Transcription Analysis of stx1, marA, and eaeA Genes in Escherichia coli O157:H7 Treated with Sodium Benzoate

2008 ◽  
Vol 71 (7) ◽  
pp. 1469-1474 ◽  
Author(s):  
FAITH J. CRITZER ◽  
DORIS H. D'SOUZA ◽  
DAVID A. GOLDEN
Keyword(s):  
Escherichia Coli ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Sodium Benzoate ◽  
Antimicrobial Agents ◽  
Transcription Analysis ◽  
E Coli ◽  
Reverse Transcriptase Pcr ◽  
One Step ◽  
K 12

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37°C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate–treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

scholarly journals The Real-Time-Based Assessment of the Microbial Killing by the Antimicrobial Compounds of Neutrophils

2011 ◽  
Vol 11 ◽  
pp. 2382-2390 ◽  
Author(s):  
J. T. Atosuo ◽  
E.-M. Lilius
Keyword(s):  
Real Time ◽  
Antimicrobial Agents ◽  
Hypochlorous Acid ◽  
Bacterial Cells ◽  
Photorhabdus Luminescens ◽  
Bacterial Luciferase ◽  
E Coli ◽  
High Concentrations ◽  
Time Basis ◽  
K 12

A recombinantEscherichia coliK-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) fromPhotorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. ThisE. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H2O2) and myeloperoxidase (MPO) was assessed. In high concentrations, H2O2alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. WhenE. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H2O2/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

Novel Approach for Detecting Prohibited Species-Specific Central Nervous System Tissue Contamination in Meat by One-Step Real-Time Reverse Transcriptase PCR

2009 ◽  
Vol 72 (5) ◽  
pp. 1063-1069 ◽  
Author(s):  
M. M. NAGARAJAN ◽  
D. LONGTIN ◽  
C. SIMARD
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Compliance Monitoring ◽  
Reverse Transcriptase Pcr ◽  
Qrt Pcr ◽  
Tissue Contamination ◽  
One Step ◽  
Species Specific

The dissemination of prohibited species-specific central nervous system (CNS) tissue contamination in meat must be tracked to mitigate human health risk associated with bovine spongiform encephalopathy. The efficiency of compliance monitoring and risk control measures taken by concerned regulatory authorities at meat production facilities to avoid such contamination depends on the ability to detect CNS tissue with a reliable and adequately sensitive quantitative method. A rapid and convenient one-step real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was developed based on the absolute quantification of glial fibrillary acidic protein (GFAP) mRNA as a marker for CNS tissue contamination in meat. The GFAP RNA quantity corresponding to a percentage of CNS tissue in artificially spiked meat was determined using an appropriate in vitro transcribed target GFAP RNA as a calibration standard in the assay. The assay had a linear dynamic range of 102 to 109 copies of target RNA and was able to detect 0.01% CNS contamination in meat. Further evaluation consisted of an analysis of 272 random meat cuts from carcasses and 109 ground meat samples received from a federally inspected abattoir and two meat processing facilities, respectively, over a 5-month period. The analyzed samples were all negative for CNS tissue contamination at an arbitrarily set lower threshold of 0.025%. Overall, the newly developed one-step qRT-PCR may be useful as an objective quantitative compliance monitoring tool and for setting an acceptable low tolerance threshold for such contamination in meat.

scholarly journals Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

2008 ◽  
Vol 8 (1) ◽  
Author(s):  
Satyendra Kumar ◽  
Ramesh S Jadi ◽  
Sudeep B Anakkathil ◽  
Babasaheb V Tandale ◽  
Akhilesh Chandra Mishra ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Reverse Transcriptase Pcr ◽  
Chandipura Virus ◽  
One Step

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

2011 ◽  
Vol 79 (12) ◽  
pp. 4819-4827 ◽  
Author(s):  
Jin-Hyung Lee ◽  
Sushil Chandra Regmi ◽  
Jung-Ae Kim ◽  
Moo Hwan Cho ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Trinitrobenzene Sulfonic Acid ◽  
Content Type ◽  
E Coli ◽  
Colon Inflammation ◽  
K 12

ABSTRACTPathogenic 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 enterohemorrhagicEscherichia coliO157:H7. The antioxidant phloretin, which is abundant in apples, markedly reducedE. coliO157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensalE. coliK-12 biofilms. Also, phloretin reducedE. coliO157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyEandstx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgAandcsgB), and dozens of prophage genes inE. coliO157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production inE. coliO157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory responsein vitrousing 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 ofE. coliO157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensalE. colibiofilms.

scholarly journals Regulation of Cell Division, Biofilm Formation, and Virulence by FlhC in Escherichia coli O157:H7 Grown on Meat

2011 ◽  
Vol 77 (11) ◽  
pp. 3653-3662 ◽  
Author(s):  
Preeti Sule ◽  
Shelley M. Horne ◽  
Catherine M. Logue ◽  
Birgit M. Prüß
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Real Time ◽  
Real Time Pcr ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Cellular Functions ◽  
Content Type ◽  
E Coli ◽  
K 12

ABSTRACTTo understand the continuous problems thatEscherichia coliO157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC ofE. coliK-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of anE. coliO157:H7 parent strain to that of its isogenicflhCmutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in theflhCmutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity ofE. coliO157:H7 on meat by interfering with the signal transduction pathways.

scholarly journals Detection of Mumps Virus RNA by Real-Time One-Step Reverse Transcriptase PCR Using the LightCycler Platform

2008 ◽  
Vol 46 (12) ◽  
pp. 4049-4051 ◽  
Author(s):  
J. J. Leblanc ◽  
J. Pettipas ◽  
R. J. Davidson ◽  
G. A. Tipples ◽  
J. Hiebert ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Mumps Virus ◽  
Reverse Transcriptase Pcr ◽  
Virus Rna ◽  
One Step

Growth Response of Escherichia coli ATCC 35218 Adapted to Several Concentrations of Sodium Benzoate and Potassium Sorbate

2009 ◽  
Vol 72 (11) ◽  
pp. 2301-2307 ◽  
Author(s):  
N. ANGÉLICA SANTIESTEBAN-LÓPEZ ◽  
MÓNICA ROSALES ◽  
ENRIQUE PALOU ◽  
AURELIO LÓPEZ-MALO
Keyword(s):  
Escherichia Coli ◽  
Succinate Dehydrogenase ◽  
Sodium Benzoate ◽  
Growth Response ◽  
Antimicrobial Agents ◽  
Weak Acid ◽  
Potassium Sorbate ◽  
E Coli ◽  
Significant Difference ◽  
Adaptation Processes

Escherichia coli ATCC 35218 growth response was evaluated after repetitive cultivation in stepwise increasing antimicrobial agent concentrations (potassium sorbate or sodium benzoate) to observe its adaptation process to high weak-acid concentrations. The effect of antimicrobial (potassium sorbate or sodium benzoate) concentration (0 to 7,000 ppm) was tested using laboratory media. Cells adapted at 1,000 ppm were inoculated in media containing the same concentration of the antimicrobial; after that, cells were transferred to media containing a higher concentration, followed by repetitive cultivations. In every case, viable cells were determined by surface plating every hour up to 48 h. Logarithmic representations of survival or growing fraction were modeled using the Gompertz equation. Adapted and nonadapted cells were analyzed for plasmid presence as well as phosphofructokinase and succinate dehydrogenase activity. Bacterial growth was observed after adaptation processes in media formulated up to 7,000 ppm of potassium sorbate or sodium benzoate. Analyses of variance demonstrated that no significant difference (P > 0.05) in lag time or growth rate was observed among adapted cells cultured in media containing the studied concentrations for each of the antimicrobials tested. These results suggest that E. coli can be adapted to high weak-acid concentrations if the exposure is performed under sublethal conditions. Furthermore, there was demonstrated inhibition of the enzymes phosphofructokinase and succinate dehydrogenase by action of sodium benzoate and potassium sorbate, respectively. E. coli adaptation to antimicrobial agents was not related to plasmid presence but appears to be due to other action mechanisms .

scholarly journals Inducement and Reversal of Tetracycline Resistance in Escherichia coli K-12 and Expression of Proton Gradient-Dependent Multidrug Efflux Pump Genes

2005 ◽  
Vol 49 (8) ◽  
pp. 3578-3582 ◽  
Author(s):  
Miguel Viveiros ◽  
Ana Jesus ◽  
Mafalda Brito ◽  
Clara Leandro ◽  
Marta Martins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reverse Transcriptase ◽  
Induced Resistance ◽  
Efflux Pump ◽  
Tetracycline Resistance ◽  
Proton Gradient ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Reverse Transcriptase Pcr ◽  
K 12

ABSTRACT Expression of eight transporter genes of Escherichia coli K-12 and its ΔacrAB mutant prior to and after induction of both strains to tetracycline resistance and after reversal of induced resistance were analyzed by quantitative reverse transcriptase PCR. All transporter genes were overexpressed after induced resistance with acrF being 80-fold more expressed in the ΔacrAB tetracycline-induced strain.

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