.7 degrees C (125 degrees F) to 71.1 degrees C (160 degrees F). Pathogen lethality varied with seasoning, temperature, and drying time (n = 288 samples). Lethality against E. coli O157:H7 ranged from 1.5 log CFU (Jerky Xpress, 57.2 degrees C [135 degrees F], 4 h) to 6.4 log CFU (Gardenmaster, 68.3 degrees C [155 degrees F], 12 h), and varied with seasoning. Lethality against Salmonella ranged from 1.7 log CFU (Jerky Xpress, 57.2 degrees C [135 degrees F], 4 h) to 6.0 log CFU (Gardenmaster, 68.3 degrees C [155 degrees F], 12 h), and also varied with seasoning. There was a > or =5-log CFU reduction in both pathogens in 0, 10, and 27 % of samples at 4, 8, and 12 h, respectively. Heating jerky for 10 min at 135 degrees C (275 degrees F) 4 or 6 h postdrying increased lethality, on average, 2.99 log CFU for Salmonella and 3.02 log CFU for E. coli O157:H7. The use of a lactic acid bacterium culture (Pediococcus spp.) as a pathogen surrogate accurately predicted safety in 28 % of samples containing E. coli O157:H7 and 78% of Salmonella-inoculated samples.

Removal of antibiotic resistant E. coli in two Norwegian wastewater treatment plants and by nano- and ultra-filtration processes.

PubMed

Schwermer, Carsten Ulrich; Krzeminski, Pawel; Wennberg, Aina Charlotte; Vogelsang, Christian; Uhl, Wolfgang

2018-02-01

The effectivity of different treatment stages at two large wastewater treatment plants (WWTPs) located in Oslo, Norway, to remove antibiotic resistant Escherichia coli from municipal wastewater was investigated. The WWTPs were effective in reducing the total cultivable E. coli. The E. coli in WWTP samples were mainly resistant to ampicillin (6-27%) and trimethoprim-sulfamethoxazole (5-24%), and, to a lesser extent, tetracycline (3-14%) and ciprofloxacin (0-7%). In the first WWTP, a clear decrease in the percentage of E. coli resistant to these antibiotics was found, with the main removal occurring during physical/chemical treatment. In the second WWTP, the percentage of cultivable resistant E. coli did not display a considerable change. During laboratory-scale membrane filtration of WWTP effluents using ultrafiltration (UF) and nanofiltration (NF) membranes, all E. coli, including those resistant to antibiotics, were removed completely. The results imply that UF and NF processes are potent measures to remove antibiotic resistant bacteria (ARB) during post-treatment of WWTP effluents, thus reducing the potential spread of antibiotic resistance in the receiving aquatic environment.

Biocidal Defeat Agents Produced by Silver-Iodine Nanoenergetic Gas Generators

NASA Astrophysics Data System (ADS)

Davila, Ivan

Nanostructured aluminum (Al), iodine pentoxide (I2O5) nano-rods, and silver oxide (Ag2O) nanoparticles, (Al-I2O5-Ag 2O) were used to compose the ternary thermite composition that serves as a Nanoenergetic Gas Generator (NGG). This composition produces biocidal gases giving the mixture the ability to destroy highly pathogenic microorganisms or bacteria. The dissemination of the biocidal gas in combustion chamber was observed using a high-speed camera. The testing of NGG combustion process against the living Escherichia coli (E.coli) K-12 strain cells, that were cultivated/placed on the sample/chamber surfaces, demonstrated that iodine and silver atoms clouds were deposited to the bacteria surface. The 10/75/15 wt % of I2O5/Ag2O/Al composition demonstrated the best performance for destroying of E.coli with efficiency over 99 %. The results of the experiments showed that gaseous silver and iodine generated from NGG combustion produces a strong biocidal environment that has a great potential to neutralize highly pathogenic microorganisms and bacteria.

[The antibacterial activity of oregano essential oil (Origanum heracleoticum L.) against clinical strains of Escherichia coli and Pseudomonas aeruginosa].

PubMed

Sienkiewicz, Monika; Wasiela, Małgorzata; Głowacka, Anna

2012-01-01

The aim of this study was to investigate the antibacterial properties of oregano (Origanum heracleoticum L.) essential oil against clinical strains of Escherichia coli and Pseudomonas aeruginosa. The antibacterial activity of oregano essential oil was investigate against 2 tested and 20 clinical bacterial strains of Escherichia coli and 20 clinical strains o Pseudomonas aeruginosa come from patients with different clinical conditions. The agar dilution method was used for microbial growth inhibition at various concentrations ofoil. Susceptibility testing to antibiotics was carried out using disc-diffusion method. The results of experiments showed that the tested oil was active against all of the clinical strains from both genus of bacteria, but strains of Escherichia coli were more sensitive to tested oil. Essential oil from Origanum heracleoticum L. inhibited the growth of Escherichia coli and Pseudomonas aeruginosa clinical strains with different patters of resistance. The obtained outcomes will enable further investigations using oregano essential oil obtained from Origanum heracleoticum L. as alternative antibacterial remedies enhancing healing process in bacterial infections and as an effective means for the prevention of antibiotic-resistant strain development.

Conductive diamond sono-electrochemical disinfection (CDSED) for municipal wastewater reclamation.

PubMed

Llanos, Javier; Cotillas, Salvador; Cañizares, Pablo; Rodrigo, Manuel A

2015-01-01

In the present work, the disinfection of actual effluents from a municipal wastewater treatment plant (WWTP) by a conductive diamond sono-electrochemical process was assessed. First, efficiency of single electrodisinfection process with diamond anodes (without the contribution of ultrasounds) was studied, finding that the total disinfection can be attained at current charges applied below 0.02kAhm(-3). It was also found that the main disinfection mechanism is the attack of Escherichia coli (E. coli) by the disinfectants produced in the electrochemical cell and that the production of chlorates is avoided when working at current densities not higher than 1.27Am(-2). Next, a marked synergistic effect was found when coupling ultrasound (US) irradiation to the electrochemical system (sono-electrochemical disinfection). This increase in the disinfection rate was found to be related to the suppression of the agglomeration of E. coli cells and the enhancement in the production of disinfectant species. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluating the role of curcum powder as a protective factor against bladder cancer--an experimental study.

PubMed

El-Mesallamy, Hala; Salman, Tarek M; Ashmawey, Abeer M; Osama, Nada

2012-01-01

Throughout human history, plant products have been used for many purposes including as medicines. Herbal products and spices can be used as preventive agents against cancer due to their antimicrobial, antioxidant and antitumorigenic properties. This study was designed to evaluate the potential protective effect of curcum in rats administered nitrosamine precursors; dibutylamine (DBA) and sodium nitrate (NaNO3); and infected with Escherichia coli (E. coli) and also to monitor changes in nuclear factor the Kappa B p65 (NF-κB p56) pathway and its downstream products, Bcl-2 and interleukin-6 (IL-6), in parallel with nitrosamine precursors, E. coli and curcum treatment. Rats were divided into three groups (n=25 each; except of control group, n+20). Group I a normal control group, group II administered DBA/NaNO3 in drinking water and infected with E. coli and group III was administered DBA/NaNO3 in drinking water, infected with E. coli and receiving standard diet containing 1% curcum powder. Histopathological examination reflected that the curcum treated group featured a lower incidence of urinary bladder lesions,and lower levels of NF-κB, Bcl-2 and IL-6, than the group receiving nitrosamine precursor and infected with E. coli. These findings suggested that curcum may have a protective role during the process of bladder carcinogenesis by inhibiting the NF-κB pathway and its downstream products.

Escherichia coli Attenuation by Fe Electrocoagulation in Synthetic Bengal Groundwater: Effect of pH and Natural Organic Matter.

PubMed

Delaire, Caroline; van Genuchten, Case M; Nelson, Kara L; Amrose, Susan E; Gadgil, Ashok J

2015-08-18

Technologies addressing both arsenic and microbial contamination of Bengal groundwater are needed. Fe electrocoagulation (Fe-EC), a simple process relying on the dissolution of an Fe(0) anode to produce Fe(III) precipitates, has been shown to efficiently remove arsenic from groundwater at low cost. We investigated Escherichia coli (E. coli) attenuation by Fe-EC in synthetic Bengal groundwater as a function of Fe dosage rate, total Fe dosed, pH, and presence of natural organic matter (NOM). A 2.5 mM Fe dosage simultaneously achieved over 4-log E. coli attenuation and arsenic removal from 450 to below 10 μg/L. E. coli reduction was significantly enhanced at pH 6.6 compared to pH 7.5, which we linked to the decreased rate of Fe(II) oxidation at lower pH. 3 mg/L-C of NOM (Suwanee River fulvic acid) did not significantly affect E. coli attenuation. Live-dead staining and comparisons of Fe-EC with chemical coagulation controls showed that the primary mechanism of E. coli attenuation is physical removal with Fe(III) precipitates, with inactivation likely contributing as well at lower pH. Transmission electron microscopy showed that EC precipitates adhere to and bridge individual E. coli cells, resulting in large bacteria-Fe aggregates that can be removed by gravitational settling. Our results point to the promising ability of Fe-EC to treat arsenic and bacterial contamination simultaneously at low cost.

Biocatalytically active silCoat-composites entrapping viable Escherichia coli.

PubMed

Findeisen, A; Thum, O; Ansorge-Schumacher, M B

2014-02-01

Application of whole cells in industrial processes requires high catalytic activity, manageability, and viability under technical conditions, which can in principle be accomplished by appropriate immobilization. Here, we report the identification of carrier material allowing exceptionally efficient adsorptive binding of Escherichia coli whole cells hosting catalytically active carbonyl reductase from Candida parapsilosis (CPCR2). With the immobilizates, composite formation with both hydrophobic and hydrophilized silicone was achieved, yielding advanced silCoat-material and HYsilCoat-material, respectively. HYsilCoat-whole cells were viable preparations with a cell loading up to 400 mg(E. coli) · g(-1)(carrier) and considerably lower leaching than native immobilizates. SilCoat-whole cells performed particularly well in neat substrate exhibiting distinctly increased catalytic activity.

Individual and combined efficacies of mild heat and ultraviolet-c radiation against Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes in coconut liquid endosperm.

PubMed

Gabriel, Alonzo A; Ostonal, Jeffrey M; Cristobal, Jannelle O; Pagal, Gladess A; Armada, John Vincent E

2018-07-20

This study determined the inactivation kinetic parameters of selected pathogens in heat, ultraviolet-C and combined heat-UV-C treated coconut liquid endosperm. Separate cocktails of Escherichia coli O157:H7, Salmonella enterica serovars, and Listeria monocytogenes strains were inoculated into coconut liquid endosperm (pH 5.15, TSS 4.4 o Bx, TA 0.062% malic acid, extinction coefficient (ε) at 254 nm of 0.0154 cm -1 ) for inactivation studies. Result showed that all organisms generally exhibited a log-linear heat inactivation behavior (R 2 0.81-0.99). The E. coli O157:H7 cocktail (D 55  = 19.75 min, D 57  = 10.79 min, D 60  = 3.38 min, and D 63  = 0.46 min) was found to be significantly more resistant (P > 0.05) than the tested cocktail of L. monocytogenes (D 55  = 11.68 min, D 57  = 4.53 min, D 60  = 1.82 min and D 63  = 0.26 min) and S. enterica cocktail (D 55  = 3.08 min, D 57  = 2.60 min, D 60  = 0.89 min and D 63  = 0.25 min). Despite the differences in D T values, computed z values for L. monocytogenes cocktail (5.12 ± 0.43 °C) and E. coli O157:H7 cocktail (4.95 ± 0.12 °C) were not significantly different (P > 0.05), but were both significantly (P < 0.05) lower than that of S. enterica cocktail (7.10 ± 0.15 °C). All test organisms also exhibited a generally log-linear UV-C inactivation behavior (R 2 0.90-0.99) with E. coli O157:H7 cocktail (D UV-C  = 25.26 mJ/cm 2 ) demonstrating greatest resistance to UV-C than S. enterica (D UV-C  = 24.65 mJ/cm 2 ) and L. monocytogenes (D UV-C  = 17.30 mJ/cm 2 ) cocktails. The D 55 values of each organism cocktail were used to calculate for the 3-log reduction heating process schedules, during which UV-C treatments were simultaneously applied. Lethal rates (F values) calculations in the combined processes revealed that within the 3-log reduction heating processes, co-exposure of UV-C resulted in 5.62 to 6.20 log reductions in the test organism populations. Heating caused 69.3, 97.2, and 67.4% of the reduction in E. coli O157:H7, S. enterica and L. monocytogenes cocktails, respectively. These results can be used as baseline data in the establishment of mild heat treatment in combination with UV-C process schedules for coconut liquid endosperm and other similar products. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase.

PubMed

Zhang, Jianzhi; Li, Xi

2018-01-01

To enhance the efficiency of phenyllactic acid (PLA) production from L-phenylalanine (L-Phe) by introducing a novel artificial pathway into Escherichia coli RESULTS: The production of PLA from L-Phe by recombinant E. coli (ldh-lpox) coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied. The new PLA synthesis pathway was confirmed to be efficient in recombinant E. coli. Subsequently, two different biocatalyst processes were carried out and optimized for PLA production. In the whole cell biosynthesis process at high cell density using collected recombinant cells as catalyst, at optimal conditions (L-Phe 6 g/l, pH 7.5, 35 °C, CDW 24.5 g/l and 200 rpm), the recombinant E. coli (ldh-lpox) produced 1.62 g PLA/l with a conversion of 28% from L-Phe. Similarly, during the two-temperature-stage fermentation process in flasks using IPTG-induced cells, the temperature in the second stage was increased to 35 °C to benefit the biocatalyst process, and comparable phenyllactic acid production of 1.47 g/l was obtained from 12 g L-Phe/l. Recombinant E. coli (ldh-lpox) was efficient in PLA production realizing a high titer of several folds compared with studies using L-Phe as substrate.

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.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

PubMed

Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

2015-01-01

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Microbiological Status and Food Safety Compliance of Commercial Basil Production Systems.

PubMed

de Bruin, Willeke; Otto, Denise; Korsten, Lise

2016-01-01

Basil has been implicated in a number of microbe-associated foodborne illnesses across the world, and the source of contamination has often been traced back to the production and/or processing stages of the supply chain. The aim of this study was to evaluate the microbiological quality of fresh basil from the point of production to the retail outlet in the Gauteng and Northwest Provinces of South Africa. A total of 463 samples were collected over a 3-month period from two large-scale commercial herb producing and processing companies and three retail outlets. The microbiological quality of the samples was assessed based on the presence or absence of Escherichia coli O157:H7 and Salmonella Typhimurium and the levels of the indicator bacteria E. coli and total coliforms. Salmonella Typhimurium was detected on four basil samples (0.9%) arriving at the processing facility and at dispatch, but no E. coli O157:H7 was detected throughout the study. Total coliform counts were 0.4 to 4.1 CFU/g for basil, 1.9 to 3.4 log CFU/ml for water, and 0.2 to 1.7 log CFU/cm(2) for contact surfaces, whereas E. coli was detected in the water samples and only once on basil. The Colilert-18 and membrane filter methods were used to analyze water samples, and a comparison of results revealed that the Colilert-18 method was more sensitive. Strong evidence suggests that high numbers of coliforms do not necessarily indicate the presence of Salmonella Typhimurium. The study results highlight the importance of effective implementation of food safety management systems in the fresh produce industry.

Inactivation of the Haemophilus ducreyi luxS gene affects the virulence of this pathogen in human subjects.

PubMed

Labandeira-Rey, Maria; Janowicz, Diane M; Blick, Robert J; Fortney, Kate R; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M; Hansen, Eric J

2009-08-01

Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.

Prevalence and Persistence of Escherichia coli Strains with Uropathogenic Virulence Characteristics in Sewage Treatment Plants▿

PubMed Central

Anastasi, E. M.; Matthews, B.; Gundogdu, A.; Vollmerhausen, T. L.; Ramos, N. L.; Stratton, H.; Ahmed, W.; Katouli, M.

2010-01-01

We investigated the prevalence and persistence of Escherichia coli strains in four sewage treatment plants (STPs) in a subtropical region of Queensland, Australia. In all, 264 E. coli strains were typed using a high-resolution biochemical fingerprinting method and grouped into either a single or a common biochemical phenotype (S-BPT and C-BPT, respectively). These strains were also tested for their phylogenetic groups and 12 virulence genes associated with intestinal and extraintestinal E. coli strains. Comparison of BPTs at various treatment stages indicated that certain BPTs were found in two or all treatment stages. These BPTs constituted the highest proportion of E. coli strains in each STP and belonged mainly to phylogenetic group B2 and, to a lesser extent, group D. No virulence genes associated with intestinal E. coli were found among the strains, but 157 (59.5%) strains belonging to 14 C-BPTs carried one or more virulence genes associated with uropathogenic strains. Of these, 120 (76.4%) strains belonged to seven persistent C-BPTs and were found in all four STPs. Our results indicate that certain clonal groups of E. coli with virulence characteristics of uropathogenic strains can survive the treatment processes of STPs. These strains were common to all STPs and constituted the highest proportion of the strains in different treatment tanks of each STP. PMID:20622128

Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.

PubMed

Li, Cynthia H; Narhi, Linda O; Wen, Jie; Dimitrova, Mariana; Wen, Zai-qing; Li, Jenny; Pollastrini, Joseph; Nguyen, Xichdao; Tsuruda, Trace; Jiang, Yijia

2012-12-18

The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Ontology-based literature mining of E. coli vaccine-associated gene interaction networks.

PubMed

Hur, Junguk; Özgür, Arzucan; He, Yongqun

2017-03-14

Pathogenic Escherichia coli infections cause various diseases in humans and many animal species. However, with extensive E. coli vaccine research, we are still unable to fully protect ourselves against E. coli infections. To more rational development of effective and safe E. coli vaccine, it is important to better understand E. coli vaccine-associated gene interaction networks. In this study, we first extended the Vaccine Ontology (VO) to semantically represent various E. coli vaccines and genes used in the vaccine development. We also normalized E. coli gene names compiled from the annotations of various E. coli strains using a pan-genome-based annotation strategy. The Interaction Network Ontology (INO) includes a hierarchy of various interaction-related keywords useful for literature mining. Using VO, INO, and normalized E. coli gene names, we applied an ontology-based SciMiner literature mining strategy to mine all PubMed abstracts and retrieve E. coli vaccine-associated E. coli gene interactions. Four centrality metrics (i.e., degree, eigenvector, closeness, and betweenness) were calculated for identifying highly ranked genes and interaction types. Using vaccine-related PubMed abstracts, our study identified 11,350 sentences that contain 88 unique INO interactions types and 1,781 unique E. coli genes. Each sentence contained at least one interaction type and two unique E. coli genes. An E. coli gene interaction network of genes and INO interaction types was created. From this big network, a sub-network consisting of 5 E. coli vaccine genes, including carA, carB, fimH, fepA, and vat, and 62 other E. coli genes, and 25 INO interaction types was identified. While many interaction types represent direct interactions between two indicated genes, our study has also shown that many of these retrieved interaction types are indirect in that the two genes participated in the specified interaction process in a required but indirect process. Our centrality analysis of these gene interaction networks identified top ranked E. coli genes and 6 INO interaction types (e.g., regulation and gene expression). Vaccine-related E. coli gene-gene interaction network was constructed using ontology-based literature mining strategy, which identified important E. coli vaccine genes and their interactions with other genes through specific interaction types.

Biofuels from E. Coli: Engineering E. coli as an Electrofuels Chassis for Isooctane Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

2010-07-16

Electrofuels Project: Ginkgo Bioworks is bypassing photosynthesis and engineering E. coli to directly use carbon dioxide (CO2) to produce biofuels. E. coli doesn’t naturally metabolize CO2, but Ginkgo Bioworks is manipulating and incorporating the genes responsible for CO2 metabolism into the microorganism. By genetically modifying E. coli, Ginkgo Bioworks will enhance its rate of CO2 consumption and liquid fuel production. Ginkgo Bioworks is delivering CO2 to E. coli as formic acid, a simple industrial chemical that provides energy and CO2 to the bacterial system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frederix, Marijke; Mingardon, Florence; Hu, Matthew

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate (C 2C 1Im][OAc] ) formore » pretreatment of switchgrass biomass. [C 2C 1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C 2C 1Im]-tolerant E. coli strain, where [C 2C 1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C 2C 1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C 2C 1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C 2C 1 Im]-tolerant E. coli strain so as to secrete this [C 2C 1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process.« less

Improved Detection of Extended Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli in Input and Output Samples of German Biogas Plants by a Selective Pre-Enrichment Procedure

PubMed Central

Schauss, Thorsten; Glaeser, Stefanie P.; Gütschow, Alexandra; Dott, Wolfgang; Kämpfer, Peter

2015-01-01

The presence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli was investigated in input (manure from livestock husbandry) and output samples of six German biogas plants in 2012 (one sampling per biogas plant) and two German biogas plants investigated in an annual cycle four times in 2013/2014. ESBL-producing Escherichia coli were cultured by direct plating on CHROMagar ESBL from input samples in the range of 100 to 104 colony forming units (CFU) per g dry weight but not from output sample. This initially indicated a complete elimination of ESBL-producing E. coli by the biogas plant process. Detected non target bacteria were assigned to the genera Acinetobacter, Pseudomonas, Bordetella, Achromobacter, Castellaniella, and Ochrobactrum. A selective pre-enrichment procedure increased the detection efficiency of ESBL-producing E. coli in input samples and enabled the detection in five of eight analyzed output samples. In total 119 ESBL-producing E. coli were isolated from input and 46 from output samples. Most of the E. coli isolates carried CTX-M-type and/or TEM-type beta lactamases (94%), few SHV-type beta lactamase (6%). Sixty-four bla CTX-M genes were characterized more detailed and assigned mainly to CTX-M-groups 1 (85%) and 9 (13%), and one to group 2. Phylogenetic grouping of 80 E. coli isolates showed that most were assigned to group A (71%) and B1 (27%), only one to group D (2%). Genomic fingerprinting and multilocus sequence typing (MLST) showed a high clonal diversity with 41 BOX-types and 19 ST-types. The two most common ST-types were ST410 and ST1210. Antimicrobial susceptibility testing of 46 selected ESBL-producing E. coli revealed that several isolates were additionally resistant to other veterinary relevant antibiotics and some grew on CHROMagar STEC but shiga-like toxine (SLT) genes were not detected. Resistance to carbapenems was not detected. In summary the study showed for the first time the presence of ESBL-producing E. coli in output samples of German biogas plants. PMID:25799434

21 CFR 129.80 - Processes and controls.

Code of Federal Regulations, 2010 CFR

2010-04-01

... determine whether any of the coliform organisms are E. coli. (2) For chemical, physical, and radiological... bactericidal action to that required in paragraph (d)(3) of this section. (e) Unit package production code...

21 CFR 129.80 - Processes and controls.

Code of Federal Regulations, 2011 CFR

2011-04-01

... determine whether any of the coliform organisms are E. coli. (2) For chemical, physical, and radiological... bactericidal action to that required in paragraph (d)(3) of this section. (e) Unit package production code...

Biological and Physicochemical Wastewater Treatment Processes Reduce the Prevalence of Virulent Escherichia coli

PubMed Central

Biswal, Basanta Kumar; Mazza, Alberto; Masson, Luke; Gehr, Ronald

2013-01-01

Effluents discharged from wastewater treatment plants are possible sources of pathogenic bacteria, including Escherichia coli, in the freshwater environment, and determining the possible selection of pathogens is important. This study evaluated the impact of activated sludge and physicochemical wastewater treatment processes on the prevalence of potentially virulent E. coli. A total of 719 E. coli isolates collected from four municipal plants in Québec before and after treatment were characterized by using a customized DNA microarray to determine the impact of treatment processes on the frequency of specific pathotypes and virulence genes. The percentages of potentially pathogenic E. coli isolates in the plant influents varied between 26 and 51%, and in the effluents, the percentages were 14 to 31%, for a reduction observed at all plants ranging between 14 and 45%. Pathotypes associated with extraintestinal pathogenic E. coli (ExPEC) were the most abundant at three of the four plants and represented 24% of all isolates, while intestinal pathogenic E. coli pathotypes (IPEC) represented 10% of the isolates. At the plant where ExPEC isolates were not the most abundant, a large number of isolates were classified as both ExPEC and IPEC; overall, 6% of the isolates were classified in both groups, with the majority being from the same plant. The reduction of the proportion of pathogenic E. coli could not be explained by the preferential loss of one virulence gene or one type of virulence factor; however, the quinolone resistance gene (qnrS) appears to enhance the loss of virulence genes, suggesting a mechanism involving the loss of pathogenicity islands. PMID:23160132

Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association.

PubMed

Chun, Carlene K; Troll, Joshua V; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E; Bonaldo, Maria de Fatima; Casavant, Thomas L; Soares, M Bento; Ruby, Edward G; McFall-Ngai, Margaret J

2008-08-12

The light-organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution.

Effects of colonization, luminescence, and autoinducer on host transcription during development of the squid-vibrio association

PubMed Central

Chun, Carlene K.; Troll, Joshua V.; Koroleva, Irina; Brown, Bartley; Manzella, Liliana; Snir, Einat; Almabrazi, Hakeem; Scheetz, Todd E.; de Fatima Bonaldo, Maria; Casavant, Thomas L.; Soares, M. Bento; Ruby, Edward G.; McFall-Ngai, Margaret J.

2008-01-01

The light–organ symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri offers the opportunity to decipher the hour-by-hour events that occur during the natural colonization of an animal's epithelial surface by its microbial partners. To determine the genetic basis of these events, a glass-slide microarray was used to characterize the light-organ transcriptome of juvenile squid in response to the initiation of symbiosis. Patterns of gene expression were compared between animals not exposed to the symbiont, exposed to the wild-type symbiont, or exposed to a mutant symbiont defective in either of two key characters of this association: bacterial luminescence or autoinducer (AI) production. Hundreds of genes were differentially regulated as a result of symbiosis initiation, and a hierarchy existed in the magnitude of the host's response to three symbiont features: bacterial presence > luminescence > AI production. Putative host receptors for bacterial surface molecules known to induce squid development are up-regulated by symbiont light production, suggesting that bioluminescence plays a key role in preparing the host for bacteria-induced development. Further, because the transcriptional response of tissues exposed to AI in the natural context (i.e., with the symbionts) differed from that to AI alone, the presence of the bacteria potentiates the role of quorum signals in symbiosis. Comparison of these microarray data with those from other symbioses, such as germ-free/conventionalized mice and zebrafish, revealed a set of shared genes that may represent a core set of ancient host responses conserved throughout animal evolution. PMID:18682555

UV Tolerance of Spoilage Microorganisms and Acid-Shocked and Acid-Adapted Escherichia coli in Apple Juice Treated with a Commercial UV Juice-Processing Unit.

PubMed

Usaga, Jessie; Padilla-Zakour, Olga I; Worobo, Randy W

2016-02-01

The enhanced thermal tolerance and survival responses of Escherichia coli O157:H7 in acid and acidified foods is a major safety concern for the production of low-pH products, including beverages. Little is known about this phenomenon when using UV light treatments. This study was conducted to evaluate the effects of strain (E. coli O157:H7 strains C7927, ATCC 35150, ATCC 43895, and ATCC 43889 and E. coli ATCC 25922) and physiological state (control-unadapted, acid adapted, and acid shocked) on the UV tolerance of E. coli in apple juice treated under conditions stipulated in current U.S. Food and Drug Administration regulations. A greater than 5-log reduction of E. coli was obtained under all tested conditions. A significant effect of strain (P < 0.05) was observed, but the physiological state did not influence pathogen inactivation (P ≥ 0.05). The UV sensitivity of three spoilage microorganisms (Aspergillus niger, Penicillium commune, and Alicyclobacillus acidoterrestris) was also determined at UV doses of 0 to 98 mJ/cm(2). Alicyclobacillus was the most UV sensitive, followed by Penicillium and Aspergillus. Because of the nonsignificant differences in UV sensitivity of E. coli in different physiological states, the use of an unadapted inoculum would be adequate to conduct challenge studies with the commercial UV unit used in this study at a UV dose of 14 mJ/cm(2). The high UV tolerance of spoilage microorganisms supports the need to use a hurdle approach (e.g., coupling of refrigeration, preservatives, and/or other technologies) to extend the shelf life of UV-treated beverages.

Lactobacillus acidophilus INMIA 9602 Er-2 strain 317/402 probiotic regulates growth of commensal Escherichia coli in gut microbiota of familial Mediterranean fever disease subjects.

PubMed

Pepoyan, A Z; Balayan, M H; Manvelyan, A M; Mamikonyan, V; Isajanyan, M; Tsaturyan, V V; Kamiya, S; Netrebov, V; Chikindas, M L

2017-04-01

Previously, we reported a positive effect the probiotic formulation, Lactobacillus acidophilus INMIA 9602 Er-2 strain 317/402 (Narine strain), had on the blood characteristics of patients with familial Mediterranean fever disease (FMF). The aim of this investigation was to evaluate the effect of the Narine probiotic on growth characteristics in the predominant commensal Escherichia coli isolates from the gut microbiota in FMF-positive study participants. Bacterial growth of 192 prevalent commensal E. coli isolates found in the volunteer participants' guts was evaluated using Verhulst's logistic function. This study showed that the duration of the preparatory growth phase for the E. coli isolates collected from FMF-positive volunteers was significantly shorter, whereas the duration of the logarithmic growth phase was significantly longer (P < 0·03) than that of the isolates collected from healthy participants. The Narine probiotic formulation caused a significant extension (P < 0·001) of the preparatory growth phase in the commensal E. coli isolated from FMF subjects a month after the Narine probiotic administration was terminated. The data suggest that the mathematical model characterizes the growth of commensal E. coli isolates from FMF-positive participants and it can be useful in a decision-making process on the practical use of probiotics during FMF. This is the first study to demonstrate the effects of Narine, containing the probiotic Lactobacillus acidophilus, on the growth of gut commensal Escherichia coli from study participants with familial Mediterranean fever disease (FMF). Verhulst's logistic function was demonstrated to act as a possible tool for the evaluation and quantification of effects produced by the probiotic formulation in FMF participants. © 2017 The Society for Applied Microbiology.

Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays

PubMed Central

Dobrindt, Ulrich; Agerer, Franziska; Michaelis, Kai; Janka, Andreas; Buchrieser, Carmen; Samuelson, Martin; Svanborg, Catharina; Gottschalk, Gerhard; Karch, Helge; Hacker, Jörg

2003-01-01

Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA “pathoarray” developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes. PMID:12618447

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

PubMed

Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

2014-01-01

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Inactivation of Escherichia coli O157:H7 and Salmonella Typhimurium in black pepper and red pepper by gamma irradiation.

PubMed

Song, Won-Jae; Sung, Hye-Jung; Kim, Sung-Youn; Kim, Kwang-Pyo; Ryu, Sangryeol; Kang, Dong-Hyun

2014-02-17

This study evaluated the efficacy of gamma irradiation to inactivate foodborne pathogens in black pepper (Piper nigrum) and red pepper (dried Capsicum annuum). Black pepper and red pepper inoculated with Escherichia coli O157:H7 and Salmonella Typhimurium were subjected to gamma irradiation in the range of 0, 1, 2, 3 and 5 kGy, and color change was evaluated after treatment. Pathogen populations decreased with increasing treatment doses. A gamma irradiation dose of 5 kGy decreased E. coli O157:H7 and S. Typhimurium populations >4.4 to >5.2 log CFU/g in black pepper without causing color change. Similarly, 5 kGy of gamma irradiation yielded reduction of 3.8 to >5.2 log CFU/g for E. coli O157:H7 and S. Typhimurium in red pepper. During gamma irradiation treatment, L*, a* and b* values of red pepper were not significantly changed except for 297 μm to 420 μm size red pepper treated with 5 kGy of gamma irradiation. Based on the D-value of pathogens in black pepper and red pepper, S. Typhimurium showed more resistant to gamma irradiation than did E. coli O157:H7. These results show that gamma irradiation has potential as a non-thermal process for inactivating foodborne pathogens in spices with minimal color changes. Copyright © 2013 Elsevier B.V. All rights reserved.

Finished Genome Sequence of Escherichia coli K-12 Strain HMS174 (ATCC 47011).

PubMed

Mairhofer, Juergen; Krempl, Peter M; Thallinger, Gerhard G; Striedner, Gerald

2014-11-20

Escherichia coli strain K-12 substrain HMS174 is an engineered descendant of the E. coli K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is used in fermentation processes for heterologous protein production. Here, we report the complete genome sequence of E. coli HMS174 (ATCC 47011). Copyright © 2014 Mairhofer et al.

Inactivation of a diverse set of shiga toxin-producing Escherichia coli in ground beef by high pressure processing

USDA-ARS?s Scientific Manuscript database

Shiga Toxin-Producing Escherichia coli (STEC) are frequently implicated in foodborne illness outbreaks and recalls of ground beef. In this study we determined the High Pressure Processing (HPP) D-10 value (the processing conditions needed to reduce the microbial population by 1 log) of 39 individua...

High-throughput process development of an alternative platform for the production of virus-like particles in Escherichia coli.

PubMed

Ladd Effio, Christopher; Baumann, Pascal; Weigel, Claudia; Vormittag, Philipp; Middelberg, Anton; Hubbuch, Jürgen

2016-02-10

The production of safe vaccines against untreatable or new diseases has pushed the research in the field of virus-like particles (VLPs). Currently, a large number of commercial VLP-based human vaccines and vaccine candidates are available or under development. A promising VLP production route is the controlled in vitro assembly of virus proteins into capsids. In the study reported here, a high-throughput screening (HTS) procedure was implemented for the upstream process development of a VLP platform in bacterial cell systems. Miniaturized cultivations were carried out in 48-well format in the BioLector system (m2p-Labs, Germany) using an Escherichia coli strain with a tac promoter producing the murine polyomavirus capsid protein (VP1). The screening procedure incorporated micro-scale cultivations, HTS cell disruption by sonication and HTS-compatible analytics by capillary gel electrophoresis. Cultivation temperatures, shaking speeds, induction and medium conditions were varied to optimize the product expression in E. coli. The most efficient system was selected based on an evaluation of soluble and insoluble product concentrations as well as on the percentage of product in the total soluble protein fraction. The optimized system was scaled up to cultivation 2.5L shaker flask scale and purified using an anion exchange chromatography membrane adsorber, followed by a size exclusion chromatography polishing procedure. For proof of concept, purified VP1 capsomeres were assembled under defined buffer conditions into empty capsids and characterized using transmission electron microscopy (TEM). The presented HTS procedure allowed for a fast development of an efficient production process of VLPs in E. coli. Under optimized cultivation conditions, the VP1 product totalled up to 43% of the total soluble protein fraction, yielding 1.63 mg VP1 per mL of applied cultivation medium. The developed production process strongly promotes the murine polyoma-VLP platform, moving towards an industrially feasible technology for new chimeric vaccines. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid fluorescence detection of pathogenic bacteria using magnetic enrichment technique combined with magnetophoretic chromatography.

PubMed

Che, Yulan; Xu, Yi; Wang, Renjie; Chen, Li

2017-08-01

A rapid and sensitive analytical method was developed to detect pathogenic bacteria which combined magnetic enrichment, fluorescence labeling with polyethylene glycol (PEG) magnetophoretic chromatography. As pathogenic bacteria usually exist in complex matrixes at low concentration, an efficient enrichment is essential for diagnosis. In order to capture series types of pathogenic bacteria in samples, amino-modified magnetic nanoparticles (Fe 3 O 4 @SiO 2 -NH 2 ) were prepared for efficient enrichment by the electrostatic interaction with pathogenic bacteria. It was shown that the capture efficiency reached up to 95.4% for Escherichia coli (E. coli). Furthermore, quantitative analysis of the bacteria was achieved by using acridine orange (AO) as a fluorescence probe for the captured E. coli due to its ability of staining series types of bacteria and rapid labeling. In order to remove the free magnetic nanoparticles and redundant fluorescent reagent, the labeled suspension was poured into a PEG separation column and was separated by applying an external magnetic field. The presence of 100 cfu mL -1 E. coli could be detected for semi-quantitative analysis by observing the separation column with the naked eye, and the concentration could be further evaluated by fluorescence detection. All the above processes were finished within 80 min. It was demonstrated that a good linear relationship existed between the fluorescence intensity and the concentration of E. coli ranging from 10 2 to 10 6  cfu mL -1 , with a detection limit of 100 cfu mL -1 when E. coli acted as target bacteria. The recovery rate of E. coli was 93.6∼102.0% in tap water and cooked meat samples, and the RSD was lower than 7% (n = 6); the result coincided with the conventional plate count method. Graphical abstract ᅟ.

The Modular Organization of Protein Interactions in Escherichia coli

PubMed Central

Peregrín-Alvarez, José M.; Xiong, Xuejian; Su, Chong; Parkinson, John

2009-01-01

Escherichia coli serves as an excellent model for the study of fundamental cellular processes such as metabolism, signalling and gene expression. Understanding the function and organization of proteins within these processes is an important step towards a ‘systems’ view of E. coli. Integrating experimental and computational interaction data, we present a reliable network of 3,989 functional interactions between 1,941 E. coli proteins (∼45% of its proteome). These were combined with a recently generated set of 3,888 high-quality physical interactions between 918 proteins and clustered to reveal 316 discrete modules. In addition to known protein complexes (e.g., RNA and DNA polymerases), we identified modules that represent biochemical pathways (e.g., nitrate regulation and cell wall biosynthesis) as well as batteries of functionally and evolutionarily related processes. To aid the interpretation of modular relationships, several case examples are presented, including both well characterized and novel biochemical systems. Together these data provide a global view of the modular organization of the E. coli proteome and yield unique insights into structural and evolutionary relationships in bacterial networks. PMID:19798435

Occurrence of antimicrobial-resistant Escherichia coli and Salmonella enterica in the beef cattle production and processing continuum

USDA-ARS?s Scientific Manuscript database

Specific concerns have been raised that 3rd-generation cephalosporin-resistant (3GCr) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr) E. coli, 3GCr Salmonella enterica, and nalidixic acid-resistant (NALr) S. enterica, may be present in cattle production environments, persist through...

A novel toolbox for E. coli lysis monitoring.

PubMed

Rajamanickam, Vignesh; Wurm, David; Slouka, Christoph; Herwig, Christoph; Spadiut, Oliver

2017-01-01

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future.

Quantum-like model for the adaptive dynamics of the genetic regulation of E. coli's metabolism of glucose/lactose.

PubMed

Asano, Masanari; Basieva, Irina; Khrennikov, Andrei; Ohya, Masanori; Tanaka, Yoshiharu; Yamato, Ichiro

2012-06-01

We developed a quantum-like model describing the gene regulation of glucose/lactose metabolism in a bacterium, Escherichia coli. Our quantum-like model can be considered as a kind of the operational formalism for microbiology and genetics. Instead of trying to describe processes in a cell in the very detail, we propose a formal operator description. Such a description may be very useful in situation in which the detailed description of processes is impossible or extremely complicated. We analyze statistical data obtained from experiments, and we compute the degree of E. coli's preference within adaptive dynamics. It is known that there are several types of E. coli characterized by the metabolic system. We demonstrate that the same type of E. coli can be described by the well determined operators; we find invariant operator quantities characterizing each type. Such invariant quantities can be calculated from the obtained statistical data.

Copper import in Escherichia coli by the yersiniabactin metallophore system

PubMed Central

Koh, Eun-Ik; Robinson, Anne E.; Bandara, Nilantha; Rogers, Buck E.; Henderson, Jeffrey P.

2017-01-01

Copper plays a dual role as nutrient and toxin during bacterial infections. While uropathogenic Escherichia coli (UPEC) strains can use the copper-binding metallophore yersiniabactin (Ybt) to resist copper toxicity, Ybt also converts bioavailable copper to Cu(II)-Ybt in low copper conditions. Although E. coli have long been considered to lack a copper import pathway, we observed Ybt-mediated copper import in UPEC using canonical Fe(III)-Ybt transport proteins. UPEC removed copper from Cu(II)-Ybt with subsequent re-export of metal-free Ybt to the extracellular space. Copper released through this process became available to an E. coli cuproenzyme (the amine oxidase TynA), linking this import pathway to a nutrient acquisition function. Ybt-expressing E. coli thus engage in nutritional passivation, a strategy of minimizing a metal ion's toxicity while preserving its nutritional availability. Copper acquisition through this process may contribute to the marked virulence defect of Ybt transport-deficient UPEC. PMID:28759019

Survival of Escherichia coli O157:H7 during manufacture and storage of white brined cheese.

PubMed

Osaili, Tareq M; Al-Nabulsi, Anas A; Olaimat, Amin N; Shaker, Reyad R; Taha, Mohammad; Holley, Richard A

2014-09-01

Escherichia coli O157:H7 is a major foodborne pathogen that causes severe disease in humans. Survival of E. coli O157:H7 during processing and storage of white brined cheese was investigated. Cheeses were prepared using pasteurized milk inoculated with a 4 strain E. coli O157:H7 cocktail (7 log(10) CFU/g) with or without yogurt starter culture (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus salivarius ssp. thermophilus) and stored in 10% or 15% NaCl brine at 10 and 21 ºC for 28 d. NaCl concentration, water activity (a(w)), pH, and numbers of E. coli O157:H7 and lactic acid bacteria (LAB) were determined in cheese and brine. E. coli O157:H7 was able to survive in cheese stored in both brines at 10 and 21 ºC regardless of the presence of starter LAB, although the latter significantly enhanced E. coli O157:H7 reduction in cheese or its brine at 10 ºC. E. coli O157:H7 numbers were reduced by 2.6 and 3.4 log(10) CFU/g in cheese stored in 10% and 15% NaCl brine, respectively, in the presence of starter LAB and by 1.4 and 2.3 log(10) CFU/g, respectively, in the absence of starter LAB at 10 ºC. The pathogen survived, but at lower numbers in the brines. The salt concentration of cheese stored in 10% brine remained about 5% during ripening, but in 15% brine, the NaCl level increased 1.6% to 8.1% (w/w) by 28 d. Values of pH and a(w) slightly decreased 1 d after exposure to brine and reached 5.5 to 6.6 and 0.88 to 0.94, respectively, in all treatments. © 2014 Institute of Food Technologists®

Ongoing Horizontal and Vertical Transmission of Virulence Genes and papA Alleles among Escherichia coli Blood Isolates from Patients with Diverse-Source Bacteremia

PubMed Central

Johnson, James R.; O'Bryan, Timothy T.; Kuskowski, Michael; Maslow, Joel N.

2001-01-01

The phylogenetic distributions of multiple putative virulence factors (VFs) and papA (P fimbrial structural subunit) alleles among 182 Escherichia coli blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coli Reference (ECOR) collection, and other collections of extraintestinal pathogenic E. coli (ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g., fyuA (yersiniabactin; 76%) and focG (F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g., iut (aerobactin; 57%) and sfaS (S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in E. coli and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process. PMID:11500406

Titanium dioxide/UV photocatalytic disinfection in fresh carrots.

PubMed

Cho, Mihee; Choi, Yoonjung; Park, Hyojin; Kim, Kwansik; Woo, Gun-Jo; Park, Jiyong

2007-01-01

Increased occurrences of fresh produce-related outbreaks of foodborne illness have focused attention on effective washing processes for fruits and vegetables. A titanium dioxide (TiO2) photocatalytic reaction under UV radiation provides a high rate of disinfection. The photo-killing effects of TiO2 on bacteria in liquid cultures under experimental conditions have been widely studied. However, the disinfection effects of the TiO2 photocatalytic reaction on fresh vegetables during a washing process have not been evaluated. Our objectives were to design a pilot-scale TiO2/UV photocatalytic reactor for fresh carrots and to compare the bactericidal effects of the TiO2/UV reaction against bacteria in liquid media and on carrots. TiO2/UV photocatalytic reactions for 40, 60, and 30 s were required for the complete killing of Escherichia coli, Salmonella Typhimurium, and Bacillus cereus (initial counts of approximately 6.7 log CFU/ml), respectively. The counts of total aerobic bacteria in fresh carrots and foodborne pathogenic bacteria in inoculated carrots were also measured. Counts of total aerobic bacteria were reduced by 1.8 log CFU/g after TiO2/UV photocatalytic disinfection for 20 min compared with a 1.1-log CFU/g reduction by UV alone. E. coli, Salmonella Typhimurium, and B. cereus (8 log CFU/ml) were inoculated onto carrots, and the number of surviving bacteria in carrots was determined after treatment. The TiO2/UV treatment exhibited 2.1-, 2.3-, and 1.8-log CFU/g reductions in the counts of E. coli, Salmonella Typhimurium, and B. cereus, respectively, compared with 1.3-, 1.2-, and 1.2-log CFU/g reductions by UV alone. The TiO2/UV photocatalyst reaction showed significant bactericidal effects, indicating that this process is applicable to nonthermal disinfection of fresh vegetables.

Characterization of Escherichia coli O157:H7 strains from contaminated raw beef trim during "high event periods".

PubMed

Arthur, Terrance M; Bono, James L; Kalchayanand, Norasak

2014-01-01

The development and implementation of effective antimicrobial interventions by the beef processing industry in the United States have dramatically reduced the incidence of beef trim contamination by Escherichia coli O157:H7. However, individual processing plants still experience sporadic peaks in contamination rates where multiple E. coli O157:H7-positive lots are clustered in a short time frame. These peaks have been referred to as "high event periods" (HEP) of contamination. The results reported here detail the characterization of E. coli O157:H7 isolates from 21 HEP across multiple companies and processing plants to gain insight regarding the mechanisms causing these incidents. Strain genotypes were determined by pulsed-field gel electrophoresis, and isolates were investigated for characteristics linking them to human illness. Through these analyses, it was determined that individual HEP show little to no diversity in strain genotypes. Hence, each HEP has one strain type that makes up most, if not all, of the contamination. This is shown to differ from the genotypic diversity of E. coli O157:H7 found on the hides of cattle entering processing plants. In addition, it was found that a large proportion (81%) of HEP are caused by strain types associated with human illness. These results pose a potential challenge to the current model for finished product contamination during beef processing.

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.

PubMed

Lauber, Jennifer; Handrick, René; Leptihn, Sebastian; Dürre, Peter; Gaisser, Sabine

2015-01-13

Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. This work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF. In the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

Detection of Escherichia coli O157:H7 and Salmonella enterica in air and droplets at three U.S. commercial beef processing plants

USDA-ARS?s Scientific Manuscript database

Bacteria are known to be present in air at beef processing plants but published data regarding the prevalences of airborne Escherichia coli O157:H7 and Salmonella enterica are very limited. To determine if airborne pathogens were present in beef processing facilities, we placed sedimentation sponges...

Methodology for modeling the disinfection efficiency of fresh-cut leafy vegetables wash water applied on peracetic acid combined with lactic acid.

PubMed

Van Haute, S; López-Gálvez, F; Gómez-López, V M; Eriksson, Markus; Devlieghere, F; Allende, Ana; Sampers, I

2015-09-02

A methodology to i) assess the feasibility of water disinfection in fresh-cut leafy greens wash water and ii) to compare the disinfectant efficiency of water disinfectants was defined and applied for a combination of peracetic acid (PAA) and lactic acid (LA) and comparison with free chlorine was made. Standardized process water, a watery suspension of iceberg lettuce, was used for the experiments. First, the combination of PAA+LA was evaluated for water recycling. In this case disinfectant was added to standardized process water inoculated with Escherichia coli (E. coli) O157 (6logCFU/mL). Regression models were constructed based on the batch inactivation data and validated in industrial process water obtained from fresh-cut leafy green processing plants. The UV254(F) was the best indicator for PAA decay and as such for the E. coli O157 inactivation with PAA+LA. The disinfection efficiency of PAA+LA increased with decreasing pH. Furthermore, PAA+LA efficacy was assessed as a process water disinfectant to be used within the washing tank, using a dynamic washing process with continuous influx of E. coli O157 and organic matter in the washing tank. The process water contamination in the dynamic process was adequately estimated by the developed model that assumed that knowledge of the disinfectant residual was sufficient to estimate the microbial contamination, regardless the physicochemical load. Based on the obtained results, PAA+LA seems to be better suited than chlorine for disinfecting process wash water with a high organic load but a higher disinfectant residual is necessary due to the slower E. coli O157 inactivation kinetics when compared to chlorine. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficacy of Peracetic Acid in Inactivating Foodborne Pathogens on Fresh Produce Surface.

PubMed

Singh, Prashant; Hung, Yen-Con; Qi, Hang

2018-02-01

Washing treatment with effective sanitizer is one of the critical steps in ensuring fresh produce safety. This study was to evaluate the efficacy of peracetic acid (PAA; VigorOx® 15 F&V), chlorine-based sanitizers (acidic electrolyzed water [AEO], near neutral electrolyzed water and bleach), lactic acid, and deionized (DI) water to reduce Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium DT104 from fresh produce surfaces. A 5-strain cocktail of E. coli O157:H7, L. monocytogenes, and S. Typhimurium DT104 was separately prepared and used for surface inoculation on produce samples (E. coli O157:H7 on romaine lettuce, lemons, tomatoes, and blueberries; L. monocytogenes on romaine lettuce and cantaloupe; S. Typhimurium DT104 on lemons, tomatoes, cantaloupe, and blueberries). PAA at 45, 85, and 100 mg/L; AEO, NNEO, and bleach at 100 mg/L of free chlorine; lactic acid at 2%; and DI water were used for washing inoculated produce in an automated produce washer for 5 min. In general, PAA at 100 mg/L achieved the highest microbial inactivation of E. coli O157:H7 (lettuce, lemon, tomato, and blueberry at 2.2, 5.7, 5.5, and 6.7 log CFU/g, respectively), S. Typhimurium DT104 (lemon, tomato, cantaloupe, blueberry at 5.4, 6.8, 4.5, and 5.9 log CFU/g, respectively), and L. monocytogenes (lettuce and cantaloupe at 2.4 and 4.4 log CFU/g, respectively). Efficacy of sanitizers on produce with coarse surface (for example, lettuce and cantaloupe) was lower than produce with smooth texture (lemon, tomato, and blueberry). Cross-contamination of E. coli O157:H7 among romaine lettuce heads during simulated retail crisping process was greatly reduced by the application of PAA and NNEO. NNEO and PAA showed high efficacy in foodborne pathogen removal from fresh produce. Produce surface texture plays an important role in pathogen removal. NNEO and PAA effectively prevented cross-contamination during the crisping process. © 2018 Institute of Food Technologists®.

Antibiotic resistant bacteria in urban sewage: Role of full-scale wastewater treatment plants on environmental spreading.

PubMed

Turolla, A; Cattaneo, M; Marazzi, F; Mezzanotte, V; Antonelli, M

2018-01-01

The presence of antibiotic resistant bacteria (ARB) in wastewater was investigated and the role of wastewater treatment plants (WWTPs) in promoting or limiting antibiotic resistance was assessed. Escherichia coli (E. coli) and total heterotrophic bacteria (THB) resistance to ampicillin, chloramphenicol and tetracycline was monitored in three WWTPs located in Milan urban area (Italy), differing among them for the operating parameters of biological process, for the disinfection processes (based on sodium hypochlorite, UV radiation, peracetic acid) and for the discharge limits to be met. Wastewater was collected from three sampling points along the treatment sequence (WWTP influent, effluent from sand filtration, WWTP effluent). Antibiotic resistance to ampicillin was observed both for E. coli and for THB. Ampicillin resistant bacteria in the WWTP influents were 20-47% of E. coli and 16-25% of THB counts. A limited resistance to chloramphenicol was observed only for E. coli, while neither for E. coli nor for THB tetracycline resistance was observed. The biological treatment and sand filtration led to a decrease in the maximum percentage of ampicillin-resistant bacteria (20-29% for E. coli, 11-21% for THB). However, the conventionally adopted parameters did not seem adequate to support an interpretation of WWTP role in ARB spread. Peracetic acid was effective in selectively acting on antibiotic resistant THB, unlike UV radiation and sodium hypochlorite. The low counts of E. coli in WWTP final effluents in case of agricultural reuse did not allow to compare the effect of the different disinfection processes on antibiotic resistance. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cloning, expression and characterization of a pectate lyase from Paenibacillus sp. 0602 in recombinant Escherichia coli

PubMed Central

2014-01-01

Background Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are considered as environmentally friendly. As such, they become promising substitutes for conventional chemical degumming process. Since applications of Pels in various fields are widening, it is necessary to explore new pectolytic microorganisms and enzymes for efficient and effective usage. Here, we describe the cloning, expression, characterization and application of the recombinant Pel protein from a pectolytic bacterium of the genus Paenibacillus in Escherichia coli. Results A Pel gene (pelN) was cloned using degenerate PCR and inverse PCR from the chromosomal DNA of Paenibacillus sp. 0602. The open reading frame of pelN encodes a 30 amino acid signal peptide and a 445 amino acid mature protein belonging to the polysaccharide lyase family 1. The maximum Pel activity produced by E. coli in shake flasks reached 2,467.4 U mL−1, and the purified recombinant enzyme exhibits a specific activity of 2,060 U mg−1 on polygalacturonic acid (PGA). The maximum activity was observed in a buffer with 5 mM Ca2+ at pH 9.8 and 65°C. PelN displays a half-life of around 9 h and 42 h at 50°C and 45°C, respectively. The biochemical treatment achieved the maximal reduction of percentage weight (30.5%) of the ramie bast fiber. Conclusions This work represents the first study that describes the extracellular expression of a Pel gene from Paenibacillus species in E. coli. The high yield of the extracellular overexpression, relevant thermostability and efficient degumming using combined treatments indicate its strong potential for large-scale industrial production. PMID:24612647

Influence of Poly(L-Lactic Acid) Nanofibers and BMP-2–Containing Poly(L-Lactic Acid) Nanofibers on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schofer, Markus D.; Fuchs-Winkelmann, Susanne; Gräbedünkel, Christian; Wack, Christina; Dersch, Roland; Rudisile, Markus; Wendorff, Joachim H.; Greiner, Andreas; Paletta, Jürgen R. J.; Boudriot, Ulrich

2008-01-01

The aim of this study was to characterize synthetic poly-(L-lactic acid) (PLLA) nanofibers concerning their ability to promote growth and osteogenic differentiation of stem cells in vitro, as well as to test their suitability as a carrier system for growth factors. Fiber matrices composed of PLLA or BMP-2–incorporated PLLA were seeded with human mesenchymal stem cells and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of alkaline phosphatase (ALP), osteocalcin (OC), and collagen I (COL-I). Furthermore, COL-I and OC deposition, as well as cell densities and proliferation, were analyzed using fluorescence microscopy. Although the presence of nanofibers diminished the dexamethasone-induced proliferation, there were no differences in cell densities or deposition of either COL-I or OC after 22 days of culture. The gene expression of ALP, OC, and COL-I decreased in the initial phase of cell cultivation on PLLA nanofibers as compared to cover slip control, but normalized during the course of cultivation. The initial down-regulation was not observed when BMP-2 was directly incorporated into PLLA nanofibers by electrospinning, indicating that growth factors like BMP-2 might survive the spinning process in a bioactive form. PMID:19112539

Characterization of biofilm-forming capacity and resistance to sanitizers of a range of E. coli O26 pathotypes from clinical cases and cattle in Australia.

PubMed

Lajhar, Salma A; Brownlie, Jeremy; Barlow, Robert

2018-05-08

The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don't produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air-liquid interface under the conditions used in this study. Biofilms provided a protective effect to E. coli O26 strains against the three sanitizers, previously shown to successfully control the growth of their planktonic counterparts. Whether the characteristics of biofilm forming and non-biofilm forming strains observed in this study reflect their attributes within the food and meat-processing environments is unknown. Further studies that represent the food and meat-processing environments are required.

PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state.

PubMed

Yan, Muxia; Xu, Ling; Jiang, Hua; Zhou, Zhenwen; Zhou, Shishui; Zhang, Li

2017-04-01

In exposure to outer pressure, microorganisms are capable of entry into the Viable But Non-Culturable (VBNC) state, and thus survive under various elimination processing. The survival microorganisms may yield negative results on culturing, and cause false negative for this golden standard methodology. In this study, a novel PMA-LAMP assay on the detection of Enterohemorrhage E. coli and shiga toxins has been developed and evaluated, with further application on a number of food borne E. coli strains. LAMP primers were designed on the target of rfbe for Enterohemorrhage E. coli and stx1with stx2 for shiga toxins. Via specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on Enterohemorrhage E. coli and shiga toxins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

In this paper, we report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradationmore » pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. Finally, in the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.« less

Solid-state voltammetry-based electrochemical immunosensor for Escherichia coli using graphene oxide-Ag nanoparticle composites as labels.

PubMed

Jiang, Xiaochun; Chen, Kun; Wang, Jing; Shao, Kang; Fu, Tao; Shao, Feng; Lu, Donglian; Liang, Jiangong; Foda, M Frahat; Han, Heyou

2013-06-21

A new electrochemical immunosensor based on solid-state voltammetry was fabricated for the detection of Escherichia coli (E. coli) by using graphene oxide-Ag nanoparticle composites (P-GO-Ag) as labels. To construct the platform, Au nanoparticles (AuNPs) were first self-assembled on an Au electrode surface through cysteamine and served as an effective matrix for antibody (Ab) attachment. Under a sandwich-type immunoassay format, the analyte and the probe (P-GO-Ag-Ab) were successively captured onto the immunosensor. Finally, the bonded AgNPs were detected through a solid-state redox process in 0.2 M of KCl solution. Combining the advantages of the high-loading capability of graphene oxide with promoted electron-transfer rate of AuNPs, this immunosensor produced a 26.92-fold signal enhancement compared with the unamplified protocol. Under the optimal conditions, the immunosensor exhibited a wide linear dependence on the logarithm of the concentration of E. coli ranging from 50 to 1.0 × 10(6) cfu mL(-1) with a detection limit of 10 cfu mL(-1). Moreover, as a practical application, the proposed immunosensor was used to monitor E. coli in lake water with satisfactory results.

MICROBIAL CONTAMINATION OF STREET VENDED FOODS FROM A UNIVERSITY CAMPUS IN BANGLADESH.

PubMed

Islam, Sufia; Nasrin, Nishat; Rizwan, Farhana; Nahar, Lutfun; Bhowmik, Adity; Esha, Sayma Afrin; Talukder, Kaisar Ali; Akter, Mahmuda; Roy, Ajoy; Ahmed, Muniruddin

2015-05-01

The microbiological quality of street vended food samples from Dhaka, Bangladesh was evaluated. The objective of the study was to identify the presence of common pathogens (Escherichia coli, Shigella spp, Salmonella and Vibrio spp) and to describe the molecular characterization of E coli, a commonly found pathogen in various street foods. Fifty food samples were collected from fixed and mobile vendors from two sampling locations (Mohakhali and Aftabnagar) in Dhaka city, Bangladesh. The tested samples included deep fried and fried snacks; quick lunch items; pickles; fruit chutney; baked items; spicy, sour and hot snacks etc: Juices, tamarind water and plain drinking water were also tested. Sterile polythene bags were used for collecting 200 g of each category of samples. They were tested for the presence of microorganisms following conventional microbiological processes. Biochemical tests followed by serology were done for the confirmation of Shigella and Salmonella. Serological reaction was carried out for confirmation of Vibrio spp. DNA was isolated for the molecular characterization to detect the pathogenic E. coli by polymerase chain reaction (PCR). Out of 50 food samples, six (12%) were confirmed to contain different species of E. coli and Shigella. Molecular characterization of E. coli revealed that three samples were contaminated with enteroaggregative E. coli (EAEC) and one was contaminated with enterotoxigenic E. coli (ETEC). Shigellaflexneri X variant was detected in one food item and Shigella flexneri 2a was found in drinking water. All these enteric pathogens could be the potential cause for foodborne illnesses.

Verocytotoxin-producing Escherichia coli O26 in raw water buffalo (Bubalus bubalis) milk products in Italy.

PubMed

Lorusso, Vanessa; Dambrosio, Angela; Quaglia, Nicoletta Cristiana; Parisi, Antonio; La Salandra, Giovanna; Lucifora, Giuseppe; Mula, Giuseppina; Virgilio, Sebastiano; Carosielli, Leonardo; Rella, Addolorata; Dario, Marco; Normanno, Giovanni

2009-08-01

Escherichia coli 026 is known as a verocytotoxin-producing E. coli (VTEC) organism that causes severe foodborne diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Although cattle are the most important reservoir of VTEC, only a few reports on the role of water buffalo (Bubalus bubalis) as a reservoir of VTEC and on the presence of these organisms in their milk are available. However, in Southern Italy, where water buffalo are intensively reared, an outbreak of hemolytic uremic syndrome due to E. coli 026 has recently been reported, in which the consumption of typical dairy products was considered to be a common risk factor. The aims of this work were to assess the prevalence of E. coli O26 in raw water buffalo milk, to characterize the virulence gene profiles of the isolates, and to evaluate their phenotypic antimicrobial resistance pattern. Of 160 analyzed samples, 1 (0.6%) tested positive for E. coli O26, and the isolate showed the stx1+/stx2+/eae-/hlyA+ genotypic profile. The strain showed resistance against glycopeptides, macrolides, and penicillins. The presence of VTEC organisms in raw water buffalo milk could be considered to be a potential threat to consumers; however, the strict adherence to the processes used in the preparation of the most common buffalo dairy products could strongly mitigate the foodborne risk. To our knowledge, this article reports the first isolation and characterization of E. coli O26 VTEC in raw water buffalo milk.

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation

PubMed Central

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-01-01

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES–TiO2 and PE–TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti4+/Ti3+ redox catalysis occurring on PES–TiO2 and PE–TiO2 during the bacterial inactivation process. On PE–TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C–H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES–TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1–5% wt PES–TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces. PMID:25657831

Effect of surface pretreatment of TiO2 films on interfacial processes leading to bacterial inactivation in the dark and under light irradiation.

PubMed

Rtimi, Sami; Nesic, Jelena; Pulgarin, Cesar; Sanjines, Rosendo; Bensimon, Michael; Kiwi, John

2015-02-06

Evidence is presented for radio-frequency plasma pretreatment enhancing the amount and adhesion of TiO2 sputtered on polyester (PES) and on polyethylene (PE) films. Pretreatment is necessary to attain a suitable TiO2 loading leading to an acceptable Escherichia coli reduction kinetics in the dark or under light irradiation for PES-TiO2 and PE-TiO2 samples. The amount of TiO2 on the films was monitored by diffuse reflectance spectroscopy and X-ray fluorescence. X-ray electron spectroscopy shows the lack of accumulation of bacterial residues such as C, N and S during bacterial inactivation since they seem to be rapidly destroyed by TiO2 photocatalysis. Evidence was found for Ti(4+)/Ti(3+) redox catalysis occurring on PES-TiO2 and PE-TiO2 during the bacterial inactivation process. On PE-TiO2 surfaces, Fourier transform infrared spectroscopy (ATR-FTIR) provides evidence for a systematic shift of the na(CH2) stretching vibrations preceding bacterial inactivation within 60 min. The discontinuous IR-peak shifts reflect the increase in the C-H inter-bond distance leading to bond scission. The mechanism leading to E. coli loss of viability on PES-TiO2 was investigated in the dark up to complete bacterial inactivation by monitoring the damage in the bacterial outer cell by transmission electron microscopy. After 30 min, the critical step during the E. coli inactivation commences for dark disinfection on 0.1-5% wt PES-TiO2 samples. The interactions between the TiO2 aggregates and the outer lipopolysaccharide cell wall involve electrostatic effects competing with the van der Waals forces.

Analysis of microbiological contamination in mixed pressed ham and cooked sausage in Korea.

PubMed

Park, Myoung-Su; Wang, Jun; Park, Joong-Hyun; Forghani, Fereidoun; Moon, Jin-San; Oh, Deog-Hwan

2014-03-01

The objective of this study was to investigate the microbial contamination levels (aerobic bacteria plate count [APC], coliforms, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes) in mixed pressed ham and cooked sausage. A total of 180 samples were collected from factories with and without hazard analysis critical control point (HACCP) systems at four steps: after chopping (AC), after mixing (AM), cooling after the first heating process, and cooling after the second heating process. For ham, APCs and coliform and E. coli counts increased when ingredients were added to the meat at the AC step. Final product APC was 1.63 to 1.85 log CFU/g, and coliforms and E. coli were not detected. S. aureus and L. monocytogenes were found in nine (15.0%) and six (10.0%) samples, respectively, but only at the AC and AM steps and not in the final product. Sausage results were similar to those for ham. The final product APC was 1.52 to 3.85 log CFU/g, and coliforms and E. coli were not detected. S. aureus and L. monocytogenes were found in 29 (24.2%) and 25 (20.8%) samples at the AC and AM steps, respectively, but not in the final product. These results indicate that the temperature and time of the first and second heating are of extreme importance to ensure the microbiological safety of the final product regardless of whether a HACCP system is in place. Microorganism contamination must be monitored regularly and regulations regarding sanitization during processing should be improved. Education regarding employee personal hygiene, environmental hygiene, prevention of cross-contamination, ingredient control, and step-by-step process control is needed to reduce the risk of food poisoning.

Microbiological sampling of swine carcasses: a comparison of data obtained by swabbing with medical gauze and data collected routinely by excision at Swedish abattoirs.

PubMed

Lindblad, M

2007-09-15

Swab sample data from a 13-month microbiological baseline study of swine carcasses at Swedish abattoirs were combined with excision sample data collected routinely at five abattoirs. The aim was to compare the numbers of total aerobic counts, Enterobacteriaceae, and Escherichia coli, recovered by swabbing four carcass sites with gauze (total area 400 cm2) with those obtained by excision at equivalent sites (total area 20 cm2). The results are considered in relation to the process hygiene criteria that are stated in Commission Regulation (EC) No 2073/2005. These criteria apply only to destructive sampling of total aerobic counts and Enterobacteriaceae, but alternative sampling schemes, as well as alternative indicator organisms such as E. coli, are allowed if equivalent guarantees of food safety can be provided. Swab sampling resulted in higher mean log numbers of total aerobic counts at four of the five abattoirs, compared with excision, and lower or equal standard deviations at all abattoirs. The percentage of swab and excision samples positive for Enterobacteriaceae at the different abattoirs ranged from 68 to 100% and 15 to 24%, respectively. Similarly, the percentages of swab samples that were positive for E. coli were higher than the percentages of positive excision samples (range 52 to 84% and 3 to 14%, respectively). Due to the low percentage of positive excision results, the mean log numbers of Enterobacteriaceae and E. coli were only compared at two and one abattoirs, respectively, using log probability regression to substitute censored observations. Higher mean log numbers of Enterobacteriaceae were recovered by swabbing compared with excision at one abattoir, whereas the numbers of Enterobacteriaceae and E. coli did not differ significantly between sampling methods at one abattoir. This study suggests that the same process hygiene criteria as those stipulated for excision can be used for swabbing with gauze without compromising food safety. For monitoring of low numbers of Enterobacteriaceae and E. coli, like those found on swine carcasses at Swedish abattoirs, the results also show that swabbing of a relatively large area is superior to excision of a smaller area.

Intestinal colonization with phylogenetic group B2 Escherichia coli related to inflammatory bowel disease: a systematic review and meta-analysis.

PubMed

Petersen, Andreas Munk; Halkjær, Sofie Ingdam; Gluud, Lise Lotte

2015-01-01

Increased numbers of Escherichia coli and, furthermore, specific subtypes of E. coli, such as E. coli of the phylogenetic groups B2 and D have been found in the intestine of patients with inflammatory bowel disease (IBD). In this review, we wanted to evaluate the relationship between B2 and D E. coli intestinal colonization and IBD. A systematic review with meta-analyses. We included studies comparing colonization with B2 and D E. coli in IBD patients and in controls. Random-effects and fixed-effect meta-analyses were performed. We included 7 studies on 163 patients with IBD and 89 controls. Among IBD patients, 57 patients had ulcerative colitis (UC) and 95 Crohn's disease (CD). Random-effects meta-analysis showed that IBD patients were more likely to have B2 E. coli intestinal colonization compared with controls (odds ratio [OR]: 2.28; 95% confidence interval [CI]: 1.25-4.16). There was little between-study heterogeneity (I(2) = 0). The result was confirmed in subgroup analyses of patients with UC (OR: 3.58; 95% CI: 1.62-7.90), but not CD (OR: 1.94; 95% CI: 0.98-3.82). Intestinal colonization with phylogenetic group D E. coli was not found to be related to IBD, UC or CD. Our study reveals that intestinal colonization with phylogenetic group B2 E. coli is associated with UC. Due to the design, we are unable to determine if the colonization with B2 E. coli leads to the development of the disease or the disease increases the risk of colonization with B2 E. coli.

Transcriptomic analysis displays the effect of (-)-roemerine on the motility and nutrient uptake in Escherichia coli.

PubMed

Ayyildiz, Dilara; Arga, Kazim Yalcin; Avci, Fatma Gizem; Altinisik, Fatma Ece; Gurer, Caglayan; Gulsoy Toplan, Gizem; Kazan, Dilek; Wozny, Katharina; Brügger, Britta; Mertoglu, Bulent; Sariyar Akbulut, Berna

2017-08-01

Among the different families of plant alkaloids, (-)-roemerine, an aporphine type, was recently shown to possess significant antibacterial activity in Escherichia coli. Based on the increasing demand for antibacterials with novel mechanisms of action, the present work investigates the potential of the plant-derived alkaloid (-)-roemerine as an antibacterial in E. coli cells using microarray technology. Analysis of the genome-wide transcriptional reprogramming in cells after 60 min treatment with 100 μg/mL (-)-roemerine showed significant changes in the expression of 241 genes (p value <0.05 and fold change >2). Expression of selected genes was confirmed by qPCR. Differentially expressed genes were classified into functional categories to map biological processes and molecular pathways involved. Cellular activities with roles in carbohydrate transport and metabolism, energy production and conversion, lipid transport and metabolism, amino acid transport and metabolism, two-component signaling systems, and cell motility (in particular, the flagellar organization and motility) were among metabolic processes altered in the presence of (-)-roemerine. The down-regulation of the outer membrane proteins probably led to a decrease in carbohydrate uptake rate, which in turn results in nutrient limitation. Consequently, energy metabolism is slowed down. Interestingly, the majority of the expressional alterations were found in the flagellar system. This suggested reduction in motility and loss in the ability to form biofilms, thus affecting protection of E. coli against host cell defense mechanisms. In summary, our findings suggest that the antimicrobial action of (-)-roemerine in E. coli is linked to disturbances in motility and nutrient uptake.

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain.

PubMed

Laing, Chad R; Zhang, Yongxiang; Gilmour, Matthew W; Allen, Vanessa; Johnson, Roger; Thomas, James E; Gannon, Victor P J

2012-01-01

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.

A Comparison of Shiga-Toxin 2 Bacteriophage from Classical Enterohemorrhagic Escherichia coli Serotypes and the German E. coli O104:H4 Outbreak Strain

PubMed Central

Laing, Chad R.; Zhang, Yongxiang; Gilmour, Matthew W.; Allen, Vanessa; Johnson, Roger; Thomas, James E.; Gannon, Victor P. J.

2012-01-01

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors. PMID:22649523

Synthetic spider silk sustainability verification by techno-economic and life cycle analysis

NASA Astrophysics Data System (ADS)

Edlund, Alan

Major ampullate spider silk represents a promising biomaterial with diverse commercial potential ranging from textiles to medical devices due to the excellent physical and thermal properties from the protein structure. Recent advancements in synthetic biology have facilitated the development of recombinant spider silk proteins from Escherichia coli (E. coli), alfalfa, and goats. This study specifically investigates the economic feasibility and environmental impact of synthetic spider silk manufacturing. Pilot scale data was used to validate an engineering process model that includes all of the required sub-processing steps for synthetic fiber manufacture: production, harvesting, purification, drying, and spinning. Modeling was constructed modularly to support assessment of alternative protein production methods (alfalfa and goats) as well as alternative down-stream processing technologies. The techno-economic analysis indicates a minimum sale price from pioneer and optimized E. coli plants at 761 kg-1 and 23 kg-1 with greenhouse gas emissions of 572 kg CO2-eq. kg-1 and 55 kg CO2-eq. kg-1, respectively. Spider silk sale price estimates from goat pioneer and optimized results are 730 kg-1 and 54 kg-1, respectively, with pioneer and optimized alfalfa plants are 207 kg-1 and 9.22 kg-1 respectively. Elevated costs and emissions from the pioneer plant can be directly tied to the high material consumption and low protein yield. Decreased production costs associated with the optimized plants include improved protein yield, process optimization, and an Nth plant assumption. Discussion focuses on the commercial potential of spider silk, the production performance requirements for commercialization, and impact of alternative technologies on the sustainability of the system.

Effectiveness of High Intensity Light Pulses (HILP) treatments for the control of Escherichia coli and Listeria innocua in apple juice, orange juice and milk.

PubMed

Palgan, I; Caminiti, I M; Muñoz, A; Noci, F; Whyte, P; Morgan, D J; Cronin, D A; Lyng, J G

2011-02-01

High Intensity Light Pulses (HILP) represent an emerging processing technology which uses short (100-400 μs) light pulses (200-1100 nm) for product decontamination. In this study, model and real foods of differing transparencies (maximum recovery diluent (MRD), apple and orange juices and milk) were exposed to HILP in a batch system for 0, 2, 4 or 8 s at a frequency of 3 Hz. After treatment, inactivation of Escherichia coli or Listeria innocua was evaluated in pre-inoculated samples. Sensory and other quality attributes (colour, pH, Brix, titratable acidity, non-enzymatic browning, total phenols and antioxidant capacity (TEAC)) were assessed in apple juice. Microbial kill decreased with decreasing transparency of the medium. In apple juice (the most transparent beverage) E. coli decreased by 2.65 and 4.5 after exposure times of 2 or 4 s, respectively. No cell recovery was observed after 48 h storage at 4°C. No significant differences were observed in quality parameters, excepting TEAC and flavour score, where 8 s exposure caused a significant decrease (p<0.05). Based on these results, HILP with short exposure times could represent a potential alternative to thermal processing to eliminate undesirable microorganisms, while maintaining product quality, in transparent fruit juices. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effects of hot water pasteurizing treatments on the microbiological condition of manufacturing beef used for hamburger patty manufacture.

PubMed

Gill, C O; Bryant, J; Badoni, M

2001-02-15

Ten 12-kg lots of manufacturing beef from a single packing plant were obtained from a hamburger patty manufacturing plant. Each lot was divided into two, 6-kg portions, one of which was not treated while the other was treated with water of 85 degrees C. A portion from one lot was treated for 15 s. A portion from each of three lots was treated for 30 s, three portions were treated for 45 s, and three were treated for 60 s. Twenty-five pieces of meat from each portion were swabbed over areas of 100 cm2. Subsequently, each portion was first coarsely ground then finely ground, with twenty-five 100-g samples being taken from each portion at each stage of grinding. Each swab and sample of ground meat was separately processed for the enumeration of total aerobic counts, coliforms and Escherichia coli at levels of detection of 1 cfu/cm2, 1 cfu/100 cm2 and 1 cfu/100 cm2, respectively, for swab samples; and at a level of detection of 1 cfu/g for all three types of bacteria in samples of ground beef. A 250-kg batch of manufacturing beef was treated with water of 85 degrees C for 60 s. The product was processed through commercial equipment for manufacturing frozen hamburger patties. The flavour of patties prepared from the pasteurized product was compared with the flavour of patties prepared during normal commercial operation of the equipment. The weight of the manufacturing beef was not affected by the treatments. Similar total numbers of coliforms or E. coli were recovered per 2500 cm2 from the 25 swab samples or per 25 g from the 25 ground beef samples from each untreated portion. As the ratio of the surface area in cm2 to the weight in g would likely be < or = 1, the similar numbers indicated that swab sampling was inefficient for recovering coliforms and E. coli from the meat. However, coliforms and E. coli were recovered more frequently from swab than from ground beef samples from treated portions. Thus, some swabs from all three portions of beef treated for 30 s yielded coliforms and E. coli, but samples from portions treated for 45 or 60 s yielded few coliforms and no E. coli. The numbers recovered from the treated and untreated portions indicated that treatments for 45 or 60 s reduced both coliform and E. coli numbers by two orders of magnitude. The flavours of cooked patties prepared from the meat pasteurized with water of 85 degrees C for 60 s were not distinguished from the normal commercial product. The data indicate that pasteurizing manufacturing beef with water of 85 degrees C for 45 s could be a practicable treatment for enhancing the microbiological safety of frozen hamburger patties.

Development of atmospheric pressure plasma needle jet for sterilization applications

NASA Astrophysics Data System (ADS)

Elfa, Rizan Rizon; Ahmad, Mohd Khairul; Soon, Chin Fhong; Sahdan, Mohd Zainizan; Lias, Jais; Wibowo, Kusnanto Mukti; Bakar, Ahmad Shuhaimi Abu; Arshad, Mohd Khairuddin Md; Hashim, Uda; Nayan, Nafarizal

2017-09-01

Inactivation of bacteria or sterilization has been a major issue in the medical field, especially regarding of human safety, whereby, in a huge scenario fatality can be caused by hazardous bacteria. Often, E-coli as gram-negative bacteria are selected as a key indicator of proper sterilization process as E-coli is tough and dormant bacteria. The technology in sterilization has moved on from chemical, wet and irradiation sterilization to a high promising device such as atmospheric pressure plasma needle jet (APPNJ). It has been reported that atmospheric pressure plasma has provided bundle of advantages over earlier sterilization process. The APPNJ is developed in our lab using high frequency and high voltage neon transformer power supply connected to copper needle and copper sheet electrodes. The gas discharge is Ar gas flowing at 40 L/min through a quartz glass tube. The E-coli bacteria are self-cultured from waste water and then treated with APPNJ. The treatment processes are run into two difference gaps between the plasma orifice and sample with various treatment times. Only 40s is required by direct treatment to achieve 100% killing of E-coli. On the other hand, indirect treatment has inactivated 50% of the E-coli in 40s. In this study, direct and indirect effect of APPNJ to the E-coli can be observed which can be utilized into sterilization of bio-compatible material applications.

Characterization of antimicrobial resistance of Escherichia coli recovered from foods of animal and fish origin in Korea.

PubMed

Koo, Hyon-Ji; Woo, Gun-Jo

2012-05-01

Antimicrobial-resistant Escherichia coli is transferred from food-producing animals to humans through the food chain. We investigated the prevalence of antimicrobial resistance and resistance determinants and characterized the integrons of foodborne E. coli in Korea. In total, 162 E. coli isolates from commercial foods (raw meat, fish, and processed foods) were collected by the National Antimicrobial Resistance Management Program from 2004 to 2006. Susceptibility to 20 antibiotics was tested by disk diffusion, and resistance determinants were detected using PCR and genomic sequence analysis. The isolates were highly resistant to antibiotics commonly used in livestock farming. Resistance to tetracycline (74.7%) was the most frequently observed, followed by streptomycin (71%) and ampicillin (51.2%). Class 1 integrons were detected in 13 isolates (8%), and nine of these integrons were located on conjugative plasmids. None of the isolates produced extended-spectrum β -lactamase. One isolate (0.6%) harbored bla(CMY-2), which was located on a conjugative plasmid. Although the qnr gene was not detected, aac(6'9)-Ib-cr was present in two isolates (1.2%). This is the first report of aac(6'9)-Ib-cr in food isolates. Three or four amino acid substitutions at positions 83 and 87 in gyrA and at positions 80 and/or 84 in parC were found in six isolates, representing high resistance to ciprofloxacin (MIC ≥ 16 mg/liter). These results suggest that E. coli isolates carrying resistance genes and integrons are present in the Korean food chain.

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba.

PubMed

Siddiqui, Ruqaiyyah; Malik, Huma; Sagheer, Mehwish; Jung, Suk-Yul; Khan, Naveed Ahmed

2011-08-01

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P<0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts. Copyright © 2011 Elsevier Inc. All rights reserved.

A droplet-merging platform for comparative functional analysis of m1 and m2 macrophages in response to e. coli-induced stimuli.

PubMed

Hondroulis, Evangelia; Movila, Alexandru; Sabhachandani, Pooja; Sarkar, Saheli; Cohen, Noa; Kawai, Toshihisa; Konry, Tania

2017-03-01

Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells, while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate. Here, we present a simple and effective method for the co-encapsulation of polarized M1 and M2 macrophages with Escherichia coli (E. coli) by passive merging in an integrated droplet generation, merging, and docking platform. This approach facilitated live cell profiling of effector immune functions in situ and quantitative functional analysis of macrophage heterogeneity. Biotechnol. Bioeng. 2017;114: 705-709. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Draft genome sequences of four uropathogenic escherichia coli 04:H5 isolates (ATCC 700414,700415,700416 and 700417)

USDA-ARS?s Scientific Manuscript database

Uropathogenic Escherichia coli O4: H5 isolates ATCC 700414, 700415, 700416, and 700417 were recovered from women with first-time urinary tract infections. Here, we report the draft genome sequences for these four E. coli isolates, which are currently being used to validate food safety processing tec...

The cold and atmospheric-pressure air surface barrier discharge plasma for large-area sterilization applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Dacheng; Department of Aeronautics, Fujian Key Laboratory for Plasma and Magnetic Resonance, School of Physics and Mechanical and Electrical Engineering, Xiamen University, Xiamen, Fujian 361005; Zhao Di

2011-04-18

This letter reports a stable air surface barrier discharge device for large-area sterilization applications at room temperature. This design may result in visually uniform plasmas with the electrode area scaled up (or down) to the required size. A comparison for the survival rates of Escherichia coli from air, N{sub 2} and O{sub 2} surface barrier discharge plasmas is presented, and the air surface plasma consisting of strong filamentary discharges can efficiently kill Escherichia coli. Optical emission measurements indicate that reactive species such as O and OH generated in the room temperature air plasmas play a significant role in the sterilizationmore » process.« less

Immobilization of antibacterial metallic cations (Ga3+, Zn2+ and Co2+) in a polypyrrole coating formed on Nitinol.

PubMed

Saugo, M; Brugnoni, L I; Flamini, D O; Saidman, S B

2018-05-01

Gallium, zinc and cobalt species were immobilized in hollow rectangular-sectioned microtubes of polypyrrole (PPy) electrosynthesized on Nitinol (NiTi) alloy by means of two different methods. One of them involved the immobilization after the PPy electropolymerization and the other one during the electrosynthesis process. The antibacterial activity of the coating against Escherichia coli (E. coli) was evaluated and the best results were obtained with gallium species. Characterization results demonstrated that gallium is incorporated into the PPy matrix as Ga 3+ ions. The PPy film with gallium species incorporated during the electropolymerization exhibited a good corrosion protection performance. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of escherichia coli O157:H7 strains from contaminated raw beef trim during “high event periods”

USDA-ARS?s Scientific Manuscript database

The development and implementation of effective antimicrobial interventions by the beef processing industry in the United States have dramatically reduced the incidence of beef trim contamination by Escherichia coli O157:H7. However, individual processing plants still experience sporadic peaks in co...

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.

Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies

USDA-ARS?s Scientific Manuscript database

Escherichia coli (E. coli) food contaminations pose serious health and food safety concerns, and have been the subject of massive food recalls. Shiga toxin 2 (Stx2)-producing E. coli has been identified as the major cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS), the most severe di...

Quantitative determination of Escherichia coli based on the electrochemical measurement of bacterial catalase activity using H2O2-selective organic/inorganic-hybrid sol-gel film-modified Pt electrode.

PubMed

Hasebe, Yasushi; Fukuzawa, Michiru; Matsuhisa, Hironori

2009-01-01

Quantitative determination of Escherichia coli (E. coli) concentration was achieved by measuring the intrinsic catalase activity of E. coli using novel H2O2-selective organic/inorganic-hybrid sol-gel film-modified platinum (Pt) wire electrode. This hybrid sol-gel film is composed of three kinds of organosilanes and two biopolymers (i.e., chitosan and bovine serum albumin), and exhibited an excellent permselectivity toward H2O2 based on a size-exclusive mechanism. The steady-state anodic current for 100 [xmol/L H2O2 at +0.6 V (vs. Ag/AgCl) in 0.1 mol/L phosphate buffer (pH 6.5) solution was apparently diminished by the addition of E. coli samples, due to the decomposition of H2O2 by intrinsic catalase activity of E. coli. The time-dependent decrease in current (-AI/At) was significantly dependent on the E. coli concentration. The -AI/At was enhanced by the permeabilization pretreatment of E. coli samples with the mixed solution of polymyxin B and lysozyme. This H2O2-selective organic/inorganic-hybrid sol-gel film-modified platinum (Pt) wire electrode allowed quantitative determination of E. coli concentration ranging from 10(6) to 10(9) CFU/mL within 30 min. This method required no label and complicated procedure, and allowed rapid, simple and cost-effective quantitative electrochemical determination of catalase-positive bacteria.

Occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern Ontario: A preliminary study

PubMed Central

Murphy, Colleen; Reid-Smith, Richard J.; Prescott, John F.; Bonnett, Brenda N.; Poppe, Cornelis; Boerlin, Patrick; Weese, J. Scott; Janecko, Nicol; McEwen, Scott A.

2009-01-01

The prevalence and patterns of antimicrobial susceptibility of fecal Escherichia coli, Salmonella spp., extended β-lactamase producing E. coli (ESBL-E. coli), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus pseudintermedius (MRSP) were determined for healthy dogs (n = 188) and cats (n = 39) from veterinary hospitals in southern Ontario that had not had recent exposure to antimicrobials. The prevalence of antimicrobial resistance in E. coli was as follows: streptomycin (dogs — 17%, cats — 2%), ampicillin (dogs — 13%, cats — 4%), cephalothin (dogs — 13%, cats — < 1%), and tetracycline (dogs — 11%, cats — 2%). Eleven percent of dogs and 15% of cats had isolates that were resistant to at least 2 antimicrobials. Cephamycinase (CMY)-2 producing E. coli was cultured from 2 dogs. No Salmonella spp., ESBL-E. coli, MRSA, or MRSP isolates were recovered. The observed prevalence of resistance in commensal E. coli from this population was lower than that previously reported in companion animals, but a small percentage of dogs may be a reservoir for CMY-2 E. coli. PMID:20046603

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

PubMed

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

2009-11-01

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

Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

PubMed Central

Even, Sergine; Pellegrini, Olivier; Zig, Lena; Labas, Valerie; Vinh, Joelle; Bréchemmier-Baey, Dominique; Putzer, Harald

2005-01-01

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation. PMID:15831787

An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of Shiga toxin-producing Escherichia coli during raw milk cheese production.

PubMed

Peng, Silvio; Tasara, Taurai; Hummerjohann, Jörg; Stephan, Roger

2011-05-01

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.

Analysis of the efficiency of recombinant Escherichia coli strain cultivation in a gas-vortex bioreactor.

PubMed

Savelyeva, Anna V; Nemudraya, Anna A; Podgornyi, Vladimir F; Laburkina, Nadezhda V; Ramazanov, Yuriy A; Repkov, Andrey P; Kuligina, Elena V; Richter, Vladimir A

2017-09-01

The levels of aeration and mass transfer are critical parameters required for an efficient aerobic bioprocess, and directly depend on the design features of exploited bioreactors. A novel apparatus, using gas vortex for aeration and mass transfer processes, was constructed in the Center of Vortex Technologies (Novosibirsk, Russia). In this paper, we compared the efficiency of recombinant Escherichia coli strain cultivation using novel gas-vortex technology with conventional bioprocess technologies such as shake flasks and bioreactors with mechanical stirrers. We demonstrated that the system of aeration and agitation used in gas-vortex bioreactors provides 3.6 times higher volumetric oxygen transfer coefficient in comparison with mechanical bioreactor. The use of gas-vortex bioreactor for recombinant E. coli strain cultivation allows to increase the efficiency of target protein expression at 2.2 times for BL21(DE3)/pFK2 strain and at 3.5 times for auxotrophic C600/pRT strain (in comparison with stirred bioreactor). © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Prevalence and counts of Salmonella spp. in minimally processed vegetables in São Paulo, Brazil.

PubMed

Sant'Ana, Anderson S; Landgraf, Mariza; Destro, Maria Teresa; Franco, Bernadette D G M

2011-09-01

Minimally processed vegetables (MPV) may be important vehicles of Salmonella spp. and cause disease. This study aimed at detecting and enumerating Salmonella spp. in MPV marketed in the city of São Paulo, Brazil. A total of 512 samples of MPV packages collected in retail stores were tested for Salmonella spp. and total coliforms and Escherichia coli as indication of the hygienic status. Salmonella spp. was detected in four samples, two using the detection method and two using the counting method, where the results were 8.8 × 10(2) CFU/g and 2.4 × 10(2) CFU/g. The serovars were Salmonella Typhimurium (three samples) and Salmonella enterica subsp. enterica O:47:z4,z23:- (one sample). Fourteen samples (2.7%) presented counts of E. coli above the maximum limit established by the Brazilian regulation for MPV (10(2) CFU/g). Therefore, tightened surveillance and effective intervention strategies are necessary in order to address consumers and governments concerns on safety of MPV. Copyright © 2011 Elsevier Ltd. All rights reserved.

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

NASA Astrophysics Data System (ADS)

Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

1992-08-01

Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

Epidemiological studies on Escherichia coli O157:H7 in Egyptian sheep.

PubMed

Kamel, Mohammed; Abo El-Hassan, Diea G; El-Sayed, Amr

2015-08-01

In the present work, the epidemiological role of apparently healthy sheep in transmission of Escherichia coli O157:H7 in different seasons was investigated. Fecal samples (convenience sampling) of apparently healthy farmed sheep (three farms, n = 70) and from 15 wandering flocks fed on city wastes (n = 80) in the Giza governorate were examined. The samples were collected in spring under mild weather conditions and during hot summer to be compared. Out of the 150 animals, 13 (8.7%) were E. coli O157 shedders. The 13 ovine sorbitol-negative E. coli O157 were characterized by different PCR sets. The eae gene was detected in 11 isolate (85%), stx1 in 3 isolates (23%), stx2 in 8 isolates (62%), and finally the hlyA in 11 isolate (85%). Among the 13 isolates, 2 strains (15%) were positive for eae, stx1, stx2, and hlyA as gene combination, one isolate (8%) for eae, stx1, and hlyA, 5 isolates (38%) for eae, stx2, and hlyA, 1 isolate (8%) for eae and stx2, 2 isolates (15%) contained eae and hlyA, 1 isolate (8%) contained hlyA only, and finally, 1 isolate (8%) did not contain any of these genes. None of the isolates showed the gene combination eae stx1, stx1 hlyA, or stx2 hlyA. The results indicated significant association of unfavorable weather and management conditions on O157:H7 shedding while the age or sex did not play any role in this process.

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

PubMed

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

2013-10-07

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

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

PubMed Central

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

2013-01-01

To clear pathogens from host tissues or biomaterial surfaces, phagocytes have to break the adhesive bacteria-substrate interactions. Here we analysed the mechanobiological process that enables macrophages to lift-off and phagocytose surface-bound Escherichia coli (E. coli). In this opsonin-independent process, macrophage filopodia hold on to the E. coli fimbriae long enough to induce a local protrusion of a lamellipodium. Specific contacts between the macrophage and E. coli are formed via the glycoprotein CD48 on filopodia and the adhesin FimH on type 1 fimbriae (hook). We show that bacterial detachment from surfaces occurrs after a lamellipodium has protruded underneath the bacterium (shovel), thereby breaking the multiple bacterium-surface interactions. After lift-off, the bacterium is engulfed by a phagocytic cup. Force activated catch bonds enable the long-term survival of the filopodium-fimbrium interactions while soluble mannose inhibitors and CD48 antibodies suppress the contact formation and thereby inhibit subsequent E. coli phagocytosis. PMID:24097079

Antimicrobial effect of herb extracts against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium associated with beef.

PubMed

Cutter, C N

2000-05-01

The effects of plant extracts against pathogenic bacteria in vitro are well known, yet few studies have addressed the effects of these compounds against pathogens associated with muscle foods. A series of experiments was conducted to determine the effectiveness of a commercially available, generally recognized as safe, herb extract dispersed in sodium citrate (Protecta One) or sodium chloride (Protecta Two) against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes associated with beef. In the first experiment, E. coli O157:H7, Salmonella typhimurium, and L. monocytogenes inoculated onto beef and subjected to surface spray treatments with 2.5% solutions of Protecta One or Protecta Two were not affected by immediate application (day 0) of the herbal extracts. However, after 7 days of storage at 4 degrees C, E. coli O157:H7 was reduced by >1.3 log10 CFU/cm2 by Protecta Two; L. monocytogenes was reduced by 1.8 and 1.9 log10 CFU/cm2 by Protecta One and Protecta Two, respectively; Salmonella typhimurium was not reduced >0.3 log10 CFU/cm2 by either extract by day 7. In the second experiment, 2.5% Protecta Two (wt/vol or wt/wt) added to inoculated lean and adipose beef trim, processed, and packaged as ground beef chubs (80% lean, 20% adipose), did not reduce pathogen populations >0.5 log10 CFU/g up to 14 days at 4 degrees C. In the third experiment, surface spray treatments of beef with 2.5% lactic acid or 2.5% solutions of Protecta One or Protecta Two, vacuum packaged, and stored up to 35 days at 4 degrees C did reduce E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium slightly. These studies suggest that the use of herb extracts may afford some reductions of pathogens on beef surfaces; however, the antimicrobial activity may be diminished in ground beef by adipose components.

Anti-quorum sensing activity of flavonoid-rich fraction from Centella asiatica L. against Pseudomonas aeruginosa PAO1.

PubMed

Vasavi, H S; Arun, A B; Rekha, P D

2016-02-01

Inhibition of quorum sensing (QS), a cell-density dependent regulation of gene expression in bacteria by autoinducers is an attractive strategy for the development of antipathogenic agents. In this study, the anti-QS activity of the ethanolic extract of the traditional herb Centella asiatica was investigated by the biosensor bioassay using Chromobacterium violaceum CV026. The effect of ethyl acetate fraction (CEA) from the bioassay-guided fractionation of ethanol extract on QS-regulated violacein production in C. violaceum ATCC12472 and pyocyanin production, proteolytic and elastolytic activities, swarming motility, and biofilm formation in Pseudomonas aeruginosa PAO1 were evaluated. Possible mechanism of QS-inhibitory action on autoinducer activity was determined by measuring the acyl homoserine lactone using C. violaceum ATCC31532. Anti-QS compounds in the CEA fraction were identified using thin layer chromatography biosensor overlay assay. Ethanol extract of C. asiatica showed QS inhibition in C. violaceum CV026. Bioassay-guided fractionation of ethanol extract revealed that CEA was four times more active than the ethanol extract. CEA, at 400 μg/mL, completely inhibited violacein production in C. violaceum ATCC12472 without significantly affecting growth. CEA also showed inhibition of QS-regulated phenotypes, namely, pyocyanin production, elastolytic and proteolytic activities, swarming motility, and biofilm formation in P. aeruginosa PAO1 in a concentration-dependent manner. Thin layer chromatography of CEA with biosensor overlay showed anti-QS spot with an Rf value that corresponded with that of standard kaempferol. The anti-QS nature of C. asiatica herb can be further exploited for the formulation of drugs targeting bacterial infections where pathogenicity is mediated through QS. Copyright © 2014. Published by Elsevier B.V.

[Antimicrobial sensitivity of Escherichia coli strains with K1 antigen isolated from pregnant women and newborns].

PubMed

Kaczmarek, Agnieszka; Budzyńska, Anna; Gospodarek, Eugenia

2011-01-01

The aim of this study was comparison of the susceptibility to antibiotics of E. coli strains with K1 antigen (E. coli K1+) and non-K1 E. coli strains (E. coli K1-). This study included 67 of E. coli K1+ and 67 of E. coli K1- strains isolated in the time period from June to September of 2008 from pregnant women and newborns hospitalized at dr. J. Biziel University Hospital number 2 L. Rydygier Collegium Medicum in Bydgoszcz Nicolaus Copernicus University in Toruń. Antimicrobial susceptibility of E. coli strains was tested by the disc-diffusion method, on the Mueller Hinton 2 Agar (Becton Dickinson). It was found that 64,2% of E. coli K1+ strains and 53,7% of E. coli K1-strains were susceptible to all tested antibiotics and chemioterapeutics. E. coli K1- strains were more often than E. coli K1+ nonsusceptible to at least one antimicrobial agent. The obtained results indicate that E. coli K1+ strains significant differed in the susceptibility to ampicillin/sulbactam (85,1% versus 95,5%) (p=0,041), cephalothin (70,1% versus 85,1%) (p=0,038) and tetracycline (91,0% versus 74,6%) (p=0,012) from E. coli K1-strains. All tested E. coli K1+ and K1-strains were sensitive to piperacillin/tazobactam, cefoperazone/sulbactam, cefotaxime, ceftazidime, cefepime, imipenem, amikacin, netilmicin and tigecycline. There weren't the ESBL-producing strains among tested E. coli K1+ and K1- rods.

Prevalence Rates of Escherichia coli O157:H7 and Salmonella at Different Sampling Sites on Cattle Hides at a Feedlot and Processing Plant

USDA-ARS?s Scientific Manuscript database

The distributions of Escherichia coli O157:H7 and Salmonella on cattle hides were mapped at a feedlot and a processing plant. Sponge samples were collected from six hide surface sites at the feedlot (left and right shoulders, left and right ribs, back, and belly) and four sites at the processing pla...

Elimination of Escherichia coli and Salmonella in Clam by Using Zeolite in a Station of Depuration.

PubMed

Gdoura, Morsi; Sellami, Hanen; Khannous, Lamia; Ketata, Najib; Neila, Idriss Ben; Traore, Al Ibrahim; Chekir, Zouhair; Gdoura, Radhouane

2017-09-01

  The application of natural zeolite for water and wastewater treatment has been carried out and is still a promising technique in environmental cleaning processes. Natural zeolite can be used to improve the purification process of clams (Ruditapes decussatus). Thus, our study aimed at improving the clam purification system in order to reduce Escherichia coli and eliminate Salmonella in samples artificially contaminated with this bacterium using a natural zeolite to replace the biological filter. The results showed that zeolite used in a depuration system improved the clam purification process. Moreover, natural zeolite exhibited high performance in the adsorption of bacteria and allowed to reduce the Escherichia coli abundance in 24 h, thus ensuring purified clams conformity with the ISO 16649-3 standard. These results indicate the beneficial effects of using zeolite in the adsorption of bacteria and the reduction in the abundance of Escherichia coli and set the Salmonella from marine organisms.

Inactivation of Uropathogenic Escherichia coli in Ground Chicken Meat Using High Pressure Processing and Gamma Radiation, and in Purge and Chicken Meat Surfaces by Ultraviolet Light.

PubMed

Sommers, Christopher H; Scullen, O J; Sheen, Shiowshuh

2016-01-01

Extraintestinal pathogenic Escherichia coli, including uropathogenic E. coli (UPEC), are common contaminants in poultry meat and may cause urinary tract infections after colonization of the gastrointestinal tract and transfer of contaminated feces to the urethra. Three non-thermal processing technologies used to improve the safety and shelf-life of both human and pet foods include high pressure processing (HPP), ionizing (gamma) radiation (GR), and ultraviolet light (UV-C). Multi-isolate cocktails of UPEC were inoculated into ground chicken which was then treated with HPP (4°C, 0-25 min) at 300, 400, or 500 MPa. HPP D10, the processing conditions needed to inactivate 1 log of UPEC, was 30.6, 8.37, and 4.43 min at 300, 400, and 500 MPa, respectively. When the UPEC was inoculated into ground chicken and gamma irradiated (4 and -20°C) the GR D10 were 0.28 and 0.36 kGy, respectively. The UV-C D10 of UPEC in chicken suspended in exudate and placed on stainless steel and plastic food contact surfaces ranged from 11.4 to 12.9 mJ/cm(2). UV-C inactivated ca. 0.6 log of UPEC on chicken breast meat. These results indicate that existing non-thermal processing technologies such as HPP, GR, and UV-C can significantly reduce UPEC levels in poultry meat or exudate and provide safer poultry products for at-risk consumers.

Drivers of Water Quality Variability in Northern Coastal Ecuador

PubMed Central

Hubbard, Alan E.; Nelson, Kara L.; Eisenberg, Joseph N.S.

2012-01-01

The microbiological safety of water is commonly measured using indicator organisms, but the spatiotemporal variability of these indicators can make interpretation of data difficult. Here we systematically explore variability in E.coli concentrations in surface source and household drinking water in a rural Ecuadorian village over one year. We observed more variability in water quality on an hourly basis (up to 2.4-log difference) than on a daily (2.2-log difference) or weekly basis (up to 1.8-log difference). E.coli counts were higher in the wet season than in the dry season for both source (0.42-log difference; p<0.0001) and household (0.11-log difference; p=0.077) samples. In the wet season, a one-cm increase in weekly rainfall was associated with a 3% decrease (p=0.006) in E.coli counts in source samples and a 6% decrease (p=0.012) in household samples. Each additional person in the river when source samples were collected was associated with a 4% increase (p=0.026) in E.coli counts in the wet season. Factors affecting household water quality included rainfall, water source, and covering the container. The variability can be understood as a combination of environmental (e.g., seasonal and soil processes) and other drivers (e.g., human river use, water practices and sanitation), each working at different timescales. PMID:19368173

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7.

PubMed

Hosein, Althea M; Breidt, Frederick; Smith, Charles E

2011-02-01

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.

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

PubMed

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

2015-09-24

To assess public health risks from environmental exposure to Extended-Spectrum β-Lactamases (ESBL)-producing bacteria, it is necessary to have insight in the proportion of relative harmless commensal variants and potentially pathogenic ones (which may directly cause disease). In the current study, 170 ESBL-producing E. coli from Dutch wastewater (n = 82) and surface water (n = 88) were characterized with respect to ESBL-genotype, phylogenetic group, resistance phenotype and virulence markers associated with enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), extraintesinal E. coli (ExPEC), and Shiga toxin-producing E. coli (STEC). Overall, 17.1% of all ESBL-producing E. coli were suspected pathogenic variants. Suspected ExPECs constituted 8.8% of all ESBL-producing variants and 8.3% were potential gastrointestinal pathogens (4.1% EAEC, 1.8% EPEC, 1.2% EIEC, 1.2% ETEC, no STEC). Suspected pathogens were significantly associated with ESBL-genotype CTX-M-15 (X(2) = 14.7, P < 0.001) and phylogenetic group B2 (X(2) = 23.5, P < 0.001). Finally, 84% of the pathogenic ESBL-producing E. coli isolates were resistant to three or more different classes of antibiotics. In conclusion, this study demonstrates that the aquatic environment is a potential reservoir of E. coli variants that combine ESBL-genes, a high level of multi-drug resistance and virulence factors, and therewith pose a health risk to humans upon exposure.

Spread of multidrug-resistant Escherichia coli harboring integron via swine farm waste water treatment plant.

PubMed

Park, Jin-Hyeong; Kim, Young-Ji; Binn-Kim; Seo, Kun-Ho

2018-03-01

Wastewater treatment plants (WWTPs) that release treated wastewater into the environment have emerged as a major threat to public health. In this study, we investigated Escherichia coli load and antibiotic-resistance profiles across different treatment processes at a swine farm WWTP. The frequency of the detection of class 1 and 2 integrons, and their association with antibiotic resistance, were also analyzed. Samples were obtained at each of five sampling sites that represented each processing step within the WWTP. The largest decrease in E. coli load was observed during the anaerobic digestion step (from 4.86 to 2.89log CFU/mL). Isolates resistant to β-lactam antibiotics were efficiently removed after a series of treatment steps, whereas the proportions of isolates resistant to non-β-lactam antibiotics and multidrug-resistant strains were maintained across treatments. The occurrence of integron-positive strains was not significantly different at the various sampling sites (43.4-70%; p>0.05). Of the class 1 integron-positive isolates, 17.9% harbored the integron-associated gene cassettes aadA2, aadA12, aadA22, and dfrA15. To the best of our knowledge, this is the first description of a class 1 integron containing the aadA12 gene cassette from a swine farm and the presence of a class 1 integron containing dfrA15 in E. coli. This suggests that novel antibiotic-resistance gene cassette arrays could be generated in swine farm WWTPs. Moreover, 75% of integron-positive strains were categorized as multidrug resistant, whereas only 15.4% of integron-negative strains were multidrug resistant (p<0.05), indicating that integrons may be responsible for mediating resistance in WWTPs. With regard to the occurrence of multidrug-resistant, integron-positive E. coli recovered from the final effluent, our results highlighted the potential risks associated with wastewater discharge from swine farm WWTPs in terms of the spread of antibiotic-resistant bacteria to the aquatic environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigation of E. coli bacteria inactivation by photocatalytic activity of TiO2 coated expanded polystyrene foam

NASA Astrophysics Data System (ADS)

Varnagiris, S.; Sakalauskaite, S.; Tuckute, S.; Lelis, M.; Daugelavicius, R.; Milcius, D.

2017-03-01

Photocatalytic properties of anatase and other TiO2 polymorphs are widely researched and applied in practical application. In current study TiO2 films on the plasma pre-treated expanded polystyrene (EPS) foam were deposited using magnetron sputtering technique. Main properties of the films were characterised using combination of XRD, XPS and SEM techniques. Photocatalytic properties of the observed crystalline anatase phase were tested by investigating bleaching of the methylene blue (MB) aqueous solution and by testing Escherichia coli (E. coli) viability after incubation under UV-B irradiation. E. coli viability experiments indicated that there are two mechanisms of E. coli bacteria inactivation. UV irradiation alone causes rapid damage to the outer membrane of E. coli bacteria. The second mechanism of E. coli inactivation is invoked only with synergistic combination of TiO2 and UV. Acting as photocatalyst TiO2 generates active radicals who initiate the chain peroxidation of organic molecules and within 45 min reduce E. coli bacteria viability by nearly 90%.

Prevalence of Escherichia coli O157:H7 in beef cattle at slaughter and beef carcasses at retail shops in Ethiopia

USDA-ARS?s Scientific Manuscript database

Background: There is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia. Methods: Various samples we...

Evidence for existence of different Escherichia coli populations in karst aquifer depending on hydrological conditions and the use of watershed. Fabienne Petit1*, Mehdy Ratajczak1, Nicolas Massei 1, Olivier Clermont 2, Erick Denamur 2, Thierry Berthe1,. 1CNRS UMR 6143 M2C, Université de Rouen, FED SCALE 4116, 76821 Mont Saint Aignan 2 INSERM U722, Université Paris 7 Denis Diderot ,75018 Paris

NASA Astrophysics Data System (ADS)

Fabienne, P.; Mehdy, R.; Massei, N.; Clermont, O.; Denamur, E.; Berthe, T.

2011-12-01

Escherichia coli (E. coli) is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, even if the aquatic environment could be considered as a secondary habitat. During turbids events consecutive to the rainfall, E.coli are released from manure and feces in karstic hydrosystem with different settling velocities, related to their association to particles. In water, survival of E. coli, was dependant to the grazing by protozoans and their ability to overcome environmental stress. In these conditions, viable but non culturable (VNC) population of E. coli, could be observed. The aim of this study was to investigate, in a small well characterized rural karstic watershed (i) the structure of E. coli population based on the survival ability, the distribution in four main phylo-groups (A, B1, B2, D), and the phenotypic characteristics, (ii) the fate and the distribution of viable non culturable E. coli according their settling velocities, from surface water to groundwater. For this purpose we combined microbiology and hydrology approaches, and solid phase cytometry (ChemScan°RDI) methodology was performed to numbered VNC E. coli. The distribution in the four main E. coli phylo-groups (A, B1, B2, D) shown that the E. coli population structure was modified not only by the hydrological conditions but also the use of the watershed (presence of cattle). Survival abilities of E. coli strains based on microcosm experiments, vary from 2 days to at least 14 days. Characterization of E. coli was performed by studying specific traits present in host-associated strains (virulence factors, antibiotic resistance) and those that could be involved in water persistence (growth temperature substrate range, biofilm formation and grazing by protozoa). Three major clusters of strains were defined by using a correspondence factor analysis. In water characterized by high level of fecal contamination a first cluster of E. coli strains was related to A and B2 phylo-group, presented a multiple-antibiotic-resistance profile, and had low survival abilities in water. In slightly contaminated water, E. coli strains were persistent in water, sensitive to antibiotics, and able to develop at low temperature (from 7°C to 20°C) and to degrade macromolecules. In the same karstic hydrosystem, whatever the hydrological conditions, a population of E. coli in VNC state was observed, even in dry period where VNC E. coli raised to 96% of the total viable E. coli population. The distribution of the E. coli VNC population according to their settling velocity varies along the transfer between the swallow hole to the spring. Thus rapid flow inside karstic aquifer supports the culturability of E. coli. In contrast, in during low-flow period with slow transport of contaminant, E. coli lose their culturability but could maintained inside in VNC state in such hydrosystem.

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

USDA-ARS?s Scientific Manuscript database

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...

Distribution of Escherichia coli and Enterococci in water, sediments, and bank soils along North Shore Channel between Bridge Street and Wilson Avenue, Metropolitan Water Reclamation District of Greater Chicago

USGS Publications Warehouse

Byappanahalli, Muruleedhara; Whitman, Richard L.; Shively, Dawn; Przybyla-Kelly, Katarzyna; Lukasik, Ashley M.

2010-01-01

The Metropolitan Water Reclamation District of Greater Chicago (MWRDGC) wished to know the distribution and potential sources of fecal indicator bacteria, E. coli and enterococci, in water, sediments, and upland soils along an upstream and downstream portion of the North Shore Channel (NSC) that is the receiving stream for the District’s North Side Water Reclamation Plant (NSWRP) outfall. Biweekly water and sediment samples were collected between August and October 2008 and included the following locations upstream of the outfall: Bridge Street (UPS-1), Oakton Street (UPS-2), the NSWRP outfall (OF), and downstream: Foster Avenue (DNS-1), and Wilson Avenue (DNS-2). E. coli and enterococci were consistently found in water and sediments at all sampling locations, with bacterial densities in water increasing below the NSWRP outfall; bacterial densities in sediment were more variable. On a relative measurement basis (i.e., 100 ml=100 g), both E. coli and enterococci densities were significantly higher in sediments than water. E. coli and enterococci were consistently recovered from bank soil along wooded, grassy, erosional, and depositional areas at two recreational parks, as well as other riparian areas along the river. Thus, soils along the river basin are likely sources of these bacteria to the NSC channel, introduced through runoff or other physical processes. Tributaries, such as the North Branch of the Chicago River (NBCR) that flow into NSC near Albany Ave, may provide a constant source of E. coli and enterococci to the NSC. Additionally, storm sewer outfalls may increase E. coli loadings to NSC during wet weather conditions. Our findings suggest that the abundance of nonpoint sources contributing to the overall fecal indicator bacteria (FIB) load in the NSC channel may complicate bacteria source determination and remediation efforts to protect the stream water quality.

Exogenous carbon monoxide suppresses Escherichia coli vitality and improves survival in an Escherichia coli-induced murine sepsis model.

PubMed

Shen, Wei-chang; Wang, Xu; Qin, Wei-ting; Qiu, Xue-feng; Sun, Bing-wei

2014-12-01

Endogenous carbon monoxide (CO) has been shown to modulate inflammation and inhibit cytokine production both in vivo and in vitro. The aim of this study was to examine whether exogenous carbon monoxide could suppress the vitality of Escherichia coli (E coli) and improve the survival rate in an E coli-induced murine sepsis model. ICR mice were infected with E coli, and immediately injected intravenously with carbon monoxide releasing molecule-2 (CORM-2, 8 mg/kg) or inactive CORM-2 (8 mg/kg). The survival rate was monitored 6 times daily for up to 36 h. The blood samples, liver and lung tissues were collected at 6 h after the infection. Bacteria in peritoneal lavage fluid, blood and tissues were enumerated following culture. Tissue iNOS mRNA expression was detected using RT-PCR. NF-κB expression was detected with Western blotting. Addition of CORM-2 (200 and 400 μmol/L) into culture medium concentration-dependently suppressed the growth of E coli and decreased the colony numbers, but inactive CORM-2 had no effect. Treatment of the infected mice with CORM-2 significantly increased the survival rate to 55%, while all the infected mice treated with inactive CORM-2 died within 36 h. E coli infection caused severe pathological changes in liver and lungs, and significantly increased serum transaminases, lipopolysaccharide, TNF-α and IL-1β levels, as well as myeloperoxidase activity, TNF-α and IL-1β levels in the major organs. Meanwhile, E coli infection significantly increased the number of colonies and the expression of iNOS mRNA and NF-κB in the major organs. All these abnormalities were significantly attenuated by CORM-2 treatment, while inactive CORM-2 was ineffective. In addition directly suppressing E coli, CORM-2 protects the liver and lungs against E coli-induced sepsis in mice, thus improving their survival.

Prevention of Escherichia coli K1 penetration of the blood-brain barrier by counteracting the host cell receptor and signaling molecule involved in E. coli invasion of human brain microvascular endothelial cells.

PubMed

Zhu, Longkun; Pearce, Donna; Kim, Kwang Sik

2010-08-01

Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A(2)alpha (cPLA(2)alpha). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA(2)alpha, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.

Identification of diarrheagenic Escherichia coli strains from avian organic fertilizers.

PubMed

Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P; Nishio, Erick K; Kobayashi, Renata K T; Nakazato, Gerson

2014-08-28

The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

Inactivation of E. coli O157:H7 on blueberries by electrolyzed water, ultraviolet light, and ozone.

PubMed

Kim, Chyer; Hung, Yen-Con

2012-04-01

Increased interest in blueberries due to their nutritional and health benefits has led to an increase in consumption. However, blueberries are consumed mostly raw or minimally processed and are susceptible to microbial contamination like other type of fresh produce. This study was, therefore, undertaken to evaluate the efficacy of electrostatic spray of electrolyzed oxidizing (EO) water, UV light, ozone, and a combination of ozone and UV light in killing Escherichia coli O157:H7 on blueberries. A 5-strain mixture of E. coli O157:H7 were inoculated on the calyx and skin of blueberries and then subjected to the treatments. Electrostatic EO water spray reduced initial populations of E. coli O157:H7 by only 0.13 to 0.24 log CFU/g and 0.88 to 1.10 log CFU/g on calyx and skin of blueberries, respectively. Ozone treatment with 4000 mg/L reduced E. coli O157:H7 by only 0.66 and 0.72 log CFU/g on calyx and skin of blueberries, respectively. UV light at 20 mW/cm² for 10 min was the most promising single technology and achieved 2.14 and greater than 4.05 log reductions of E. coli O157:H7 on the calyx and skin of blueberries, respectively. The combination treatment of 1 min ozone and followed by a 2 min UV achieved more than 1 and 2 log additional reductions on blueberry calyx than UV or ozone alone, respectively. Outbreaks of foodborne illnesses have been associated with consumption of fresh produce. Many methods for removing pathogens as well as minimizing their effect on quality of treated produce have been investigated. UV technology and its combination with ozone used in this study to inactive E. coli O157:H7 on blueberries was found effective. Results from this study may help producers and processors in developing hurdle technologies for the delivery of safer blueberries to consumers. © 2012 Institute of Food Technologists®

[Analysis of Pathogenic Bacteria in Reclaimed Water and Impact of UV Disinfection on the Removal of Pathogenic Bacteria].

PubMed

Jing, Ming; Wang, Lei

2016-02-15

In the study, 454-pyrosequencing technology was employed to investigate the species of pathogenic bacteria and the proportion of each pathogen in secondary effluent. Culture-based, qPCR and Q-RT-PCR methods were employed to analyze the removal of indicator (E. coli) and pathogen (Salmonella and Mycobacterium) by ultraviolet (UV) disinfection at a dose of 60 mJ x Cm(-2). The results showed that 11 kinds of pathogenic bacteria were found and the most abundant potentially pathogenic bacteria in the secondary effluent were affiliated with the genera of Clostridium (2.96%), Arcobacter (0.82%) and Mycobacterium (0.36%). 99.9% of culturable E. coli and Salmonella were removed by UV disinfection (60 mJ x cm(-2), however, less than 90% of culturable Mycobacterium were removed. The removal efficiencies of viable E. coli, Salmonella and Mycobacterium were low. Q-RT-PCR seemed to be a promising method for evaluating viable microorganisms in samples. Besides, pathogenic bacteria entered into VBNC state at a UV dose of 60 mJ x cm(-2). Other advanced treatment processes were needed to ensure safe utilization of reclaimed water.

Inactivation of Escherichia Coli O157:H7 and Salmonella Enterica on Blueberries in Water Using Ultraviolet Light.

PubMed

Liu, Chuhan; Huang, Yaoxin; Chen, Haiqiang

2015-07-01

Ultraviolet light (UV) has antimicrobial effects, but the shadowing effect has limited its application. In this study, a novel setup using UV processing in agitated water was developed to inactivate Escherichia coli O157:H7 and Salmonella on blueberries. Blueberries were dip- or spot-inoculated with E. coli or Salmonella. Blueberries inoculated with E. coli were treated for 2 to 10 min with UV directly (dry UV) or immersed in agitated water during UV treatment (wet UV). E. coli was most easily killed on spot-inoculated blueberries with a 5.2-log reduction after 10-min wet UV treatment. Dip-inoculated blueberries were the most difficult to be decontaminated with only 1.6-log reduction after 10-min wet UV treatment. Wet UV treatment generally showed higher efficacies than dry UV treatment, achieving an average of 1.4 log more reduction for spot-inoculated blueberries. For dip-inoculated blueberries, chlorine washing and UV treatments were less effective, achieving <2 log reductions of E. coli. Thus, the efficacy of combinations of wet UV with sodium dodecyl sulfate (SDS), levulinic acid, or chlorine was evaluated. Inoculated blueberries were UV-treated while being immersed in agitated water containing 100 ppm SDS, 0.5% levulinic acid or 10 ppm chlorine. The 3 chemicals did not significantly enhance the wet UV treatment. Findings of this study suggest that UV treatment could be used as an alternative to chlorine washing for blueberries and potentially for other fresh produce. A novel UV light system for decontamination of blueberries in water was developed and evaluated. Results demonstrated that the decontamination efficacy of this system was generally as effective as chlorine washing, indicating that it could potentially be used as an alternative to chlorine washing for blueberries and other fresh produce. © 2015 Institute of Food Technologists®

Removal of bacteria from stallion semen by colloid centrifugation.

PubMed

Morrell, J M; Klein, C; Lundeheim, N; Erol, E; Troedsson, M H T

2014-02-01

Bacteria (environmental contaminants and occasionally potential pathogens) are found in most stallion ejaculates and may negatively affect sperm quality during storage. Since the use of antibiotics can lead to the development of resistance, an alternative means of microbial control is desirable. The removal of bacteria from stallion semen using Single Layer Centrifugation through Androcoll-E was investigated. Known doses of cultured bacteria were added to freshly collected ejaculates (15mL aliquots) before processing by Single Layer Centrifugation. The resulting sperm pellets and controls (not processed by Single Layer Centrifugation) were cultured and the bacteria identified. In experiment 1, doses of E. coli from 2×10(2) to 2×10(7) colony forming units were added to aliquots of semen. In experiment 2, Taylorella equigenitalis or a mix of E. coli, Klebsiella pneumoniae and Streptococcus equi subsp. zooepidemicus (approximately 7×10(6), 5×10(6), and 6×10(6)cfu, respectively) were added to 15mL aliquots of semen. In experiment 1, more than 90% of the bacteria were removed where loading doses were >×10(4)cfu/mL. In experiment 2, varying proportions of different bacteria were removed, ranging from 68% for naturally occurring Corynebacterium spp. to >97% for added cultured E. coli. Thus, Single Layer Centrifugation can separate spermatozoa from many, but not all bacteria in stallion ejaculates and could be a useful alternative to adding antibiotics to semen extenders to control bacterial contamination. However, further research is needed to determine the effect of small numbers of bacteria on sperm quality. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2018-07-20

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

Fecal contamination of drinking water supplies in and around Chandigarh and correlation with acute gastroenteritis.

PubMed

Neelam, Taneja; Malkit, Singh; Pooja, Rao; Manisha, Biswal; Shiva, Priya; Ram, Chander; Meera, Sharma

2012-12-01

Acute gastroenteritis due to Vibrio cholerae and Enterotoxigenic E. coli is a common problem faced in the hot and humid summer months in north India. The study was undertaken to evaluate drinking water supplies for fecal coliforms, V. cholerae and Enterotoxigenic E. coli in urban, semiurban and rural areas in and around Chandigarh and correlate with occurrence of acute gastroenteritis occurring from the same region. Drinking water sample were collected from various sources from April to October 2004 from a defined area. Samples were tested for fecal coliforms and E. coli count. E. coli were screened for heat labile toxin (LT) also. Stool samples from cases of acute gastroenteritis from the same region and time were collected and processed for V. cholerae, Enterotoxigenic E. coli (ETEC) and others like Salmonella, Shigella and Aeromonas spp. A total of 364 water samples were collected, (251 semi urban, 41 rural and 72 from urban areas). 116 (31.8%) samples were contaminated with fecal coliforms (58.5% rural, 33.4% semiurban and 11.1% of samples from urban areas). E. coli were grown from 58 samples. Ninety two isolates of E. coli were tested for enterotoxins of which 8 and 24 were positive for LT and ST respectively. V. cholerae were isolated from 2 samples during the outbreak investigation. Stored water samples showed a significantly higher level of contamination and most of Enterotoxigenic E. coli were isolated from stored water samples. A total of 780 acute gastroenteritis cases occurred; 445 from semiurban, 265 rural and 70 from urban areas. Out of 189 stool samples submitted, ETEC were the commonest (30%) followed by V. cholerae (19%), Shigellae (8.4%), Salmonellae (2.1%) and Aeromonas (2.6%). ST-ETEC (40/57) were commoner than LT-ETEC (17/57). In the present study, high levels of contamination of drinking water supplies (32.1%) correlated well with cases of acute gastroenteritis. Majority of cases of acute gastroenteritis occurred in the semi urban corresponding with high level of contamination (33.4%). The highest level of water contamination was seen in rural areas (58.5%) but the number of acute gastroenteritis cases were lesser (33.9%) as ponds were infrequently used for drinking purpose. Safer household water storage and treatment is recommended to prevent acute gastroenteritis, together with point-of-use water quality monitoring.

Engineering E. coli–E. coli cocultures for production of muconic acid from glycerol

DOE PAGES

Zhang, Haoran; Li, Zhengjun; Pereira, Brian; ...

2015-09-15

cis, cis-Muconic acid is an important chemical that can be biosynthesized from simple substrates in engineered microorganisms. Recently, it has been shown that engineering microbial cocultures is an emerging and promising approach for biochemical production. In this study, we aim to explore the potential of the E. coli–E. coli coculture system to use a single renewable carbon source, glycerol, for the production of value-added product cis, cis-muconic acid. As a result, two coculture engineering strategies were investigated. In the first strategy, an E. coli strain containing the complete biosynthesis pathway was co-cultivated with another E. coli strain containing only amore » heterologous intermediate-to-product biosynthetic pathway. In the second strategy, the upstream and downstream pathways were accommodated in two separate E. coli strains, each of which was dedicated to one portion of the biosynthesis process. Compared with the monoculture approach, both coculture engineering strategies improved the production significantly. Using a batch bioreactor, the engineered coculture achieved a 2 g/L muconic acid production with a yield of 0.1 g/g. In conclusion, our results demonstrate that coculture engineering is a viable option for producing muconic acid from glycerol. Moreover, microbial coculture systems are shown to have the potential for converting single carbon source to value-added products.« less

Microbiological quality and safety assessment of lettuce production in Brazil.

PubMed

Ceuppens, Siele; Hessel, Claudia Titze; de Quadros Rodrigues, Rochele; Bartz, Sabrina; Tondo, Eduardo César; Uyttendaele, Mieke

2014-07-02

The microbiological quality and safety of lettuce during primary production in Brazil were determined by enumeration of hygiene indicators Escherichia coli, coliforms and enterococci and detection of enteric pathogens Salmonella and E. coli O157:H7 in organic fertilizers, soil, irrigation water, lettuce crops, harvest boxes and worker's hands taken from six different lettuce farms throughout the crop growth cycle. Generic E. coli was a suitable indicator for the presence of Salmonella and E. coli O157:H7, while coliforms and enterococci were not. Few pathogens were detected: 5 salmonellae and 2 E. coli O157:H7 from 260 samples, of which only one was lettuce and the others were manure, soil and water. Most (5/7) pathogens were isolated from the same farm and all were from organic production. Statistical analysis revealed the following environmental and agro-technical risk factors for increased microbial load and pathogen prevalence in lettuce production: high temperature, flooding of lettuce fields, application of contaminated organic fertilizer, irrigation with water of inferior quality and large distances between the field and toilets. Control of the composting process of organic fertilizers and the irrigation water quality appear most crucial to improve and/or maintain the microbiological quality and safety during the primary production of lettuce. Copyright © 2014 Elsevier B.V. All rights reserved.

Persistence of Escherichia coli O157:H7 in dairy fermentation systems.

PubMed

Dineen, S S; Takeuchi, K; Soudah, J E; Boor, K J

1998-12-01

We examined (i) the persistence of Escherichia coli O157:H7 as a postpasteurization contaminant in fermented dairy products; (ii) the ability of E. coli O157:H7 strains with and without the general stress regulatory protein, RpoS, to compete with commercial starter cultures in fermentation systems; and (iii) the survival of E. coli O157:H7 in the yogurt production process. In commercial products inoculated with 10(3) CFU/ml, E. coli O157:H7 was recovered for up to 12 days in yogurt (pH 4.0), 28 days in sour cream (pH 4.3), and at levels > 10(2) CFU/ml at 35 days in buttermilk (pH 4.1). For the starter culture competition trials, the relative inhibition of E. coli O157:H7 in the experimental fermentation systems was, in decreasing order, thermophilic culture mixture, Lactobacillus delbrueckii subsp. bulgaricus R110 alone, Lactococcus lactis subsp. lactis D280 alone, Lactococcus lactis subsp. cremoris D62 alone, and Streptococcus thermophilus C90 alone showing the least inhibition. Recovery of the rpoS mutant was lower than recovery of its wild-type parent by 72 h or earlier in the presence of individual starter cultures. No E. coli O157:H7 were recovered after the curd formation step in yogurt manufactured with milk inoculated with 10(5) CFU/ml. Our results show that (i) postprocessing entry of E. coli O157:H7 into fermented dairy products represents a potential health hazard; (ii) commercial starter cultures differ in their ability to reduce E. coli O157:H7 CFU numbers in fermentation systems; and (iii) the RpoS protein appears to most effectively contribute to bacterial survival in the presence of conditions that are moderately lethal to the cell.

Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12.

PubMed

Mikhailov, A M; Smirnova, E A; Tsuprun, V L; Tagunova, I V; Vainshtein, B K; Linkova, E V; Komissarov, A A; Siprashvili, Z Z; Mironov, A S

1992-03-01

Uridine phosphorylase (UPH) from Escherichia coli K-12 has been purified to near homogeneity from a strain harbouring the udp gene, encoding UPH, on a multicopy plasmid. UPH was purified to electrophoretic homogeneity with the specific activity 230 units/mg with a recovery of 80%, yielding 120 mg of enzyme from 3g cells. Crystals of enzyme suitable for X-ray diffraction analysis were obtained in a preparative ultracentrifuge. The packing of the molecules in the crystals may be described by the space group P2(1)2(1)2(1) with the unit cell constants a = 90.4; b = 128.8; c = 136.8 A. There is one molecule per asymmetric unit, Vm = 2.4. These crystals diffract to at least 2.5-2.7 A resolution. The hexameric structure of UPH was directly demonstrated by electron microscopy study and image processing.

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

Fate of naturally occurring Escherichia coli O157:H7 and other zoonotic pathogens during minimally managed bovine feedlot manure composting processes

USDA-ARS?s Scientific Manuscript database

Reducing Escherichia coli O157:H7 in livestock manures before application to cropland is critical for reducing the risk of foodborne illness associated with produce. Our objective was to determine the fate of naturally occurring E. coli O157:H7 and other pathogens during minimally managed on-farm bo...

Effect of plant essential oils against foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in raw cookie dough

USDA-ARS?s Scientific Manuscript database

Cookie dough can be contaminated by raw ingredients, mishandling, package contamination, etc. Considering the recent outbreak of E. coli O157:H7 with commercial raw cookie dough, the ability of E. coli O157:H7 to survive in the raw cookie dough production and processing environments, it raised conce...

Determining thermal inactivation of Escherichia coli O157:H7 in fresh compost by simulating early phases of the composting process.

PubMed

Singh, Randhir; Kim, Jinkyung; Shepherd, Marion W; Luo, Feng; Jiang, Xiuping

2011-06-01

A three-strain mixture of Escherichia coli O157:H7 was inoculated into fresh dairy compost (ca. 10(7) CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The surviving E. coli O157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix, E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall, E. coli O157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well.

Determining Thermal Inactivation of Escherichia coli O157:H7 in Fresh Compost by Simulating Early Phases of the Composting Process ▿

PubMed Central

Singh, Randhir; Kim, Jinkyung; Shepherd, Marion W.; Luo, Feng; Jiang, Xiuping

2011-01-01

A three-strain mixture of Escherichia coli O157:H7 was inoculated into fresh dairy compost (ca. 107 CFU/g) with 40 or 50% moisture and was placed in an environmental chamber (ca. 70% humidity) that was programmed to ramp from room temperature to selected composting temperatures in 2 and 5 days to simulate the early composting phase. The surviving E. coli O157:H7 population was analyzed by direct plating and enrichment. Optimal and suboptimal compost mixes, with carbon/nitrogen (C/N) ratios of 25:1 and 16:1, respectively, were compared in this study. In the optimal compost mix, E. coli O157:H7 survived for 72, 48, and 24 h in compost with 40% moisture and for 72, 24, and 24 h with 50% moisture at 50, 55, and 60°C, respectively, following 2 days of come-up time (rate of heating up). However, in the suboptimal compost mix, the pathogen survived for 288, 72, and 48 h in compost with 40% moisture and for 240, 72, 24 h in compost with 50% moisture at the same temperatures, respectively. Pathogen survival was longer, with 5 days of come-up time compared with 2 days of come-up. Overall, E. coli O157:H7 was inactivated faster in the compost with 50% moisture than in the compost with 40% at 55 and 60°C. Both moisture and come-up time were significant factors affecting Weibull model parameters. Our results suggest that slow come-up time at the beginning of composting can extend pathogen survival during composting. Additionally, both the C/N ratio and the initial moisture level in the compost mix affect the rate of pathogen inactivation as well. PMID:21498743

Evidence for Transfer of CMY-2 AmpC β-Lactamase Plasmids between Escherichia coli and Salmonella Isolates from Food Animals and Humans

PubMed Central

Winokur, P. L.; Vonstein, D. L.; Hoffman, L. J.; Uhlenhopp, E. K.; Doern, G. V.

2001-01-01

Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like β-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans. PMID:11557460

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

PubMed

Srivastava, Preeti; Deb, J K

2002-07-02

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Simultaneous E. coli inactivation and NOM degradation in river water via photo-Fenton process at natural pH in solar CPC reactor. A new way for enhancing solar disinfection of natural water.

PubMed

Moncayo-Lasso, Alejandro; Sanabria, Janeth; Pulgarin, César; Benítez, Norberto

2009-09-01

Bacteria inactivation and natural organic matter oxidation in river water was simultaneously conducted via photo-Fenton reaction at "natural" pH ( approximately 6.5) containing 0.6 mg L(-1) of Fe(3+) and 10 mg L(-1) of H(2)O(2). The experiments were carried out by using a solar compound parabolic collector on river water previously filtered by a slow sand filtration system and voluntarily spiked with Escherichia coli. Fifty five percent of 5.3 mg L(-1) of dissolved organic carbon was mineralized whereas total disinfection was observed without re-growth after 24h in the dark.

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

PubMed Central

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P.; Segal, Ester

2016-01-01

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps. PMID:27901131

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry.

PubMed

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P; Segal, Ester

2016-11-30

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >10 7  cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

Porous Silicon-Based Biosensors: Towards Real-Time Optical Detection of Target Bacteria in the Food Industry

NASA Astrophysics Data System (ADS)

Massad-Ivanir, Naama; Shtenberg, Giorgi; Raz, Nitzan; Gazenbeek, Christel; Budding, Dries; Bos, Martine P.; Segal, Ester

2016-11-01

Rapid detection of target bacteria is crucial to provide a safe food supply and to prevent foodborne diseases. Herein, we present an optical biosensor for identification and quantification of Escherichia coli (E. coli, used as a model indicator bacteria species) in complex food industry process water. The biosensor is based on a nanostructured, oxidized porous silicon (PSi) thin film which is functionalized with specific antibodies against E. coli. The biosensors were exposed to water samples collected directly from process lines of fresh-cut produce and their reflectivity spectra were collected in real time. Process water were characterized by complex natural micro-flora (microbial load of >107 cell/mL), in addition to soil particles and plant cell debris. We show that process water spiked with culture-grown E. coli, induces robust and predictable changes in the thin-film optical interference spectrum of the biosensor. The latter is ascribed to highly specific capture of the target cells onto the biosensor surface, as confirmed by real-time polymerase chain reaction (PCR). The biosensors were capable of selectively identifying and quantifying the target cells, while the target cell concentration is orders of magnitude lower than that of other bacterial species, without any pre-enrichment or prior processing steps.

Plant lesions promote the rapid multiplication of Escherichia coli O157:H7 on post-harvest lettuce

USDA-ARS?s Scientific Manuscript database

Several outbreaks of Escherichia coli O157:H7 (EcO157) infections have been associated with minimally processed leafy vegetables in the U.S. Harvesting and processing cause plant tissue damage. In order to assess the role of plant tissue damage in the contamination of leafy greens with EcO157, the e...

Inactivation of uropathogenic Escherichia coli in ground chicken meat using high pressure processing and gamma radiation, and in purge and chicken meat surfaces by ultraviolet light

USDA-ARS?s Scientific Manuscript database

Uropathogenic Escherichia coli (UPEC) are common contaminants in meat and poultry. Nonthermal food safety intervention technologies used to improve safety and shelf-life of both human and pet foods can include high pressure processing (HPP), ionizing (gamma) radiation (GR), and ultraviolet light (UV...

Regulation of Toll-like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells

PubMed Central

Krishnan, Subramanian; Chen, Shuang; Turcatel, Gianluca; Arditi, Moshe; Prasadarao, Nemani V.

2012-01-01

SUMMARY The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β) is critical for the pathogenesis of E. coli K1 meningitis. Since Hsp90 chaperones Toll-like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2−/− mice are resistant to E. coli K1 meningitis, while TLR4−/− mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA− E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C-termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 and TLR2 induces a bipartite signal, one from Ecgp96 through PKC-α while the other from TLR2 through MyD88, ERK1/2 and NF-κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC. PMID:22963587

Regulation of Toll-like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells.

PubMed

Krishnan, Subramanian; Chen, Shuang; Turcatel, Gianluca; Arditi, Moshe; Prasadarao, Nemani V

2013-01-01

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90β), is critical for the pathogenesis of Escherichia coli K1 meningitis. Since Hsp90 chaperones Toll-like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2(-/-) mice are resistant to E. coli K1 meningitis, while TLR4(-/-) mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA- E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC-α while the other from TLR2 through MyD88, ERK1/2 and NF-κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC. © 2012 Blackwell Publishing Ltd.

Killing of Escherichia coli by Crohn's Disease Monocyte-derived Macrophages and Its Enhancement by Hydroxychloroquine and Vitamin D

PubMed Central

Flanagan, Paul K.; Chiewchengchol, Direkrit; Wright, Helen L.; Edwards, Steven W.; Alswied, Abdullah; Satsangi, Jack; Subramanian, Sreedhar; Rhodes, Jonathan M.

2015-01-01

Background: Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli. Methods: Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D. Results: Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39–1.78); HC: N = 9, 1.50 (95% CI, 1.02–1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31–2.80]; HC = 2.65 [95% CI, 2.46–2.85], P = 0.42). HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH2-vitamin D3: 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin. Conclusions: CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics. PMID:25839777

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

DOE PAGES

Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G.; ...

2016-03-22

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits,more » the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe 2O 3 (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.« less

CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G.

Introducing extracellular electron transfer pathways into heterologous organisms offers the opportunity to explore fundamental biogeochemical processes and to biologically alter redox states of exogenous metals for various applications. While expression of the MtrCAB electron nanoconduit from Shewanella oneidensis MR-1 permits extracellular electron transfer in Escherichia coli, the low electron flux and absence of growth in these cells limits their practicality for such applications. In this paper, we investigate how the rate of electron transfer to extracellular Fe(III) and cell survival in engineered E. coli are affected by mimicking different features of the S. oneidensis pathway: the number of electron nanoconduits,more » the link between the quinol pool and MtrA, and the presence of flavin-dependent electron transfer. While increasing the number of pathways does not significantly improve the extracellular electron transfer rate or cell survival, using the native inner membrane component, CymA, significantly improves the reduction rate of extracellular acceptors and increases cell viability. Strikingly, introducing both CymA and riboflavin to Mtr-expressing E. coli also allowed these cells to couple metal reduction to growth, which is the first time an increase in biomass of an engineered E. coli has been observed under Fe 2O 3 (s) reducing conditions. Overall and finally, this work provides engineered E. coli strains for modulating extracellular metal reduction and elucidates critical factors for engineering extracellular electron transfer in heterologous organisms.« less

Escherichia coli attachment to model particulates: The effects of bacterial cell characteristics and particulate properties.

PubMed

Liang, Xiao; Liao, Chunyu; Soupir, Michelle L; Jarboe, Laura R; Thompson, Michael L; Dixon, Philip M

2017-01-01

E. coli bacteria move in streams freely in a planktonic state or attached to suspended particulates. Attachment is a dynamic process, and the fraction of attached microorganisms is thought to be affected by both bacterial characteristics and particulate properties. In this study, we investigated how the properties of cell surfaces and stream particulates influence attachment. Attachment assays were conducted for 77 E. coli strains and three model particulates (ferrihydrite, Ca-montmorillonite, or corn stover) under environmentally relevant conditions. Surface area, particle size distribution, and total carbon content were determined for each type of particulate. Among the three particulates, attachment fractions to corn stover were significantly larger than the attachments to 2-line ferrihydrite (p-value = 0.0036) and Ca-montmorillonite (p-value = 0.022). Furthermore, attachment to Ca-montmorillonite and corn stover was successfully modeled by a Generalized Additive Model (GAM) using cell characteristics as predictor variables. The natural logarithm of the net charge on the bacterial surface had a significant, positive, and linear impact on the attachment of E. coli bacteria to Ca-montmorillonite (p-value = 0.013), but it did not significantly impact the attachment to corn stover (p-value = 0.36). The large diversities in cell characteristics among 77 E. coli strains, particulate properties, and attachment fractions clearly demonstrated the inadequacy of using a static parameter or linear coefficient to predict the attachment behavior of E. coli in stream water quality models.

Evaluation of Combined Disinfection Methods for Reducing Escherichia coli O157:H7 Population on Fresh-Cut Vegetables

PubMed Central

Petri, Eva; Rodríguez, Mariola; García, Silvia

2015-01-01

Most current disinfection strategies for fresh-cut industry are focused on the use of different chemical agents; however, very little has been reported on the effectiveness of the hurdle technology. The effect of combined decontamination methods based on the use of different sanitizers (peroxyacetic acid and chlorine dioxide) and the application of pressure (vacuum/positive pressure) on the inactivation of the foodborne pathogen E. coli O157:H7 on fresh-cut lettuce (Lactuca sativa) and carrots (Daucus carota) was studied. Fresh produce, inoculated with E. coli O157:H7, was immersed (4 °C, 2 min) in tap water (W), chlorine water (CW), chlorine dioxide (ClO2: 2 mg/L) and peroxyacetic acid (PAA: 100 mg/L) in combination with: (a) vacuum (V: 10 mbar) or (b) positive pressure application (P: 3 bar). The product quality and antimicrobial effects of the treatment on bacterial counts were determined both in process washing water and on fresh-cut produce. Evidence obtained in this study, suggests that the use of combined methods (P/V + sanitizers) results in a reduction on the microorganism population on produce similar to that found at atmospheric pressure. Moreover, the application of physical methods led to a significant detrimental effect on the visual quality of lettuce regardless of the solution used. Concerning the process water, PAA proved to be an effective alternative to chlorine for the avoidance of cross-contamination. PMID:26213954

Evaluation of Combined Disinfection Methods for Reducing Escherichia coli O157:H7 Population on Fresh-Cut Vegetables.

PubMed

Petri, Eva; Rodríguez, Mariola; García, Silvia

2015-07-23

Most current disinfection strategies for fresh-cut industry are focused on the use of different chemical agents; however, very little has been reported on the effectiveness of the hurdle technology. The effect of combined decontamination methods based on the use of different sanitizers (peroxyacetic acid and chlorine dioxide) and the application of pressure (vacuum/positive pressure) on the inactivation of the foodborne pathogen E. coli O157:H7 on fresh-cut lettuce (Lactuca sativa) and carrots (Daucus carota) was studied. Fresh produce, inoculated with E. coli O157:H7, was immersed (4 °C, 2 min) in tap water (W), chlorine water (CW), chlorine dioxide (ClO2: 2 mg/L) and peroxyacetic acid (PAA: 100 mg/L) in combination with: (a) vacuum (V: 10 mbar) or (b) positive pressure application (P: 3 bar). The product quality and antimicrobial effects of the treatment on bacterial counts were determined both in process washing water and on fresh-cut produce. Evidence obtained in this study, suggests that the use of combined methods (P/V + sanitizers) results in a reduction on the microorganism population on produce similar to that found at atmospheric pressure. Moreover, the application of physical methods led to a significant detrimental effect on the visual quality of lettuce regardless of the solution used. Concerning the process water, PAA proved to be an effective alternative to chlorine for the avoidance of cross-contamination.

Oxaloacetate and malate production in engineered Escherichia coli by expression of codon-optimized phosphoenolpyruvate carboxylase2 gene from Dunaliella salina.

PubMed

Park, Soohyun; Chang, Kwang Suk; Jin, Eonseon; Pack, Seung Pil; Lee, Jinwon

2013-01-01

A new phosphoenolpyruvate carboxylase (PEPC) gene of Dunaliella salina is identified using homology analysis was conducted using PEPC gene of Chlamydomonas reinhardtii and Arabidopsis thaliana. Recombinant E. coli SGJS115 with increased production of malate and oxaloacetate was developed by introducing codon-optimized phosphoenolpyruvate carboxylase2 (OPDSPEPC2) gene of Dunaliella salina. E. coli SGJS115 yielded a 9.9 % increase in malate production. In addition, E. coli SGJS115 exhibited two times increase in the yield of oxaloacetate over the E. coli SGJS114 having identified PEPC2 gene obtained from Dunaliella salina.

Microbial safety and overall quality of cantaloupe fresh-cut pieces prepared from whole fruit after wet steam treatment.

PubMed

Ukuku, Dike O; Geveke, David J; Chau, Lee; Niemira, Brendan A

2016-08-16

Fresh-cut cantaloupes have been associated with outbreaks of Salmonellosis. Minimally processed fresh-cut fruits have a limited shelf life because of deterioration caused by spoilage microflora and physiological processes. The objectives of this study were to use a wet steam process to 1) reduce indigenous spoilage microflora and inoculated populations of Salmonella, Escherichia coli O157:H7 and Listeria monocytogenes on the surface of cantaloupes, and 2) reduce the populations counts in cantaloupe fresh-cut pieces after rind removal and cutting. The average inocula of Salmonella, E. coli O157:H7 and Listeria monocytogenes was 10(7)CFU/ml and the populations recovered on the cantaloupe rind surfaces after inoculation averaged 4.5, 4.8 and 4.1logCFU/cm(2), respectively. Whole cantaloupes were treated with a wet steam processing unit for 180s, and the treated melons were stored at 5°C for 29days. Bacterial populations in fresh-cut pieces prepared from treated and control samples stored at 5 and 10°C for up to 12days were determined and changes in color (CIE L*, a*, and b*) due to treatments were measured during storage. Presence and growth of aerobic mesophilic bacteria and Salmonella, E. coli O157:H7 and L. monocytogenes were determined in fresh-cut cantaloupe samples. There were no visual signs of physical damage on all treated cantaloupe surfaces immediately after treatments and during storage. All fresh-cut pieces from treated cantaloupes rind surfaces were negative for bacterial pathogens even after an enrichment process. Steam treatment significantly (p<0.05) changed the color of the fresh-cut pieces. Minimal wet steam treatment of cantaloupes rind surfaces designated for fresh-cut preparation will enhance the microbial safety of fresh-cut pieces, by reducing total bacterial populations. This process holds the potential to significantly reduce the incidence of foodborne illness associated with fresh-cut fruits. Published by Elsevier B.V.

Evaluation of CO2-based cold sterilization of a model hydrogel.

PubMed

Jiménez, A; Zhang, J; Matthews, M A

2008-12-15

The purpose of the present work is to evaluate a novel CO(2)-based cold sterilization process in terms of both its killing efficiency and its effects on the physical properties of a model hydrogel, poly(acrylic acid-co-acrylamide) potassium salt. Suspensions of Staphylococcus aureus and Escherichia coli were prepared for hydration and inoculation of the gel. The hydrogels were treated with supercritical CO(2) (40 degrees C, 27.6 MPa). The amount of bacteria was quantified before and after treatment. With pure CO(2), complete killing of S. aureus and E. coli was achieved for treatment times as low as 60 min. After treatment with CO(2) plus trace amounts of H(2)O(2) at the same experimental conditions, complete bacteria kill was also achieved. For times less than 30 min, incomplete kill was noted. Several physical properties of the gel were evaluated before and after SC-CO(2) treatment. These were largely unaffected by the CO(2) process. Drying curves showed no significant change between treated (pure CO(2) and CO(2) plus 30% H(2)O(2)) and untreated samples. The average equilibrium swelling ratios were also very similar. No changes in the dry hydrogel particle structure were evident from SEM micrographs.

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterization of antimicrobial resistant Escherichia coli isolated from processed bison carcasses.

PubMed

Li, Q; Sherwood, J S; Logue, C M

2007-12-01

To determine the phenotypic and genotypic antimicrobial susceptibility profiles of Escherichia coli from bison carcasses. The antimicrobial resistance of 138 E. coli isolates recovered from processed bison carcasses was determined by using the National Antimicrobial Resistance Monitoring System panels, polymerase chain reaction assays, plasmid analysis and conjugation studies. Resistance to 14 of the 16 antimicrobials was observed. Twenty-three (16.7%) isolates displayed resistance to at least one antimicrobial agent. The most prevalent resistances were to tetracycline (13.0%), sulfamethoxazole (7.9%) and streptomycin (5.8%). No resistance was observed to amikacin and ciprofloxacin. Further analysis of 23 antimicrobial-resistant E. coli isolates showed the presence of resistance genes corresponding to their phenotypic profiles. Results of conjugation studies carried out showed most isolates tested were able to transfer their resistance to recipients. This study indicated that multidrug-resistant E. coli isolates are present in bison. However, the resistance rate is lower than that reported in other meat species. The beneficial effects of antimicrobial-free feeding practice in bison may be promoting a reduction in the prevalence of antimicrobial resistance in commensal flora of bison.

The Campylobacter jejuni/coli cjaA (cj0982c) gene encodes an N-glycosylated lipoprotein localized in the inner membrane.

PubMed

Wyszyńska, Agnieszka; Zycka, Joanna; Godlewska, Renata; Jagusztyn-Krynicka, Elzbieta K

2008-09-01

The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor.

A quarantine process for the resolution of duodenoscope-associated transmission of multidrug-resistant Escherichia coli.

PubMed

Ross, Andrew S; Baliga, Christopher; Verma, Punam; Duchin, Jeffrey; Gluck, Michael

2015-09-01

Because of their complex design, duodenoscopes have been long recognized to be difficult to fully disinfect and may play a role in transmission of bacteria between patients. Recent reports of duodenoscope-associated carbapenem-resistant enterobacteriaceae transmission have confirmed these suspicions. An outbreak of a multidrug resistant strain of Escherichia coli was recently reported at our institution. Herein we report the results of our investigation and the process improvements that we deployed in an effort to contain the outbreak. A full investigation into the environment, endoscopists, infection control practices, high-level disinfection process as well as endoscopes was undertaken in conjunction with the local county health authority and the Centers for Disease Control and Prevention. Duodenoscopes were cultured and quarantined for 48 hours until negative cultures were obtained. Ergonomic changes were made to the endoscope reprocessing area, duodenoscopes were returned for routine maintenance, and surveillance cultures were obtained from all patients undergoing ERCP. Between November 2012 and August 2013, 32 patients were found to harbor 1 of 2 clonal strains of multidrug-resistant E coli, all of whom had undergone ERCP or duodenoscopy. A total of 1149 ERCPs were performed during this time period. Seven patients died within 31 days of the organism being identified in culture, 16 patients died overall by March 2015. The exact contribution of E coli to death is unclear because most patients had underlying late-stage malignancy or other severe medical comorbidities. No breach in high-level disinfection protocol or infection control practices was identified. The clonal strain of E coli was identified in culture on 4 of 8 duodenoscopes, 3 of which required critical repairs despite lack of obvious malfunction. The defect rate in high-level disinfection of duodenoscopes was 2% over a 1-year period. The implemented quality improvements, subsequent to which 1625 ERCPs have been performed, were successful in halting the outbreak. The existing manufacturer-recommended high-level disinfection protocols for duodenoscopes are inadequate. Although the ultimate solution may be a design change to the instrument, the timeline for such a change appears long and potentially difficult to exact. In the interim, a reliable method to ensure that bacterial pathogens are not present on the duodenoscope after high-level disinfection is needed. Copyright © 2015 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.

The effects on the microbiological condition of product of carcass dressing, cooling, and portioning processes at a poultry packing plant.

PubMed

Gill, C O; Moza, L F; Badoni, M; Barbut, S

2006-07-15

The log mean numbers of aerobes, coliforms, Escherichia coli and presumptive staphylococci plus listerias on chicken carcasses and carcass portions at various stages of processing at a poultry packing plant were estimated from the numbers of those bacteria recovered from groups of 25 randomly selected product units. The fractions of listerias in the presumptive staphylococci plus listerias groups of organisms were also estimated. Samples were obtained from carcasses by excising a strip of skin measuring approximately 5 x 2 cm(2) from a randomly selected site on each selected carcass, or by rinsing each selected carcass portion. The log mean numbers of aerobes, coliforms, E. coli and presumptive staphylococci plus listerias on carcasses after scalding at 58 degrees C and plucking were about 4.4, 2.5, 2.2 and 1.4 log cfu/cm(2), respectively. The numbers of bacteria on eviscerated carcasses were similar. After the series of operations for removing the crop, lungs, kidneys and neck, the numbers of aerobes were about 1 log unit less than on eviscerated carcasses, but the numbers of the other bacteria were not substantially reduced. After cooling in water, the numbers of coliforms and E. coli were about 1 log unit less and the numbers of presumptive staphylococci plus listerias were about 0.5 log unit less than the numbers on dressed carcasses, but the numbers of aerobes were not reduced. The numbers of aerobes were 1 log unit more on boneless breasts, and 0.5 log units more on skin-on thighs and breasts that had been tumbled with brine than on cooled carcasses; and presumptive staphylococci plus listerias were 0.5 log unit more on thighs than on cooled carcasses. Otherwise the numbers of bacteria on the product were not substantially affected by processing. Listerias were <20% of the presumptive staphylococci plus listerias group of organisms recovered from product at each point in the process except after breasts were tumbled with brine, when >40% of the organisms were listerias.

Antimicrobial resistance and genetic profiling of Escherichia coli from a commercial beef packing plant.

PubMed

Aslam, Mueen; Service, Cara

2006-07-01

The objective of this study was to investigate the extent of antimicrobial resistance and to genetically characterize resistant Escherichia coli recovered from a commercial beef packing plant. E. coli isolates were recovered by a hydrophobic grid membrane filtration method by direct plating on SD-39 medium. A total of 284 isolates comprising 71, 36, 55, 52, and 70 isolates from animal hides, washed carcasses, conveyers, beef trimmings, and ground beef, respectively, were analyzed. The susceptibility of E. coli isolates to 15 antimicrobial agents was evaluated with an automated broth microdilution system, and the genetic characterization of these isolates was performed by the random amplified polymorphic DNA (RAPD) method. Of the 284 E. coli isolates, 56% were sensitive to all 15 antimicrobial agents. Resistance to tetracycline, ampicillin, and streptomycin was observed in 38, 9, and 6% of the isolates, respectively. Resistance to one or more antimicrobial agents was observed in 51% of the E. coli isolates recovered from the hides but in only 25% of the E. coli from the washed carcasses. Resistance to one or more antimicrobial agents was observed in 49, 50, and 37% of the isolates recovered from conveyers, beef trimmings, and ground beef, respectively. The RAPD pattern data showed that the majority of resistant E. coli isolates were genetically diverse. Only a few RAPD types of resistant strains were shared among various sample sources. The results of this study suggest that antimicrobial-resistant E. coli isolates were prevalent during all stages of commercial beef processing and that considerably higher numbers of resistant E. coli were present on conveyers, beef trimmings, and ground beef than on dressed carcasses. This stresses the need for improving hygienic conditions during all stages of commercial beef processing and meatpacking to avoid the risks of transfer of antimicrobial-resistant bacteria to humans.

Biased expression, under the control of single promoter, of human interferon α-2b and Escherichia coli methionine amino peptidase genes in E. coli, irrespective of their distance from the promoter.

PubMed

Arif, Amina; Rashid, Naeem; Aslam, Farheen; Mahmood, Nasir; Akhtar, Muhammad

2016-03-01

Human interferon α-2b and Escherichia coli methionine amino peptidase genes were cloned independently as well as bicistronically in expression plasmid pET-21a (+). Production of human interferon α-2b was comparable to that of E. coli methionine amino peptidase when these genes were expressed independently in E. coli BL21-CodonPlus (DE3)-RIL. However, human interferon α-2b was produced in a much less amount whereas there was no difference in the production of methionine amino peptidase when the encoding genes were expressed bicistronically. It is important to note that human interferon α-2b was the first gene in order, after the promoter and E. coli methionine amino peptidase was the next with a linker sequence of 27 nucleotides between them.

A direct plating method for estimating populations of Escherichia coli O157 in bovine manure and manure-based materials.

PubMed

Berry, Elaine D; Wells, James E

2008-11-01

Escherichia coli O157:H7 outbreaks associated with produce consumption have brought attention to livestock manures and manure-based soil amendments as potential sources of pathogens for the contamination of these crops. Procedures for enumeration of E. coli O157:H7 are needed to assess the risks of transmission from these manures and their by-products. A direct plating method employing spiral plating onto CHROMagar O157 was investigated for enumeration of E. coli O157:H7 in feedlot surface material, aged bovine manure, bovine manure compost, and manure-amended soil. In studies utilizing samples spiked with a five-strain cocktail of E. coli O157:H7 at levels ranging from 102 to 10(5) CFU/g of sample, there were strong correlations between the observed and predicted levels of this pathogen. Although the addition of 2.5 mg/liter potassium tellurite and 5 mg/liter novobiocin made the medium more restrictive, these amendments enhanced the ability to identify and enumerate E. coli O157:H7 in feedlot surface material, which contained a higher proportion of fresh feces than did the other three sample types and therefore higher levels of interfering bacterial microflora. The spiral plating method was further assessed to determine its ability to enumerate E. coli O157:H7 in naturally contaminated feedlot surface material. Comparison of E. coli O157:H7 counts in feedlot surface material obtained by the spiral plating method and a most probable number technique were well correlated. We conclude that direct spiral plating onto CHROMagar O157 is effective for estimating E. coli O157:H7 levels in a variety of manures and manure-containing sample types to a lower detection limit of 200 CFU/g. The method has application for determining E. coli O157:H7 concentrations in manures and composts before their sale and use as soil amendments and for measuring the effectiveness of manure treatment processes to reduce or inactivate this pathogen.

Physical Covering to control Escherichia coli O157:H7 and Salmonella in Static and Windrow Composting Process

USDA-ARS?s Scientific Manuscript database

This study investigated the effect of 30-cm covering of finished compost on survival of E. coli O157:H7 and Salmonella in active static and windrow composting systems. Feedstock inoculated with E. coli O157:H7 (7.41 log CFU/g) and Salmonella (6.46 log CFU/g) were placed in biosentry tubes (7.5 cm di...

Draft Genome Sequences of Five Neonatal Meningitis-Causing Escherichia coli Isolates (SP-4, SP-5, SP-13, SP-46, and SP-65)

PubMed Central

Xu, Aixia; Johnson, James R.; Sheen, Shiowshuh

2018-01-01

ABSTRACT Neonatal meningitis-causing Escherichia coli isolates (SP-4, SP-5, SP-13, SP-46, and SP-65) were recovered between 1989 and 1997 from infants in the Netherlands. Here, we report the draft genome sequences of these five E. coli isolates, which are currently being used to validate food safety processing technologies. PMID:29674529

Draft genome sequences of six neonatal meningitis-causing escherichia coli isolates (SP-4, SP-5, SP-13, SP-16, SP-46, and SP-65)

USDA-ARS?s Scientific Manuscript database

Neonatal meningitis Escherichia coli isolates (SP-4, SP-5, SP-13, SP-16, SP-46, and SP-65) were recovered from infants in the Netherlands from 1989 to 1997. Here, we report the draft genome sequences for these six E. coli isolates, which are currently being used to validate food safety processing te...

Factors Involved in the Persistence of a Shiga Toxin-Producing Escherichia coli O157:H7 Strain in Bovine Feces and Gastro-Intestinal Content

PubMed Central

Segura, Audrey; Auffret, Pauline; Bibbal, Delphine; Bertoni, Marine; Durand, Alexandra; Jubelin, Grégory; Kérourédan, Monique; Brugère, Hubert; Bertin, Yolande; Forano, Evelyne

2018-01-01

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment. PMID:29593666

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

PubMed

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

2015-01-01

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

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

PubMed Central

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

2015-01-01

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis. PMID:25549220

Assessing Pathogen Levels in Dairy Lagoon Wastewater and Potential Evaporation Losses

NASA Astrophysics Data System (ADS)

Pandey, P.; Biswas, S.; Souza, A.; Silva-del-Rio, N.; Vaddella, V. K.; Castillo, A. R.

2014-12-01

The ongoing drought in California necessitates the conservation of existing water resources while protecting the water quality. There is a critical need to improve the understanding of evaporation losses from dairy lagoons in California, and their corresponding changes in pathogen levels in the lagoon wastewater. We have carried out preliminary studies involving extensive dairy lagoon water sampling, and batch-scale experiments. The dairy wastewater was collected from both primary and secondary lagoons in three counties of California (Merced, Tulare, and Glenn Counties) for enumerating the levels of E. coli O157:H7, Salmonella spp., and Listeria monocytogenes. Subsequently, we conducted batch-scale experiments at mesophilic (31 and 37 ⁰C) and thermophilic temperatures (43 and 49 ⁰C) to understand the E. coli O157:H7 inactivation process and potential evaporative water losses. In addition, we compared the evaporation losses under the environment of biological activity and the environment of restricted biological activity (extreme environment with low pH). For E. coli O157:H7 enumeration, we used MacConkey agar, while for Salmonella spp., XLD agar was used. Listeria monocytogenes levels were measured using PALCAM agar (with selective supplement). In flushed dairy wastewater (fresh) samples, the average of E. coli O157:H7 levels were 2 × 104 CFU/mL. The average Listeria monocytogenes levels in flushed manure were 9 × 101. The levels of Salmonella spp. were non-detectable. In mesophilic condition (37 ⁰C) after 5 days of incubation, 8% of total water loss was observed, while at thermophilic temperature (49 ⁰C), 70% of total water loss was observed. After 5 days of incubation at 37 ⁰C, E. coli O157:H7 levels in flushed dairy manure were increased from 2.8 × 103 to 5.2 × 104 CFU/mL, while at 49 ⁰C, E. coli O157:H7 levels were reduced from 2.8 × 103 to 5 × 101 CFU/mL after 4 days of incubation. We anticipate that the results of this study would be useful in understanding the dynamics of water losses and pathogen inactivation in dairy lagoon wastewater.

[Effects of exogenous carbon monoxide-releasing molecules 2 on the vitality and toxicity of E.coli].

PubMed

Qiu, Xue-feng; Liu, Da-dong; Sun, Bing-wei; Liang, Feng; Cao, Jie

2013-04-01

To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on the vitality and toxicity of E. coli ATCC 25922, and to analyze the potential mechanism. (1) In vitro experiments. Standard strains of E. coli ATCC 25922 were divided into groups A (without addition), B, C, D, and E according to the random number table, and then the latter 4 groups were respectively cultured with 1.2 mmol/L CORM-2, 1.6 mmol/L CORM-2, 1.2 mmol/L inactive CORM-2 (iCORM-2), 1.6 mmol/L iCORM-2, with six samples in each group. After being cultured for 0, 3, 5, 8, 10, 12, 16, 20, 24, 27, 30, 48 hours, proliferative vitality of E. coli was examined (denoted as absorption value under 600 nm wavelength), and bacteria number was counted. Other standard strains of E. coli ATCC 25922 were divided into groups F (without addition) and G (cultured with 0.8 mmol/L CORM-2), the expressions of genes fliA, dnaK, marA, and waaQ related to E. coli were detected by quantitative real-time (qRT)-PCR. (2) In vivo experiments. Other standard strains of E. coli ATCC 25922 were grouped as A', B', C', D', and E' and treated with the same method as that in groups A, B, C, D, and E, and 0.5 mL bacterial liquid of each group were collected when the absorption value of bacterial liquid in group A' was equal to 0.4 (under 600 nm wavelength). Seventy-two C57BL/6 mice were divided into groups, namely blank control (without treatment), H, I, J, K, and L according to the random number table, with 12 mice in each group. The mice in the latter 5 groups were intraperitoneally injected with 0.5 mL bacterial suspension of groups A', B', C', D', and E' respectively. After injection, general condition of mice in groups H, I, J, K, and L was observed. The serum levels of TNF-α and IL-6 were determined at post injection hour (PIH) 6, 12. The liver and lung samples were harvested for determination of myeloperoxidase (MPO) activity at PIH 12. The same process was carried out in blank control group. Data were processed with repeated measure analysis of variance (ANOVA), factorial design ANOVA, one-way ANOVA, and t test. (1) In vitro experiments. Compared with those of groups A and D, the proliferative vitality and bacteria number of E. coli in group B were all decreased (with F values respectively 1170.80, 217.52, P values all below 0.01). Compared with those of groups A and E, the proliferative vitality and bacteria number of E. coli in group C were also obviously decreased (with F values respectively 7948.34, 14 432.85, P values all below 0.01). Compared with those in group F, the expression of fliA was downregulated, while the expressions of dnaK, marA, and waaQ were upregulated in group G (with t values 30.28, -165.54, -168.88, -187.28, P values all below 0.01). (2) In vivo experiments. Symptoms including listlessness and tachypnea were observed in mice in groups H, K, and L, and they were ameliorated or not obvious in groups I and J. At PIH 6, the serum levels of TNF-α and IL-6 in groups H and K were respectively (647.3 ± 3.8) pg/mL, (3.44 ± 0.22) ng/mL and (639.3 ± 0.8) pg/mL, (2.47 ± 0.32) ng/mL, which were obviously higher than those in group I [(124.6 ± 10.7) pg/mL, (1.03 ± 0.16) ng/mL, with t values from 15.22 to 84.03, P values all below 0.01]. The serum levels of TNF-α and IL-6 in group J at PIH 6, 12 were also obviously decreased as compared with those in groups H and L (with t values from 19.27 to 245.34, P values all below 0.01). MPO activity of liver and lung tissues were significantly attenuated in group I at PIH 12 as compared with those in groups H and K, and it was also attenuated in group J when compared with those in groups H and L (with t values respectively from 17.36 to 18.92 and 2.35 to 3.61, P values all below 0.01). CORM-2 can obviously inhibit the vitality and toxicity of E. coli, which might be attributable to regulation of expressions of genes fliA, dnaK, marA, and waaQ of E. coli.

A Fast, Reliable, and Sensitive Method for Detection and Quantification of Listeria monocytogenes and Escherichia coli O157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR

PubMed Central

Russo, Pasquale; Botticella, Giuseppe; Capozzi, Vittorio; Massa, Salvatore; Spano, Giuseppe; Beneduce, Luciano

2014-01-01

In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype. PMID:24949460

Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase.

PubMed

Yoshida, Wataru; Kezuka, Aki; Murakami, Yoshiyuki; Lee, Jinhee; Abe, Koichi; Motoki, Hiroaki; Matsuo, Takafumi; Shimura, Nobuaki; Noda, Mamoru; Igimi, Shizunobu; Ikebukuro, Kazunori

2013-11-01

An automatic polymerase chain reaction (PCR) product detection system for food safety monitoring using zinc finger (ZF) protein fused to luciferase was developed. ZF protein fused to luciferase specifically binds to target double stranded DNA sequence and has luciferase enzymatic activity. Therefore, PCR products that comprise ZF protein recognition sequence can be detected by measuring the luciferase activity of the fusion protein. We previously reported that PCR products from Legionella pneumophila and Escherichia coli (E. coli) O157 genomic DNA were detected by Zif268, a natural ZF protein, fused to luciferase. In this study, Zif268-luciferase was applied to detect the presence of Salmonella and coliforms. Moreover, an artificial zinc finger protein (B2) fused to luciferase was constructed for a Norovirus detection system. In the luciferase activity detection assay, several bound/free separation process is required. Therefore, an analyzer that automatically performed the bound/free separation process was developed to detect PCR products using the ZF-luciferase fusion protein. By means of the automatic analyzer with ZF-luciferase fusion protein, target pathogenic genomes were specifically detected in the presence of other pathogenic genomes. Moreover, we succeeded in the detection of 10 copies of E. coli BL21 without extraction of genomic DNA by the automatic analyzer and E. coli was detected with a logarithmic dependency in the range of 1.0×10 to 1.0×10(6) copies. Copyright © 2013 Elsevier B.V. All rights reserved.

Equilibrium Isotherm, Kinetic Modeling, Optimization, and Characterization Studies of Cadmium Adsorption by Surface-Engineered Escherichia coli

PubMed Central

Tafakori, Vida; Zadmard, Reza; Tabandeh, Fatemeh; Amoozegar, Mohammad Ali; Ahmadian, Gholamreza

2017-01-01

Background: Amongst the methods that remove heavy metals from environment, biosorption approaches have received increased attention because of their environmentally friendly and cost-effective feature, as well as their superior performances. Methods: In the present study, we investigated the ability of a surface-engineered Escherichia coli, carrying the cyanobacterial metallothionein on the cell surface, in the removal of Ca (II) from solution under different experimental conditions. The biosorption process was optimized using central composite design. In parallel, the kinetics of metal biosorption was studied, and the rate constants of different kinetic models were calculated. Results: Cadmium biosorption is followed by the second-order kinetics. Freundlich and Langmuir equations were used to analyze sorption data; characteristic parameters were determined for each adsorption isotherm. The biosorption process was optimized using the central composite design. The optimal cadmium sorption capacity (284.69 nmol/mg biomass) was obtained at 40°C (pH 8) and a biomass dosage of 10 mg. The influence of two elutants, EDTA and CaCl2, was also assessed on metal recovery. Approximately, 68.58% and 56.54% of the adsorbed cadmium were removed by EDTA and CaCl2 during desorption, respectively. The Fourier transform infrared spectrophotometer (FTIR) analysis indicated that carboxyl, amino, phosphoryl, thiol, and hydroxyl are the main chemical groups involved in the cadmium bioadsorption process. Conclusion: Results from this study implied that chemical adsorption on the heterogeneous surface of E. coli E and optimization of adsorption parameters provides a highly efficient bioadsorbent. PMID:28555492

Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples

PubMed Central

Morcatti Coura, Fernanda; Diniz, Soraia de Araújo; Silva, Marcos Xavier; Mussi, Jamili Maria Suhet; Barbosa, Silvia Minharro; Lage, Andrey Pereira; Heinemann, Marcos Bryan

2015-01-01

This study analyzes the occurrence and distribution of phylogenetic groups of 391 strains of Escherichia coli isolated from poultry, cattle, and water buffalo. The frequency of the phylogroups was A = 19%, B1 = 57%, B2 = 2.3%, C = 4.6%, D = 2.8%, E = 11%, and F = 3.3%. Phylogroups A (P < 0.001) and F (P = 0.018) were associated with E. coli strains isolated from poultry, phylogroups B1 (P < 0.001) and E (P = 0.002) were associated with E. coli isolated from cattle, and phylogroups B2 (P = 0.003) and D (P = 0.017) were associated with E. coli isolated from water buffalo. This report demonstrated that some phylogroups are associated with the host analyzed and the results provide knowledge of the phylogenetic composition of E. coli from domestic animals. PMID:26421310

Impact of UV and peracetic acid disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli in wastewater effluents.

PubMed

Biswal, Basanta Kumar; Khairallah, Ramzi; Bibi, Kareem; Mazza, Alberto; Gehr, Ronald; Masson, Luke; Frigon, Dominic

2014-06-01

Wastewater discharges may increase the populations of pathogens, including Escherichia coli, and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli (UPEC), the most abundant E. coli pathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766 E. coli isolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm(2) and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters.

Impact of UV and Peracetic Acid Disinfection on the Prevalence of Virulence and Antimicrobial Resistance Genes in Uropathogenic Escherichia coli in Wastewater Effluents

PubMed Central

Biswal, Basanta Kumar; Khairallah, Ramzi; Bibi, Kareem; Mazza, Alberto; Gehr, Ronald; Masson, Luke

2014-01-01

Wastewater discharges may increase the populations of pathogens, including Escherichia coli, and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli (UPEC), the most abundant E. coli pathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766 E. coli isolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm2 and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters. PMID:24727265

Associations between intrauterine bacterial infection, reproductive tract inflammation, and reproductive performance in pasture-based dairy cows.

PubMed

de Boer, Melvin; Buddle, Bryce M; Heuer, Cord; Hussein, Hassan; Zheng, Tao; LeBlanc, Stephen J; McDougall, Scott

2015-06-01

Reproductive tract bacterial infections, particularly those caused by Escherichia coli and Trueperella pyogenes, can have a negative impact on reproductive performance. It has been hypothesized that the presence of E coli early postpartum may increase the risk of isolation of T pyogenes later postpartum. The objective of the present study was to examine associations between intrauterine bacterial infections with E coli and T pyogenes and any bacterial growth (irrespective of bacterial species), purulent vaginal discharge (PVD), cytologic evidence of endometritis (an increased proportion of polymorphonuclear cells [PMNs]), and reproductive performance. Dairy cows (n = 272) from six herds were examined at Days 0 (median, 2 days in milk), 21 and 42 postpartum. From each cow two intrauterine samples were collected via triple-guarded cytobrush at Days 0 and 21. The first cytobrush was used for bacteriologic culture. Escherichia coli and T pyogenes were isolated by culture, and E coli isolates were assigned to one of four phylogenetic groups using a two-step triplex polymerase chain reaction. In addition, T pyogenes was confirmed by polymerase chain reaction. The second cytobrush was used to prepare a cytology slide. Nucleated cells (n = 200) were categorized as epithelial cells, PMNs, or macrophages. Cows were also assessed for body condition score, PVD score, the presence of a CL, and pregnancy. Statistical analysis was performed using multivariable models. There was no association between the presence of E coli at Day 0 and probability of isolation of T pyogenes 3 weeks later; however, E coli positive cows at Day 0 were more likely to be diagnosed with E coli at Day 21 (relative risk [RR] = 2.0, P < 0.01). Escherichia coli at Day 0 or T pyogenes at Day 21 increased the risk of PVD diagnosis 3 weeks later (RR = 1.9; P = 0.04 and RR = 3.0; P = 0.05, respectively). Cows with any bacterial growth at Day 21, irrespective of species, were less likely to conceive within 3 weeks after the start of the seasonal breeding program (RR = 0.8; P = 0.05). Interestingly, cows with 25% PMNs or greater at Day 0 had shorter time to pregnancy (hazard ratio = 1.32; P = 0.05). Intrauterine bacterial infection may impair reproductive performance but the presence of E coli was not associated with isolation of T pyogenes 3 weeks later. Increased endometrial flux of PMNs in cows early postpartum may be a physiological process and improve reproductive performance. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipocalin 2 regulates intestine bacterial survival by interplaying with siderophore in a weaned piglet model of Escherichia coli infection.

PubMed

Guo, Bing-Xiu; Wang, Qian-Qian; Li, Jia-Hui; Gan, Zhen-Shun; Zhang, Xiao-Feng; Wang, Yi-Zhen; Du, Hua-Hua

2017-09-12

Iron is an essential nutrient that facilitates cell proliferation and growth, which plays a pivotal role in modulating the battle for survival between mammalian hosts and their pathogens. Pathogenic bacteria secrete siderophores to acquire iron from the host. However, lipocalin 2 (Lcn2), a siderophore-binding antimicrobial protein, binds to siderophores to prevent bacterial uptake of iron, which is critical for the control of systemic infection with Escherichia coli ( E. coli ). But few studies focus on the anti-infective response of Lcn2 in the intestines by inhibiting bacterial proliferation based on microbial iron metabolism. In this study, we showed that iron was sequestrated within cells in a piglet model of E. coli K88 infection. Siderophores was produced following E. coli K88 infection and siderophore-related genes expression was upregulated in iron-deficiency environment in vitro . Meanwhile, we found that Lcn2 expression was rapidly and robustly induced in jejunum by E. coli K88 infection and could be stimulated by IL-17 and IL-22. Furthermore, both Lcn2 induced in epithelial cells IPEC-1 and added exogenously as a recombinant protein could inhibit the growth of E. coli . We can conclude that Lcn2 is a crucial component of mucosal immune defense against intestinal infection with E. coli K88.

Parallel steady state studies on a milliliter scale accelerate fed-batch bioprocess design for recombinant protein production with Escherichia coli.

PubMed

Schmideder, Andreas; Cremer, Johannes H; Weuster-Botz, Dirk

2016-11-01

In general, fed-batch processes are applied for recombinant protein production with Escherichia coli (E. coli). However, state of the art methods for identifying suitable reaction conditions suffer from severe drawbacks, i.e. direct transfer of process information from parallel batch studies is often defective and sequential fed-batch studies are time-consuming and cost-intensive. In this study, continuously operated stirred-tank reactors on a milliliter scale were applied to identify suitable reaction conditions for fed-batch processes. Isopropyl β-d-1-thiogalactopyranoside (IPTG) induction strategies were varied in parallel-operated stirred-tank bioreactors to study the effects on the continuous production of the recombinant protein photoactivatable mCherry (PAmCherry) with E. coli. Best-performing induction strategies were transferred from the continuous processes on a milliliter scale to liter scale fed-batch processes. Inducing recombinant protein expression by dynamically increasing the IPTG concentration to 100 µM led to an increase in the product concentration of 21% (8.4 g L -1 ) compared to an implemented high-performance production process with the most frequently applied induction strategy by a single addition of 1000 µM IPGT. Thus, identifying feasible reaction conditions for fed-batch processes in parallel continuous studies on a milliliter scale was shown to be a powerful, novel method to accelerate bioprocess design in a cost-reducing manner. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1426-1435, 2016. © 2016 American Institute of Chemical Engineers.

Inactivation of Uropathogenic Escherichia coli in Ground Chicken Meat Using High Pressure Processing and Gamma Radiation, and in Purge and Chicken Meat Surfaces by Ultraviolet Light

PubMed Central

Sommers, Christopher H.; Scullen, O. J.; Sheen, Shiowshuh

2016-01-01

Extraintestinal pathogenic Escherichia coli, including uropathogenic E. coli (UPEC), are common contaminants in poultry meat and may cause urinary tract infections after colonization of the gastrointestinal tract and transfer of contaminated feces to the urethra. Three non-thermal processing technologies used to improve the safety and shelf-life of both human and pet foods include high pressure processing (HPP), ionizing (gamma) radiation (GR), and ultraviolet light (UV-C). Multi-isolate cocktails of UPEC were inoculated into ground chicken which was then treated with HPP (4°C, 0–25 min) at 300, 400, or 500 MPa. HPP D10, the processing conditions needed to inactivate 1 log of UPEC, was 30.6, 8.37, and 4.43 min at 300, 400, and 500 MPa, respectively. When the UPEC was inoculated into ground chicken and gamma irradiated (4 and -20°C) the GR D10 were 0.28 and 0.36 kGy, respectively. The UV-C D10 of UPEC in chicken suspended in exudate and placed on stainless steel and plastic food contact surfaces ranged from 11.4 to 12.9 mJ/cm2. UV-C inactivated ca. 0.6 log of UPEC on chicken breast meat. These results indicate that existing non-thermal processing technologies such as HPP, GR, and UV-C can significantly reduce UPEC levels in poultry meat or exudate and provide safer poultry products for at-risk consumers. PMID:27148167

Dual Functional Core-Shell Fluorescent Ag2S@Carbon Nanostructure for Selective Assay of E. coli O157:H7 and Bactericidal Treatment.

PubMed

Wang, Ning; Wei, Xing; Zheng, An-Qi; Yang, Ting; Chen, Ming-Li; Wang, Jian-Hua

2017-03-24

A dual functional fluorescent core-shell Ag 2 S@Carbon nanostructure is prepared by a hydrothermally assisted multi-amino synthesis approach with folic acid (FA), polyethylenimine (PEI), and mannoses (Mans) as carbon and nitrogen sources (FA-PEI-Mans-Ag 2 S nanocomposite shortly as Ag 2 S@C). The nanostructure exhibits strong fluorescent emission at λ ex /λ em = 340/450 nm with a quantum yield of 12.57 ± 0.52%. Ag 2 S@C is bound to E. coli O157:H7 via strong interaction with the Mans moiety in Ag 2 S@C with FimH proteins on the fimbriae tip in E. coli O157:H7. Fluorescence emission from Ag 2 S@C/E. coli conjugate is closely related to the content of E. coli O157:H7. Thus, a novel procedure for fluorescence assay of E. coli O157:H7 is developed, offering a detection limit of 330 cfu mL -1 . Meanwhile, the Ag 2 S@C nanostructure exhibits excellent antibacterial performance against E. coli O157:H7. A 99.9% sterilization rate can be readily achieved for E. coli O157:H7 at a concentration of 10 6 -10 7 cfu mL -1 with 3.3 or 10 μg mL -1 of Ag 2 S@C with an interaction time of 5 or 0.5 min, respectively.

Combining sun-based technologies (microalgae and solar disinfection) for urban wastewater regeneration.

PubMed

Gutiérrez-Alfaro, Sergio; Rueda-Márquez, Juan J; Perales, José A; Manzano, Manuel A

2018-04-01

Solar disinfection (SODIS) of urban wastewater can be a suitable technology for improving the microbiological quality of reclaimed water as a complement to other extensive and environmentally friendly technologies such as microalgae biotreatment. The objective of this work is to evaluate the feasibility of incorporating the SODIS technology at the end of a pilot scale urban wastewater treatment plant (WWTP) where the processes are based on microalgae biotechnology and comprising three Upflow Anaerobic Sludge Blanket (UASB, 20m 3 each one) reactor, six High Rate Algal Ponds (HRAP, 32m 2 each one), and a Dissolved Air Flotation (DAF, 1m 3 ) unit. E. coli concentration was monitored at the effluent of the different units (UASB, HRAP, DAF) of the pilot WWTP. The efficiency of the SODIS process was studied for the inactivation of three of the commonly employed indicator microorganisms (Escherichia coli, Enterococcus spp. and Clostridium perfringens) using a compound parabolic collector (CPC) for five months under various conditions of irradiance and temperature. E. coli and Enterococcus spp. were more effectively disinfected by the SODIS unit (2.9 and 2.5 logarithms of reduction on average, respectively) than by the HRAP (2 and 1.1) or the DAF (0.9 and 0.1). On the contrary, the DAF technology achieved better reduction rates of C. perfringens (1.7) than the SODIS (0.9) and the HRAP (0.1). No regrowth of any microorganisms was detected during dark storage after the SODIS treatment. Incorporating a SODIS unit after the non-conventional WWTP processes substantially increases the possibilities for reuse of the treated water after receiving a cumulative UV radiation dose of 25W·h/m 2 (50min of normalized time of solar illumination). The surface requirement of the SODIS equipment would be 3.5 times smaller than the HRAP's surface. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation of bacteriophages against non-O157 and O157 Shiga toxin-producing Escherichia coli (STEC) from composting of non-fecal materials and the potential impact on produce safety

USDA-ARS?s Scientific Manuscript database

Composting is a complex process to produce fertilizers used to improve crop yields. A complete composting process usually confers bactericidal effect due to change of temperature and pH However, some produce outbreaks associated with Shiga toxin-producing E. coli (STEC) contamination were linked to ...

Evaluation of the Live Biotherapeutic Product, Asymptomatic Bacteriuria Escherichia coli 2-12, in Healthy Dogs and Dogs with Clinical Recurrent UTI.

PubMed

Segev, G; Sykes, J E; Klumpp, D J; Schaeffer, A J; Antaki, E M; Byrne, B A; Yaggie, R E; Westropp, J L

2018-01-01

Antimicrobial resistance is an emerging problem. To investigate the safety and efficacy of a live biotherapeutic product, ASB E. coli 2-12 for UTI treatment. Six healthy research dogs; nine client-owned dogs with recurrent UTI. Prospective noncontrolled clinical trial. For safety data, research dogs were sedated, a urinary catheter was inserted into the bladder; 10 10 CFU/mL of ASB E. coli 2-12 was instilled. Urine was cultured on days 1, 3, and 8 post-instillation and dogs were observed for lower urinary tract signs (LUTS). For client-owned dogs, ASB E. coli 2-12 was instilled similarly and urine cultures analyzed on days 1, 7, and 14 days postinstillation. No LUTS were noted in any of the 6 research dogs after ASB E. coli 2-12 infusion. Pulse field gel electrophoresis (PFGE) studies confirmed the bacterial strains isolated matched that ASB E. coli 2-12 strain. Four of the nine client-owned dogs had complete or nearly complete clinical cures by day 14. Of these four dogs, 3 also had microbiologic cures at day 14; one of these dogs had subclinical bacteriuria (in addition to ASB E. coli 2-12). Three of these four dogs had ASB E. coli 2-12 isolated from their urine at day 14. With the exception of mild, temporary, self-limiting, hyporexia in two dogs on the day of biotherapeutic administration, there were no major adverse effects. These results suggest ASB E. coli 2-12 is safe and should be investigated in a larger controlled study evaluating clinical UTI in dogs. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium.

PubMed

Genís, Sandra; Sánchez-Chardi, Alejandro; Bach, Àlex; Fàbregas, Francesc; Arís, Anna

2017-01-01

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1β, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Enteroaggregative, Shiga Toxin-Producing Escherichia coli O111:H2 Associated with an Outbreak of Hemolytic-Uremic Syndrome

PubMed Central

Morabito, Stefano; Karch, Helge; Mariani-Kurkdjian, Patrizia; Schmidt, Herbert; Minelli, Fabio; Bingen, Edouard; Caprioli, Alfredo

1998-01-01

Shiga toxin-producing Escherichia coli O111:H2 strains from an outbreak of hemolytic-uremic syndrome showed aggregative adhesion to HEp-2 cells and harbored large plasmids which hybridized with the enteroaggregative E. coli probe PCVD432. These strains present a novel combination of virulence factors and might be as pathogenic to humans as the classic enterohemorrhagic E. coli. PMID:9508328

Bacteria attenuation by iron electrocoagulation governed by interactions between bacterial phosphate groups and Fe(III) precipitates.

PubMed

Delaire, Caroline; van Genuchten, Case M; Amrose, Susan E; Gadgil, Ashok J

2016-10-15

Iron electrocoagulation (Fe-EC) is a low-cost process in which Fe(II) generated from an Fe(0) anode reacts with dissolved O2 to form (1) Fe(III) precipitates with an affinity for bacterial cell walls and (2) bactericidal reactive oxidants. Previous work suggests that Fe-EC is a promising treatment option for groundwater containing arsenic and bacterial contamination. However, the mechanisms of bacteria attenuation and the impact of major groundwater ions are not well understood. In this work, using the model indicator Escherichia coli (E. coli), we show that physical removal via enmeshment in EC precipitate flocs is the primary process of bacteria attenuation in the presence of HCO3(-), which significantly inhibits inactivation, possibly due to a reduction in the lifetime of reactive oxidants. We demonstrate that the adhesion of EC precipitates to cell walls, which results in bacteria encapsulation in flocs, is driven primarily by interactions between EC precipitates and phosphate functional groups on bacteria surfaces. In single solute electrolytes, both P (0.4 mM) and Ca/Mg (1-13 mM) inhibited the adhesion of EC precipitates to bacterial cell walls, whereas Si (0.4 mM) and ionic strength (2-200 mM) did not impact E. coli attenuation. Interestingly, P (0.4 mM) did not affect E. coli attenuation in electrolytes containing Ca/Mg, consistent with bivalent cation bridging between bacterial phosphate groups and inorganic P sorbed to EC precipitates. Finally, we found that EC precipitate adhesion is largely independent of cell wall composition, consistent with comparable densities of phosphate functional groups on Gram-positive and Gram-negative cells. Our results are critical to predict the performance of Fe-EC to eliminate bacterial contaminants from waters with diverse chemical compositions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Significance of Viable but Nonculturable Escherichia coli: Induction, Detection, and Control.

PubMed

Ding, Tian; Suo, Yuanjie; Xiang, Qisen; Zhao, Xihong; Chen, Shiguo; Ye, Xingqian; Liu, Donghong

2017-03-28

Diseases caused by foodborne or waterborne pathogens are emerging. Many pathogens can enter into the viable but nonculturable (VBNC) state, which is a survival strategy when exposed to harsh environmental stresses. Pathogens in the VBNC state have the ability to evade conventional microbiological detection methods, posing a significant and potential health risk. Therefore, controlling VBNC bacteria in food processing and the environment is of great importance. As the typical one of the gram-negatives, Escherichia coli ( E. coli ) is a widespread foodborne and waterborne pathogenic bacterium and is able to enter into a VBNC state in extreme conditions (similar to the other gram-negative bacteria), including inducing factors and resuscitation stimulus. VBNC E. coli has the ability to recover both culturability and pathogenicity, which may bring potential health risk. This review describes the concrete factors (nonthermal treatment, chemical agents, and environmental factors) that induce E. coli into the VBNC state, the condition or stimulus required for resuscitation of VBNC E. coli , and the methods for detecting VBNC E. coli . Furthermore, the mechanism of genes and proteins involved in the VBNC E. coli is also discussed in this review.

Production of caffeoylmalic acid from glucose in engineered Escherichia coli.

PubMed

Li, Tianzhen; Zhou, Wei; Bi, Huiping; Zhuang, Yibin; Zhang, Tongcun; Liu, Tao

2018-07-01

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli. We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli-E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L. Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

Escherichia coli K1-induced cytopathogenicity of human brain microvascular endothelial cells.

PubMed

Khan, Naveed Ahmed; Iqbal, Junaid; Siddiqui, Ruqaiyyah

2012-01-01

Pathophysiology of Escherichia coli sepsis is complex involving circulating bacterial products, cytokine release, and sustained bacteremia resulting in the damage of vascular endothelium. Here, it is shown that E. coli K1 produced cytopathogenicity of human brain microvascular endothelial cells (HBMEC), that constitute the blood-brain barrier. Whole bacteria or their conditioned medium produced severe HBMEC damage suggesting E. coli K1-cytopathogenicity is a contact-independent process. Using lipopolysaccharide (LPS) inhibitor, polymyxin B, purified LPS extracted from E. coli K1 as well as LPS mutant derived from E. coli K1, we showed that LPS is not the sole determinant of E. coli K1-mediated HBMEC death. Bacterial product(s) for HBMEC cytopathogenicity was heat-labile suggesting LPS-associated proteins. Several isogenic gene-deletion mutants (ΔompA, ΔibeA, ΔibeB, Δcnf1) exhibited HBMEC cytopathogenicity similar to that produced by wild type E. coli K1. E. coli K1-mediated HBMEC death was independent of phosphatidylinositol 3-kinase (PI3K) but dependent partially on focal adhesion kinase (FAK) using HBMEC expressing dominant negative FAK and PI3K. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preliminary plant design of Escherichia coli BPPTCC-EgRK2 cell culture for recombinant cellulase production using Oil Palm Empty Fruit Bunch (OPEFB) as substrate

NASA Astrophysics Data System (ADS)

Surya, E. A.; Rahman, S. F.; Zulamraini, S.; Gozan, M.

2018-03-01

An economic analysis of recombinant cellulase production from E. coli BPPTCC Eg-RK2 was conducted to support the fulfilling of Indonesia’s energy roadmap for ethanol production. The plant use oil palm empty fruit bunch (OPEFB) as primary substrate in cellulase production, with the expected lifetime of 12 years. The plant is assumed to be built in Indonesia and will fulfill 1% of total market demand. The effect of different pretreatment process (alkaline, steam explosion, and sequential acid-alkaline) on the economic value was also studied. A simulation using SuperPro Designer was used to calculate the mass and energy balance based on the kinetic parameter of E. coli BPPTCC-EgRK2. Technology evaluation show that alkaline pretreatment gave the highest yield with no known inhibitors formed. The steam explosion show the lowest lignin and hemicellulose removal and known to form known fermentation inhibitors. The net present value of alkaline, steam explosion, and sequential acid-alkaline pretreatment were USD 7,118,000; - USD 73,411,000 and USD -114,013,000 respectively, which mean alkaline pretreatment is the only economically feasible pretreatment method for recombinant cellulase production.

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

PubMed

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

2016-08-01

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

Quantitative assessment of human exposure to extended spectrum and AmpC β-lactamases bearing E. coli in lettuce attributable to irrigation water and subsequent horizontal gene transfer.

PubMed

Njage, P M K; Buys, E M

2017-01-02

The contribution of the fresh produce production environment to human exposure with bacteria bearing extended spectrum β-lactamases and AmpC β-lactamases (ESBL/AmpC) has not been reported. High prevalence of ESBLs/AmpC bearing E. coli as well as a high gene transfer efficiency of lettuce and irrigation water E. coli isolates was previously reported. This stochastic modeling was aimed at quantitatively assessing human exposure to ESBL/AmpC bearing E. coli through lettuce attributable to irrigation water and subsequent horizontal gene transfer. Modular process risk approach was used for the quantitative exposure assessment and models were constructed in Ms. Excel spreadsheet with farm to consumption chain accounted for by primary production, processing, retail and consumer storage. Probability distributions were utilised to take into account the variability of the exposure estimates. Exposure resulting from ESBL/AmpC positive E. coli and gene transfer was taken into account. Monte Carlo simulation was carried out using @Risk software followed by sensitivity and scenario analysis to assess most effective single or combinations of mitigation strategies for the ESBL/AmpC positive E. coli events from farm to fork. Three percent of South African lettuce consumers are exposed to lettuce contaminated with about 10 6.4 ±10 6.7 (95% CI: 10 5.1 -10 7 ) cfu of ESBL/AmpC positive E. coli per serving. The contribution of originally positive isolates and conjugative genetic transfer was 10 6 ±10 6.7 (95% CI: 10 5 -10 7 ) and 10 5.2 ±10 5.6 (95% CI: 10 3.9 -10 5.8 ) cfu per serving respectively. Proportion of ESBL/AmpC positive E. coli (Spearman's correlation coefficient (ρ)=0.85), conjugative gene transfer (ρ=0.05-0.14), washing in chlorine water (ρ=0.18), further rinsing (ρ=0.15), and prevalence of E. coli in irrigation water (ρ=0.16) had highest influence on consumer exposure. The most effective single methods in reducing consumer exposure were reduction in irrigation water microbial quality variation (87.4% reduction), storage period (49.9-87.4% reduction) and growth rate reduction by 75% (90% reduction). Reduction in growth rate together with storage time (92.1-99.4%) and reduction in storage time combined with E. coli concentration in irrigation water (95-96% reduction) were most effective combinations of mitigation measures. The high variation in exposure reflected the high irrigation water quality variation. The exposure levels may impose higher consumer risk than acceptable for irrigation water risk. E. coli contamination and growth related measures, as well as measures to reduce contamination with antimicrobial resistant E. coli from lettuce production environment are recommended. This exposure model could form a basis for the development of similar models assessing the impact of contaminated irrigation water and gene transfer in other microbial hazards, antimicrobial resistance types and fresh produce types. Copyright © 2016 Elsevier B.V. All rights reserved.

The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.

PubMed Central

Jones, S; Yu, B; Bainton, N J; Birdsall, M; Bycroft, B W; Chhabra, S R; Cox, A J; Golby, P; Reeves, P J; Stephens, S

1993-01-01

Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively. E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups. Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent. Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta. Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant. Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups. Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P. aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production. HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants. This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes. Images PMID:8508773

Production of quorum-sensing signals by bacteria in the coral mucus layer

NASA Astrophysics Data System (ADS)

Li, Jie; Kuang, Weiqi; Long, Lijuan; Zhang, Si

2017-12-01

Quorum sensing is an integral part of bacterial communication and interaction, but has not been well characterized in coral mucus microbiota. In this study, of 61 coral mucus isolates, five alphaproteobacteria and one Vibrio species were found to produce N-acyl homoserine lactone (AHL), a quorum-sensing signal in bacteria. Eight gammaproteobacteria isolates were found to produce autoinducer-2 (AI-2) quorum-sensing signals along with two actinobacteria of the genus Rothia. Coral mucus is rich in the antioxidant dimethylsulfoniopropionate (DMSP), the concentration of which has been found to increase under heat stress. Neither AHL nor AI-2 activity was induced by DMSP in those coral mucus isolates that did not initially produce quorum-sensing signals. However, the AI-2 activities of one Rothia isolate (SCSIO 13017) from coral mucus and of Vibrio shilonii (DSM 13774 isolated from a bleached coral) were found to increase in response to 5 μM DMSP but decreased in response to 50 μM DMSP for the first time. These findings suggest that the production of quorum-sensing signals in the coral mucus microbiota may play a role in structuring the surface microbial community as they respond to environmental stress.

Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation

PubMed Central

Raisch, Jennifer; Buc, Emmanuel; Bonnet, Mathilde; Sauvanet, Pierre; Vazeille, Emilie; de Vallée, Amélie; Déchelotte, Pierre; Darcha, Claude; Pezet, Denis; Bonnet, Richard; Bringer, Marie-Agnès; Darfeuille-Michaud, Arlette

2014-01-01

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients. METHODS: Phylogroups and the presence of cyclomodulin-encoding genes of mucosa-associated E. coli from colon cancer and diverticulosis specimens were determined by PCR. Adhesion and invasion experiments were performed with I-407 intestinal epithelial cells using gentamicin protection assay. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) expression in T84 intestinal epithelial cells was measured by enzyme-linked immunosorbent assay and by Western Blot. Gut colonization, inflammation and pro-carcinogenic potential were assessed in a chronic infection model using CEABAC10 transgenic mice. Cell proliferation was analyzed by real-time mRNA quantification of PCNA and immunohistochemistry staining of Ki67. RESULTS: Analysis of mucosa-associated E. coli from colon cancer and diverticulosis specimens showed that whatever the origin of the E. coli strains, 86% of cyclomodulin-positive E. coli belonged to B2 phylogroup and most harbored polyketide synthase (pks) island, which encodes colibactin, and/or cytotoxic necrotizing factor (cnf) genes. In vitro assays using I-407 intestinal epithelial cells revealed that mucosa-associated B2 E. coli strains were poorly adherent and invasive. However, mucosa-associated B2 E. coli similarly to Crohn’s disease-associated E. coli are able to induce CEACAM6 expression in T84 intestinal epithelial cells. In addition, in vivo experiments using a chronic infection model of CEACAM6 expressing mice showed that B2 E. coli strain 11G5 isolated from colon cancer is able to highly persist in the gut, and to induce colon inflammation, epithelial damages and cell proliferation. CONCLUSION: In conclusion, these data bring new insights into the ability of E. coli isolated from patients with colon cancer to establish persistent colonization, exacerbate inflammation and trigger carcinogenesis. PMID:24914378

Contamination of Canadian private drinking water sources with antimicrobial resistant Escherichia coli.

PubMed

Coleman, Brenda L; Louie, Marie; Salvadori, Marina I; McEwen, Scott A; Neumann, Norman; Sibley, Kristen; Irwin, Rebecca J; Jamieson, Frances B; Daignault, Danielle; Majury, Anna; Braithwaite, Shannon; Crago, Bryanne; McGeer, Allison J

2013-06-01

Surface and ground water across the world, including North America, is contaminated with bacteria resistant to antibiotics. The consumption of water contaminated with antimicrobial resistant Escherichia coli (E. coli) has been associated with the carriage of resistant E. coli in people who drink it. To describe the proportion of drinking water samples submitted from private sources for bacteriological testing that were contaminated with E. coli resistant to antibiotics and to determine risk factors for the contamination of these water sources with resistant and multi-class resistant E. coli. Water samples submitted for bacteriological testing in Ontario and Alberta Canada were tested for E. coli contamination, with a portion of the positive isolates tested for antimicrobial resistance. Households were invited to complete questionnaires to determine putative risk factors for well contamination. Using multinomial logistic regression, the risk of contamination with E. coli resistant to one or two classes of antibiotics compared to susceptible E. coli was higher for shore wells than drilled wells (odds ratio [OR] 2.8) and higher for farms housing chickens or turkeys (OR 3.0) than properties without poultry. The risk of contamination with multi-class resistant E. coli (3 or more classes) was higher if the properties housed swine (OR 5.5) or cattle (OR 2.2) than properties without these livestock and higher if the wells were located in gravel (OR 2.4) or clay (OR 2.1) than in loam. Housing livestock on the property, using a shore well, and having a well located in gravel or clay soil increases the risk of having antimicrobial resistant E. coli in E. coli contaminated wells. To reduce the incidence of water borne disease and the transmission of antimicrobial resistant bacteria, owners of private wells need to take measures to prevent contamination of their drinking water, routinely test their wells for contamination, and use treatments that eliminate bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transcriptome-enabled discovery and functional characterization of enzymes related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum and their application for metabolic engineering.

PubMed

Guo, Lei; Chen, Xi; Li, Li-Na; Tang, Wei; Pan, Yi-Ting; Kong, Jian-Qiang

2016-02-04

(2S)-Pinocembrin is a chiral flavanone with versatile pharmacological and biological activities. Its health-promoting effects have spurred on research effects on the microbial production of (2S)-pinocembrin. However, an often-overlooked salient feature in the analysis of microbial (2S)-pinocembrin is its chirality. Here, we presented a full characterization of absolute configuration of microbial (2S)-pinocembrin from engineered Escherichia coli. Specifically, a transcriptome-wide search for genes related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum, a plant rich in flavonoids, was first performed in the present study. A total of 104,180 unigenes were finally generated with an average length of 520 bp. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping assigned 26 unigenes, representing three enzyme families of 4-coumarate:coenzyme A ligase (4CL), chalcone synthase (CHS) and chalcone isomerase(CHI), onto (2S)-pinocembrin biosynthetic pathway. A total of seven, three and one full-length candidates encoding 4CL, CHS and CHI were then verified by reverse transcription polymerase chain reaction, respectively. These candidates were screened by functional expression in E. coli individual or coupled multienzyme reaction systems based on metabolic engineering processes. Oc4CL1, OcCHS2 and OcCHI were identified to be bona fide genes encoding respective pathway enzymes of (2S)-pinocembrin biosynthesis. Then Oc4CL1, OcCHS2 and MsCHI from Medicago sativa, assembled as artificial gene clusters in different organizations, were used for fermentation production of (2S)-pinocembrin in E. coli. The absolute configuration of the resulting microbial pinocembrin at C-2 was assigned to be 2S-configured by combination of retention time, UV spectrum, LC-MS, NMR, optical rotation and circular dichroism spectroscopy. Improvement of (2S)-pinocembrin titres was then achieved by optimization of gene organizations, using of codon-optimized pathway enzymes and addition of cerulenin for increasing intracellular malonyl CoA pools. Overall, the optimized strain can produce (2S)-pinocembrin of 36.92 ± 4.1 mg/L. High titre of (2S)-pinocembrin can be obtained from engineered E. coli by an efficient method. The fermentative production of microbial (2S)-pinocembrin in E. coli paved the way for yield improvement and further pharmacological testing.

Microbiological quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments.

PubMed

Abadias, M; Usall, J; Anguera, M; Solsona, C; Viñas, I

2008-03-31

A survey of fresh and minimally-processed fruit and vegetables, and sprouts was conducted in several retail establishments in the Lleida area (Catalonia, Spain) during 2005-2006 to determine whether microbial contamination, and in particular potentially pathogenic bacteria, was present under these commodities. A total of 300 samples--including 21 ready-to-eat fruits, 28 whole fresh vegetables, 15 sprout samples and 237 ready-to-eat salads containing from one to six vegetables--were purchased from 4 supermarkets. They were tested for mesophilic and psychrotrophic aerobic counts, yeasts and moulds, lactic acid bacteria, Enterobacteriaceae, presumptive E. coli and Listeria monocytogenes counts as well as for the presence of Salmonella, E. coli O157:H7, Yersinia enterocolitica and thermotolerant Campylobacter. Results for the fresh-cut vegetables that we analyzed showed that, in general, the highest microorganism counts were associated with grated carrot, arugula and spinach (7.8, 7.5 and 7.4 log cfu g(-1) of aerobic mesophilic microorganisms; 6.1, 5.8 and 5.2 log cfu g(-1) of yeast and moulds; 5.9, 4.0 and 5.1 log cfu g(-1) lactic acid bacteria and 6.2, 5.3 and 6.0 log cfu g(-1) of Enterobacteriaceae). The lowest counts were generally associated with fresh-cut endive and lettuce (6.2 and 6.3 log cfu g(-1) of aerobic mesophilic microorganisms; 4.4 and 4.6 log cfu g(-1) of yeast and moulds; 2.7 and 3.8 log cfu g(-1) lactic acid bacteria and 4.8 and 4.4 log cfu g(-1) of Enterobacteriaceae). Counts of psychrotrophic microorganisms were as high as those of mesophilic microorganisms. Microbiological counts for fresh-cut fruit were very low. Sprouts were highly contaminated with mesophilic (7.9 log cfu g(-1)), psychrotrophic microorganisms (7.3 log cfu g(-1)) and Enterobacteriaceae (7.2 log cfu g(-1)) and showed a high incidence of E. coli (40% of samples). Of the samples analyzed, four (1.3%) were Salmonella positive and two (0.7%) harboured L. monocytogenes. None of the samples was positive for E. coli O157:H7, pathogenic Y. enterocolitica or thermotolerant Campylobacter.

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells.

PubMed

Yao, Yufeng; Xie, Yi; Perace, Donna; Zhong, Yi; Lu, Jie; Tao, Jing; Guo, Xiaokui; Kim, Kwang Sik

2009-11-01

Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.

Guava Leaf Extract Inhibits Quorum-Sensing and Chromobacterium violaceum Induced Lysis of Human Hepatoma Cells: Whole Transcriptome Analysis Reveals Differential Gene Expression

PubMed Central

Tiwary, Bipransh Kumar; Kumar, Anoop

2014-01-01

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value≤0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum. PMID:25229331

Guava leaf extract inhibits quorum-sensing and Chromobacterium violaceum induced lysis of human hepatoma cells: whole transcriptome analysis reveals differential gene expression.

PubMed

Ghosh, Runu; Tiwary, Bipransh Kumar; Kumar, Anoop; Chakraborty, Ranadhir

2014-01-01

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value ≤ 0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum.

Enhanced Inactivation of Escherichia coli and MS2 Coliphage by Cupric Ion in the Presence of Hydroxylamine: Dual Microbicidal Effects.

PubMed

Kim, Hyung-Eun; Nguyen, Thuy T M; Lee, Hongshin; Lee, Changha

2015-12-15

The inactivation of Escherichia coli and MS2 coliphage by Cu(II) is found to be significantly enhanced in the presence of hydroxylamine (HA). The addition of a small amount of HA (i.e., 5-20 μM) increased the inactivation efficacies of E. coli and MS2 coliphage by 5- to 100-fold, depending on the conditions. Dual effects were anticipated to enhance the biocidal activity of Cu(II) by the addition of HA, viz. (i) the accelerated reduction of Cu(II) into Cu(I) (a stronger biocide) and (ii) the production of reactive oxidants from the reaction of Cu(I) with dissolved oxygen (evidenced by the oxidative transformation of methanol into formaldehyde). Deaeration enhanced the inactivation of E. coli but slightly decreased the inactivation efficacy of MS2 coliphage. The addition of 10 μM hydrogen peroxide (H2O2) greatly enhanced the MS2 inactivation, whereas the same concentration of H2O2 did not significantly affect the inactivation efficacy of E. coli Observations collectively indicate that different biocidal actions lead to the inactivation of E. coli and MS2 coliphage. The toxicity of Cu(I) is dominantly responsible for the E. coli inactivation. However, for the MS2 coliphage inactivation, the oxidative damage induced by reactive oxidants is as important as the effect of Cu(I).

Dynamics of CMY-2 producing E. coli in a broiler parent flock.

PubMed

Dame-Korevaar, Anita; Fischer, Egil A J; Stegeman, Arjan; Mevius, Dik; van Essen-Zandbergen, Alieda; Velkers, Francisca; van der Goot, Jeanet

2017-05-01

Extended-spectrum β-lactamase and plasmid mediated AmpC β-lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC-Escherichia coli in a broiler parent flock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen (n=228), and in the laying period two groups of 33 chickens were individually sampled (n=66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC-E. coli presence was determined by selective culturing. In the samples of week 16-19 the concentration of ESBL/pAmpC-E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene bla CMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2-E. coli prevalence decreased from 91% (95%CI 86-94%) at day 7 (week 1) to 0% (95%CI 0-5%) in week 21. However, CMY-2-E. coli remained present in the environmental samples during the whole study. CMY-2-E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2-E. coli in this broiler parent flock in absence of antibiotics suggests a selective disadvantage of bla CMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain. Copyright © 2017 Elsevier B.V. All rights reserved.

Escherichia coli pathotypes in Pakistan from consecutive floods in 2010 and 2011.

PubMed

Bokhari, Habib; Shah, Muhammad Ali; Asad, Saba; Akhtar, Sania; Akram, Muhammad; Wren, Brendan W

2013-03-01

This study compares Escherichia coli pathotypes circulating among children in Pakistan during the floods of 2010 and 2011 and from sporadic cases outside flood affected areas. Using multiplex polymerase chain reaction 115 of 205 stool samples (56.29%) were positive for diarrheagenic E. coli from specimens taken during the floods compared with 50 of 400 (12.5%) stool samples being positive for sporadic cases. The E. coli pathotypes were categorized as Enteropathogenic E. coli 33 (28.69%) and 13 (26%), Enterotoxigenic E. coli 29 (25.21%) and 15 (30%), Enteroaggregative E. coli 21 (18.2%) and 18 (36%), Enterohemorrhagic E. coli 5 (4.34%) and 1 (2%) from flood and sporadic cases, respectively. Furthermore, patients co-infected with more than one pathotype were 26 (22.60%) and 3 (6%) from flood and sporadic cases, respectively. The study shows an unexpectedly high rate of isolation of E. coli pathotypes suggesting Pakistan as an endemic region that requires active surveillance particularly during flood periods.

Escherichia coli Pathotypes in Pakistan from Consecutive Floods in 2010 and 2011

PubMed Central

Bokhari, Habib; Shah, Muhammad Ali; Asad, Saba; Akhtar, Sania; Akram, Muhammad; Wren, Brendan W.

2013-01-01

This study compares Escherichia coli pathotypes circulating among children in Pakistan during the floods of 2010 and 2011 and from sporadic cases outside flood affected areas. Using multiplex polymerase chain reaction 115 of 205 stool samples (56.29%) were positive for diarrheagenic E. coli from specimens taken during the floods compared with 50 of 400 (12.5%) stool samples being positive for sporadic cases. The E. coli pathotypes were categorized as Enteropathogenic E. coli 33 (28.69%) and 13 (26%), Enterotoxigenic E. coli 29 (25.21%) and 15 (30%), Enteroaggregative E. coli 21 (18.2%) and 18 (36%), Enterohemorrhagic E. coli 5 (4.34%) and 1 (2%) from flood and sporadic cases, respectively. Furthermore, patients co-infected with more than one pathotype were 26 (22.60%) and 3 (6%) from flood and sporadic cases, respectively. The study shows an unexpectedly high rate of isolation of E. coli pathotypes suggesting Pakistan as an endemic region that requires active surveillance particularly during flood periods. PMID:23358642

Growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7 on beef extract medium as influenced by package atmosphere and storage temperature.

PubMed

Semanchek, J J; Golden, D A; Williams, R C

1999-03-01

The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.

Adenosine monophosphate affects competence development and plasmid DNA transformation in Escherichia coli.

PubMed

Zhang, Yan; Li, Wenhua; Wang, Liming; Shen, Ping; Xie, Zhixiong

2013-11-01

Artificial plasmid DNA transformation of Escherichia coli induced by calcium chloride is a routine technique in molecular biology and genetic engineering processes, but its mechanism has remained elusive. Because adenosine monophosphate (AMP) has been found to regulate natural transformation in Haemophilus influenza, we aimed to investigate the effects of AMP and its derivatives on E. coli transformation by treating competence with different concentrations of them. Analysis of the transformation efficiencies revealed that AMP inhibited the artificial plasmid DNA transformation of E. coli in a concentration- and time-dependent manner. Furthermore, we found that AMP had no effect on the expression of the transformed gene but that the intracellular AMP level of the competent cells rose after a 6 h treatment. These results suggested that the intracellular AMP level had an important role in E. coli transformation. And these have useful implications for the further investigation of the mechanism of E. coli transformation.

Modelling Escherichia coli concentrations in the tidal Scheldt river and estuary.

PubMed

de Brauwere, Anouk; de Brye, Benjamin; Servais, Pierre; Passerat, Julien; Deleersnijder, Eric

2011-04-01

Recent observations in the tidal Scheldt River and Estuary revealed a poor microbiological water quality and substantial variability of this quality which can hardly be assigned to a single factor. To assess the importance of tides, river discharge, point sources, upstream concentrations, mortality and settling a new model (SLIM-EC) was built. This model was first validated by comparison with the available field measurements of Escherichia coli (E. coli, a common fecal bacterial indicator) concentrations. The model simulations agreed well with the observations, and in particular were able to reproduce the observed long-term median concentrations and variability. Next, the model was used to perform sensitivity runs in which one process/forcing was removed at a time. These simulations revealed that the tide, upstream concentrations and the mortality process are the primary factors controlling the long-term median E. coli concentrations and the observed variability. The tide is crucial to explain the increased concentrations upstream of important inputs, as well as a generally increased variability. Remarkably, the wastewater treatment plants discharging in the study domain do not seem to have a significant impact. This is due to a dilution effect, and to the fact that the concentrations coming from upstream (where large cities are located) are high. Overall, the settling process as it is presently described in the model does not significantly affect the simulated E. coli concentrations. Copyright © 2011 Elsevier Ltd. All rights reserved.

The effect of regions of interest and spectral pre-processing on the detection of non-O157 shiga-toxin producing escherichia coli serogroups on agar media by hyperspectral imaging

USDA-ARS?s Scientific Manuscript database

Food borne infection caused by Shiga toxin-producing Escherichia coli (STEC) is a major worldwide health concern. The best known STEC serotype is E. coli O157:H7, which can be easily identified when cultured on sorbitol-MacConkey (SMAC) agar. Recently, six non-O157 STEC serotypes have been found t...

Identification of Diarrheagenic Escherichia coli Strains from Avian Organic Fertilizers

PubMed Central

Puño-Sarmiento, Juan; Gazal, Luis Eduardo; Medeiros, Leonardo P.; Nishio, Erick K.; Kobayashi, Renata K. T.; Nakazato, Gerson

2014-01-01

The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections. PMID:25170683

Antibacterial Activity of Emulsified Pomelo (Citrus grandis Osbeck) Peel Oil and Water-Soluble Chitosan on Staphylococcus aureus and Escherichia coli.

PubMed

Chen, Guan-Wen; Lin, Yu-Hsin; Lin, Chia-Hua; Jen, Hsiao-Chin

2018-04-06

This study utilized pomelo steam distillation to isolate pomelo peel essential oil. The constituents were then analyzed through gas chromatography-mass spectrometry (GC-MS), and the antibacterial activity of the essential oil emulsions at different homogenizer speed conditions and concentrations of water-soluble chitosan (degree of acetylation, DA = 54.8%) against S. aureus and E. coli was examined. Analysis of the essential oil composition identified a total of 33 compounds with the main constituent, limonene accounting for 87.5% (940.07 mg/g) of the total. The pomelo peel oil was emulsified through homogenization at 24,000 rpm, resulting in a minimal inhibitory concentration (MIC) for E. coli that was 1.9 times lower than that of the essential oil without homogenization. In addition, a mixture of 0.4% essential oil emulsion and 0.03% water-soluble chitosan had the strongest synergetic antibacterial effect on S. aureus and E. coli at pH 7.4. In comparison with chitosan alone, the MIC value of this mixture was significantly 2.4 and 2.5 times lower. Hence, this study suggests using a mixture of emulsified pomelo peel oil and water-soluble chitosan to develop a novel natural food preservative, and that the processability of food, as well as the economic value of the byproducts of the Taiwan Matou pomelo and chitosan, could be increased.

Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar

PubMed Central

Andualem, Berhanu; Gessesse, Amare

2013-01-01

Objective To investigate and optimize microbial media that substitute peptone agar using brebra seed defatted flour. Methods 'Defatted process, inoculums preparation, evaluation of bacterial growth, preparation of cooked and hydrolyzed media and growth turbidity of tested bacteria were determined. Results Two percent defatted flour was found to be suitable concentration for the growth of pathogenic bacteria: Escherichia coli (ATCC 25922) (E. coli), Pseudomonas aeruginosa (ATCC 27853), Salmonella (NCTC 8385) and Shigella flexneri (ATCC 12022) (S. flexneri), while 3% defatted flour was suitable for Staphylococcus aureus (ATCC 25923) (S. aureus). E. coli (93±1) and S. flexneri (524±1) colony count were significantly (P≤0.05) greater in defatted flour without supplement than in supplemented medium. E. coli [(3.72×109±2) CFU/mL], S. aureus [(7.4×109±2) CFU/mL], S. flexneri [(4.03×109±2) CFU/mL] and Salmonella [(2.37×109±1) CFU/mL] in non-hydrolyzed sample were statistically (P≤0.05) greater than hydrolyzed one and commercial peptone agar. Colony count of Salmonella [(4.55×109±3) CFU/mL], S. flexneri [(5.40×109±3) CFU/mL] and Lyesria moncytogenes (ATCC 19116) [(5.4×109±3) CFU/mL] on raw defatted flour agar was significantly (P≤0.05) greater than cooked defatted flour and commercial peptone agar. Biomass of E. coli, S. aureus, Salmonella and Enterococcus faecalis in non-hydrolyzed defatted flour is highly increased over hydrolyzed defatted flour and commercial peptone broth. Conclusions The defatted flour agar was found to be better microbial media or comparable with peptone agar. The substances in it can serve as sources of carbon, nitrogen, vitamins and minerals that are essential to support the growth of microorganisms without any supplements. Currently, all supplements of peptone agar are very expensive in the market. PMID:24075344